genetics and functions of the sars coronavirus spike protein
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Louisiana State UniversityLSU Digital Commons
LSU Doctoral Dissertations Graduate School
2005
Genetics and functions of the SARS coronavirusspike proteinChad Michael PetitLouisiana State University and Agricultural and Mechanical College, [email protected]
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Recommended CitationPetit, Chad Michael, "Genetics and functions of the SARS coronavirus spike protein" (2005). LSU Doctoral Dissertations. 653.https://digitalcommons.lsu.edu/gradschool_dissertations/653
GENETICS AND FUNCTIONS OF THE SARS CORONAVIRUS SPIKE PROTEIN
A Dissertation
Submitted to the Graduate Faculty of the Louisiana State University and
Agricultural and Mechanical College in partial fulfillment of the
requirements for the degree of Doctor of Philosophy
in
The Interdepartmental Program in Veterinary Medical Sciences through the
Department of Pathobiological Sciences
By Chad M. Petit
B.S. Louisiana State University, 2001 B.S. Louisiana State University, 2001 B.S. Louisiana State University, 2001
December, 2005
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© Copyright 2005 Chad M. Petit
All Rights Reserved
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ACKNOWLEDGEMENTS
First, and foremost, I would like to thank my loving wife Lauren for her patience,
understanding and support that she has given me throughout my graduate career. I can
only hope that I will be able to reciprocate all that she has given me. I would also like to
thank my family especially my parents (Mom &Gene, Dad & Kim, Tammy & Mike,
Randy & Josie) for their support. Also, I would like to thank my grandparents (Joyce &
Arnie, Billy & Betty) and in memory of Leander & Betty Petit and William Gassen. I
would also especially like to thank my grandmother Mona Gassen for being a second
mother to me and who, along with my mother Gwyneth Duhe, made many sacrifices in
order to make sure I had every encouragement and opportunity growing up. Also, I
would like to express my gratitude to my father, Kevin Petit, who provided his support,
advice, and company whenever needed throughout my professional career. I also would
like to thank all of my life long friends, without whom my life would not be the same.
I would like to express my gratitude to Dr. Vladimir Chouljenko and Dr. Jeff
Melancon, both of whom provided invaluable assistance, suggestions, and instruction to
aid my research presented in my dissertation research. In addition, I owe thanks to all
members of the Kousoulas laboratory who gave helpful advice whenever asked. Special
thanks to Arun Iyer, Preston Fulmer, and Rafael Luna for all their advice and friendship
that we sharded in pursuit of our professional goals. I would also like to thank the
members of Genelab, Thaya Geudry, Ryan Stiles, and Mamie Burrell, for their assistance
in DNA sequencing and primer synthesis.
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I would like thank the faculty members that served on my graduate committee,
Dr. Ding Shih, Dr. Elmer Godeny, Dr. Patrick DiMario, and Dr. Dennis Ingram, for their
advice and guidance throughout my graduate career. Finally, I would like thanks Dr.
Konstantin G. Kousoulas for providing guidance, encouragement, support, and all of the
rope that he has given me throughout my stint in his laboratory. Without his “sink or
swim” method of mentorship, I would not have developed as much professionally as I
have.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ............................................................................................. iii
LIST OF TABLES .......................................................................................................... viii
LIST OF FIGURES .......................................................................................................... ix
ABSTRACT..................................................................................................................... xvi
CHAPTER I: INTRODUCTION .....................................................................................1 STATEMENT OF RESEARCH PROBLEM AND HYPOTHESIS..........................1 STATEMENT OF RESEARCH OBJECTIVES .........................................................2 LITERATURE REVIEW ..............................................................................................3
Taxonomy of Coronaviridae ...........................................................................3 Coronavirus Architecture ..............................................................................5
Virion Classification and Morphology .........................................................5 Structural Proteins.........................................................................................8
Nucleocapsid Protein ................................................................................8 Membrane Protein...................................................................................10 Envelope Protein.....................................................................................11 Hemagglutinin-Esterase Glycoprotein....................................................13 Spike Glycoprotein .................................................................................15
Nonstructural Proteins ................................................................................17 The Replicase Protein .............................................................................17
Novel SARS-CoV Specific Proteins...........................................................19 U274........................................................................................................19 U122........................................................................................................20
Protein Antigenicity ....................................................................................20 Organization of the Viral Genome ..............................................................21 The Coronavirus Lifecycle ...........................................................................25
Virus Attachment and Entry .......................................................................25 Viral Receptors ...........................................................................................28
Angiotensin Converting Enzyme 2.........................................................29 CD209L (L-SIGN)..................................................................................30
Genome Expression ....................................................................................31 Replication of Genomic RNA.....................................................................38 Translation of Viral Proteins.......................................................................40 Assembly and Release of Virions ...............................................................41 Cellular Proteins Involved in Coronavirus Replication ..............................44
HNRNP A1 .............................................................................................44 PTB .........................................................................................................45 PABP ......................................................................................................46 Mitochondrial Aconitase.........................................................................47
Pathology and Disease ..................................................................................47
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REFERENCES..............................................................................................................50
CHAPTER II: GENETIC ANALYSIS OF THE SARS-CORONAVIRUS SPIKE GLYCOPROTEIN FUNCTIONAL DOMAINS INVOLVED IN CELL-SURFACE EXPRESSION AND CELL-TO-CELL FUSION .................................................................................................................79
INTRODUCTION ........................................................................................................79 RESULTS ......................................................................................................................82
Genetic Analysis of S Glycoprotein Functional Domains .....................82 Effect of Mutations on S Synthesis ..........................................................86 Ability of Mutant S Glycoproteins to be Expressed on the Cell
Surface .................................................................................................89 Effect of Mutations on S-mediated Cell-to-Cell Fusion.........................92 Detection of the Intracellular Distribution of S Mutant
Glycoproteins Via Confocal Microscopy ..........................................98 Time-dependent Endocytotic Profiles of Wild-type and Mutant
S Proteins. ..........................................................................................101 DISCUSSION ..............................................................................................................104
Functional Domains of the S Ectodomain ............................................105 Functional Domains of the Endodomain of S.......................................107
MATERIALS AND METHODS ...............................................................................114 Cells ..........................................................................................................114 Plasmids ...................................................................................................114 Production of SARS-CoV S Monoclonal Antibodies ...........................115 Western Blot and S Oligomerization Analysis .....................................115 Cell Surface Immunohistochemistry.....................................................116 Determination of Cell-surface to Total Cell S Glycoprotein
Expression..........................................................................................117 Confocal Microscopy ..............................................................................117 S Glycoprotein Endocytosis Assay ........................................................118 Quantitation of the Extent of S-mediated Cell Fusion. .......................119
REFERENCES............................................................................................................120
CHAPTER III: GENETIC ANALYSIS OF THE CYSTEINE RICH DOMAINS IN THE CARBOXYL TERMINUS OF THE SARS-COV SPIKE GLYCOPROTEIN.................................................................................127
INTRODUCTION ......................................................................................................127 RESULTS ....................................................................................................................132
Genetic Analysis of S Glycoprotein Cysteine Rich Domain................132 Effects of Mutations on S Synthesis ......................................................133 Ability of Mutant S Glycoproteins to Be Expressed on the Cell
Surface. ..............................................................................................133 Effect of Mutations of S-mediated Cell-to-Cell Fusion. ......................138
DISSCUSSION............................................................................................................145 MATERIALS AND METHODS ...............................................................................147
Cells ..........................................................................................................147 Plasmids ...................................................................................................147
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Production of SARS-CoV S Monoclonal Antibodies ...........................148 Western Blot Analysis.............................................................................148 Cell Surface Immunohistochemistry.....................................................149 Determination of Cell-surface to Total Cell S Glycoprotein
Expression..........................................................................................150 Quantitation of the Extent of S-mediated Cell Fusion ........................150
REFERENCES............................................................................................................151
CHAPTER IV: GENETIC COMPARISON OF THE SARS-COV AND BCOV S GLYCOPROTEINS ...........................................................................158
INTRODUCTION ......................................................................................................158 RESULTS ....................................................................................................................166
Comparison of the Ectodomain of the SARS-CoV and BCoV Spike Glycoproteins ..........................................................................166
Comparison of the Endodomain of the SARS-CoV and BCoV Spike Glycoproteins ..........................................................................169
Effect of Mutations of S-mediated Cell-to-Cell Fusion .......................172 DISCUSSION ..............................................................................................................179
Comparison Between the SARS-CoV and BCoV Spike Proteins.......179 S-mediated Cell-to-Cell Fusion ..............................................................181
MATERIALS AND METHODS ...............................................................................185 Cells ..........................................................................................................185 Plasmids ...................................................................................................185 Total Protein Immunohistochemistry ...................................................186 Quantitation of the Extent of S-mediated Cell Fusion ........................186
REFERENCES............................................................................................................187
CHAPTER V: CONCLUDING REMARKS...............................................................195 SUMMARY .................................................................................................................195 CURRENT AND FUTURE RESEARCH CHALLENGES....................................198 REFERENCES............................................................................................................199
APPENDIX A: ADDITIONAL WORK ......................................................................203 VIRUS LIKE PARTICLE AND PACKAGING SYSTEM ....................................203 BOVINE CORONAVIRUS INFECTIOUS CLONE ..............................................207 REFERENCES............................................................................................................210
APPENDIX B: PERMISSION TO INCLUDE PUBLISHED WORK .....................215
VITA.................................................................................................................................218
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LIST OF TABLES
Table 1.1: Coronaviruses listed by their respective antigenic group with their host organisms and diseases they cause indicated..............................................4
Table 2.1: Grouping of S mutants by their cell-surface expression and their cell-fusion properties...........................................................................................97
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LIST OF FIGURES
Figure 1.1: A schematic diagram of a typical coronavirion structure. S, spike glycoprotein; HE hemagglutinin-esterase glycoprotein (found only in a subset of coronaviruses); M, membrane glycoprotein; E, small envelope protein; N nucleocapsid phosphoprotein .................................................6
Figure 1.2: Genomic Organization of several serotypes of coronavirus. The genome structure for five previously sequenced coronavirus RNAs are shown. All genes, except for gene 1 (pp1ab) are drawn approximately to scale. The severe acute respiratory syndrome (SARS-CoV), mouse hepatitis virus (MHV), avian infectious bronchitis virus (IBV), porcine transmissible gastroenteritis virus (TGEV), and human respiratory coronavirus (HCoV-229E) are composed of positive stranded RNA genomes of approximately 29.7, 31.2, 27.6, 28.5, and 27.2 kb respectively. The 5' end of RNA genome is capped and contains a 65- to 98-base long leader sequence (L). Structural proteins represented by shaded boxes while nonstructural proteins are shown as unshaded boxes. Vertical lines shown between open reading frames (ORFs) represent intergenic sequences. The area found bracketed by two intergenic sequences represents a single gene. Each separate ORF within that single gene is translated from a single mRNA species. Several variations exist between the serotypes of coronaviruses in the number, location, and sequence of ORFs encoding nonstructural proteins. The positive stranded RNAs encoding the coronavirus genomes are polyadenylated at their 3' end .................22
Figure 1.3: Schematic diagram of a typical coronavirus replication cycle. Interactions between the spike protein on the virion particle and specific receptor glycoproteins or glycans found on the surface of the target host cell facilitate virion binding to the plasma membrane of the host cell. Spike protein mediated fusion between plasma membrane or endosomal membrane with the viral envelop allows penetration of the cell by the virus. Once inside the cell, gene one encoded by the positive sensed RNA genome is translated into a large polyprotein. Cotranslational or posttranslational processing of this large polyprotein produces an RNA-dependent RNA polymerase as well as several other proteins that are involved in viral RNA synthesis. The genomic RNA is then used as a template by the RNA-dependent RNA polymerase and the other polyprotein products to synthesize negative stranded RNAs. These negative stranded RNAs are subsequently used to produce genomic and subgenomic mRNAs. These mRNAs have an overlapping nested set of 3' coterminal RNAs that possess a common leader sequence at the 5' end. Each of the subgenomic mRNAs only have their 5' most ORF translated into viral proteins effectively making them monocistronic. Proteins produced include the S, spike glycoprotein; M, membrane glycoprotein; E, small envelope protein; and N nucleocapsid phosphoprotein as well as
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several nonstructural proteins. Also the HE (hemagglutinin-esterase glycoprotein) is also produced in a small subset of coronaviruses. Once translated, the N protein interacts in the cytoplasm with newly synthesized genomic RNA in order to form helical nucleocapsids. The M and E proteins are inserted in the endoplasmic reticulum and anchored in the Golgi apparatus. The helical nucleocapsid, produced through N protein and RNA interactions, most likely first joins with M at the budding compartment which is located between the rough endoplasmic reticulum and the Golgi apparatus. Interactions between the M and E proteins elicit the budding of virions which encloses the helical nucleocapsid. Also associated in the Golgi, the S and HE structural proteins are translated on membrane-bound polysomes, inserted into the rough endoplasmic reticulum and then transported to the Golgi complex. During protein transport, some of the S and HE proteins interact with the M protein and are incorporated into maturing virion particles. S and HE proteins not incorporated into virions are transported to the cellular surface where they may play a role in mediating cell-cell fusion or hemaadsorption, respectively. Virions seem to be released by exocytosis-like fusion of smooth-walled vesicles that contain the virion particles with the plasma membrane. Virions also may remain attached to the plasma membranes of infected cells. The entire coronavirus replication cycle takes place solely in the cytoplasm of the host cell............................................................................26
Figure 1.4: Schematic diagram of the three proposed models of coronavirus mRNA transcription. Positive stranded RNA is represented by solid black lines while negative stranded RNA is represented by dashed grey lines. Leader RNA and antileader RNA is represented by black and grey boxes respectively. Arrows are used to indicate direction of transcription. ........33
Figure 2.1: Schematic diagram of the SARS-CoV S glycoprotein. (A) Graphical representation of the S glycoprotein showing the approximate location of the cluster to lysine mutations CL1-CL5 relative to known and indicated functional domains. (B) Shown on the top of the diagram is a graphical representation of the SARS-CoV S glycoprotein. The predicted fusion peptide and the HR1 region are enlarged below to show the sets of amino acids replaced by lysines in the cluster mutations. The heptad repeat a and d positions are labeled above the corresponding amino acid. Amino acids changed to lysine are demarcated by arrows with the name of that particular mutation shown in brackets. (C) Amino acid sequences of the carboxyl termini of the truncation and acidic cluster associated mutations. Cysteine clusters (CRM1 and CRM2) are denoted by underlined italicized text as well as a bracket encompassing their respective regions. The charged cluster is bracketed over the region. Amino acids mutated to alanines for the CL6 and CL7 cluster mutations are in bold.................................83
Figure 2.2: Western blot analysis of the expressed mutant SARS-CoV S mutant glycoproteins. (A,B) Immunoblots of wild-type (3xFLAG So
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(WT)), cluster to lysine, cluster to alanine, and carboxyl truncation mutant S glycoproteins probed with monoclonal anti-SARS S antiserum. "Cells only" represents a negative control in which Vero cells with no protein transfected into them were probed with the monoclonal antibody to SARS S glycoprotein. (B) In order to detect trimer formation more efficiently, the protein extracts of the mutants were neither boiled nor subject to treatment with beta mercaptoethanol ....................................................................87
Figure 2.3: Immunohistochemical detection of cell-surface and total expression of the SARS-CoV S wild-type and mutant proteins. Vero cells were transfected with the wild-type SARS-CoV optimized S (3xFLAG So (WT)) (F1, F2), CL1 (A1, A2), CL2 (B1, B2), CL3 (C1, C2), CL4 (D1, D2), CL5 (E1, E2), CL6 (L1, L2), CL7 (M1, M2), T1214 (K1, K2), T1229 (J1, J2), T1238 (I1, I2), T1247(H1, H2) and a wild-type SARS-CoV optimized S labeled with a 3xFLAG carboxyl tag (G1, G2), which served as a negative control. At 48 hours post-transfection, cells were immunhistochemically processed either under live conditions to show surface expression. (A2, B2, C2, D2, E2, F2, G2, H2, I2, J2, K2, L2, and M2) or fixed and permeabilized conditions to show total expression(A1, B1, C1, D1, E1, F1, G1, H1, I1, J1, K1, L1, and M1) with anti-FLAG antibody ..............................................................................................90
Figure 2.4: Ratios of cell-surface to total cellular expression of mutant SARS-CoV S glycoproteins. Detection of cell surface and total glycoprotein distribution was determined by immunohistochemistry and ELISA (see Materials and Methods). Cell-surface expression of the S glycoprotein was measured by incubating the transfected cell monolayers with anti-FLAG antibody at room temperature before permeabilization. For total S glycoprotein detection, cells were fixed and permeabilized prior to incubation with the anti-FLAG antibody. A ratio between the surface localization and the total expression was calculated and normalized to the wild type protein, then set to a percentage of the wild-type. The error bars represent the maximum and minimum surface to total ratios obtained from three independent experiments, and the bar height represents the average surface to total ratio ..........................................................93
Figure 2.5: Quantitation of the extent of S-mediated cell fusion. The average size of syncytia for each mutant was determined by digitally analyzing the area of approximately 300 syncytia stained by immunohistochemistry for S glycoprotein expression using the Image Pro Plus 5.0 software package (see Materials and Methods). Error bars shown represent the standard deviations calculated through comparison of the data from each of three experiments. ..........................................................................................................95
Figure 2.6: Confocal microscopic visualization of endocytosed and intracellular distribution of SARS-CoV S glycoprotein mutants. Vero cells expressing wild-type SARS-CoV S glycoprotein (3xFLAG So (WT))
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(A and B), CL6 (C and D), CL7 (E and F), T1229 (G and H), T1238 (I and J) and T1247 (K and L) were processed for confocal microscopy using two different methods in order to assess different properties of the mutants. Endocytosis patterns (A,C,E,G,I, and K) were visualized by adding anti-FLAG (green) antibody into the media 12 h prior to processing, enabling detection of the mutant protein after endocytosis from cellular surfaces. Early endosomes were also detected for these panels using a polyclonal anti-early endosomal antigen I antibody (red). For total glycoprotein detection (B, D, F, H, J, and L), cells were fixed and permeabilized prior to labeling with anti-FLAG (green). .....................................99
Figure 2.7: Analysis of the endocytotic kinetic profile of the truncation mutants and the acidic cluster mutants using confocal microscopy. After transfection, SARS-CoV S glycoprotein wild-type (3xFLAG So (WT)) (A1-A4), T1229 (B1-B4), T1238 (C1-C4), T1247 (D1-D4), CL6 (E1-E4), and CL7 (F1-F4) expressing cells were incubated with an anti-FLAG monoclonal antibody (green) for 1 hr and then returned to 37ºC for different times. Cell nuclei were labeled with To-Pro-3 Iodide (blue). Panels A1-A4, B1-B4, C1-C4, D1-D4, E1-E4, and F1-F4 correspond to 0-, 5-, 15-, and 60-min incubation times at 37ºC, respectively. ...............................102
Figure 2.8: Cluster alignment of the carboxyl terminus of the SARS-CoV S and the MHV S glycoproteins. The shaded residues indicate the position of the cysteine rich motif in their respective protein. The cysteine residues are bolded. The charged clusters are indicated by a bracket over the corresponding regions. ........................................................................................111
Figure 3.1: Alignment of the membrane spanning domain and endodomain of the spike glycoprotein from ten different coronaviruses (Abraham et al., 1990; Binns et al., 1985; Delmas et al., 1992; Kunkel and Herrler, 1993; Luytjes et al., 1987; Marra et al., 2003; Mounir and Talbot, 1993; Parker, Gallagher, and Buchmeier, 1989; Raabe, Schelle-Prinz, and Siddell, 1990; Rasschaert and Laude, 1987). A schematic diagram of the SARS-CoV S protein is shown on top from amino acid 1 to amino acid 1255. A vertical line demarcates the approximate location of the division between the S1 and S2 subunits of the protein. The carboxyl terminus (amino acids 1193 to 1255) of the SARS-CoV S glycoprotein is shown enlarged below and is aligned with the same region of the S glycoprotein from nine other coronaviruses. Viruses from antigenic group I (feline infectious peritonitis virus [FIPV], transmissible gastroenteritis virus [TGEV’, human coronavirus 229E [HCoV-229E]), antigenic group II (three different mouse hepatitis virus strains [A59, JHM, and MHV2], bovine coronavirus [BCoV], and human coronavirus OC43 [HCoV-OC43]), and antigenic group III (infectious bronchitis virus [IBV]) are represented in the alignment. The membrane spanning domain and the cytoplasmic tail are denoted with arrows above the alignment. Residues conserved in at least eight of the ten coronaviruses represented are
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indicated by the shaded residues. Cysteines that are highly conserved throughout all of the S proteins are noted by asterisks. ......................................130
Figure 3.2: Schematic diagram of the SARS-CoV S glycoprotein endodomain and the cysteine cluster to alanine mutations. Amino acid sequences of the carboxyl termini and the cysteine cluster to alanine mutations are shown for the wild-type as well as the mutant proteins. The cysteine cluster and the charged rich regions of the S proteins are encompassed in brackets and appropriately labeled. The transmembrane portion of the endodomain is italicized and underlined. Amino acids mutated to alanines for the C1217A, C1223A, C1230A, and C1235A cluster mutations are in bold. ..............................................................................134
Figure 3.3: Western blot analysis of the expressed mutant SARS-CoV mutant glycoproteins. Immunoblots of wild-type [So-3xF(WT)] and cysteine to alanine mutant S glycoproteins probed with monoclonal anti-SARS S antiserum. “Cells only” represents a negative control in which Vero cells with no protein transfected into them were probed with the monoclonal antibody to the SARS-CoV S glycoprotein.....................................136
Figure 3.4: Immunohistochemical detection of cell-surface and total expression of the SARS-CoV S wild-type and mutant proteins. Vero cells were transfected with the wild-type SARS-CoV optimized S (SARS So 3xF) (E1, E2), C1217A (A1, A2), C1223A (B1,B2), C1230A (C1,C2), C1235A (D1,D2), and a wild-type SARS-CoV optimized S labeled with a 3xFLAG carboxyl tag (F1, F2), which served as a negative control. At 48 hours post-transfection, cells were immunohistochemically processed wither under live conditions to show surface expression (A1, B1, C1, D1, E1, and F1) and permeablilized conditions to show total expression (A2, B2, C2, D2, E2, and F2) with anti-FLAG antibody. ...........................................139
Figure 3.5: Ratios of cell-surface to total cellular expression of mutant SARS-CoV S glycoproteins. Detection of cell surface and total glycoprotein distribution was determined by immunohistochemistry and ELISA (see materials and methods). Cell-surface expression of the S glycoprotein was measured by incubating the transfected cell monolayers with anti-FLAG antibody at room temperature before permeabilization. For total S glycoprotein detection, cells were fixed and permeabilized prior to incubation with the anti-FLAG antibody. A ratio between the surface localization and the total expression was calculated and normalized to the wild-type protein, then set to a percentage of the wild-type. The error bars represent the maximum and minimum surface to total ratios obtained from three independent experiments, and the bar height represents the average surface to total ratio as a percentage of the wild-type. .....................................................................................................................141
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Figure 3.6: Quantitation of the extent of S-mediated cell fusion. The average size of syncytia for each mutant was determined by digitally analyzing the area of approximately 300 syncytia stained by immunohistochemistry for S glycoprotein expression using the Image Pro Plus 5.0 software package (see Materials and Methods). Error bars shown represent the standard deviation calculated through comparison of the data from each of three experiments. ........................................................................................................143
Figure 4.1: Schematic diagram of the BCoV S glycoprotein denoting the differences between RBCoV and EBCoV. (Chouljenko et al., 2001) A schematic diagram of the BCoV S protein is shown on top from amino acid 1 to amino acid 1363. The transmembrane region of the protein is noted with a grey box labeled “Tm”. The two heptad repeat regions are indicated with grey boxes labeled HR1 and HR2 respectively. A vertical line demarcates the location of the division between the S1 and S2 subunits of the protein with the amino acid number between S1 and S2 shown. Amino acid differences are denoted with the amino acid number shown and the corresponding residues listed below. ..........................................160
Figure 4.2: Schematic diagram of the SARS-CoV and BCoV S glycoprotein endodomains. Amino acid sequences of the carboxyl termini are shown for the wild-type S glycoproteins. The cysteine cluster and the charged rich regions of the S proteins are encompassed in brackets and appropriately labeled. The shaded residues represent the transmembrane portion of the endodomain. Amino acids that serve as predicted phosphorylation sites are denoted by asterisks ...................................................164
Figure 4.3. Alignment of the membrane spanning domain and endodomain of the spike glycoprotein from ten different coronaviruses (Abraham et al., 1990; Binns et al., 1985; Delmas et al., 1992; Kunkel and Herrler, 1993; Luytjes et al., 1987; Marra et al., 2003; Mounir and Talbot, 1993; Parker, Gallagher, and Buchmeier, 1989; Raabe, Schelle-Prinz, and Siddell, 1990; Rasschaert and Laude, 1987). A schematic diagram of the SARS-CoV S protein is shown on top from amino acid 1 to amino acid 1255. A vertical line demarcates the approximate location of the division between the S1 and S2 subunits of the protein. The carboxyl terminus (amino acids 1193 to 1255) of the SARS-CoV S glycoprotein is shown enlarged below and is aligned with the same region of the S glycoprotein from nine other coronaviruses. Viruses from antigenic group I (feline infectious peritonitis virus [FIPV], transmissible gastroenteritis virus [TGEV’, human coronavirus 229E [HCoV-229E]), antigenic group II (three different mouse hepatitis virus strains [A59, JHM, and MHV2], bovine coronavirus [BCoV], and human coronavirus OC43 [HCoV-OC43]), and antigenic group III (infectious bronchitis virus [IBV]) are represented in the alignment. The membrane spanning domain and the cytoplasmic tail are denoted with arrows above the alignment. Residues conserved in at least eight of the ten coronaviruses represented are
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indicated by the shaded residues. Cysteines that are highly conserved throughout all of the S proteins are noted by asterisks .......................................167
Figure 4.4. Alignment heptad repeat regions of the S1 subunit of the spike glycoprotein from SARS-CoV and BCoV (Abraham et al., 1990; Marra et al., 2003)A schematic diagram of the SARS-CoV S protein is shown on top from amino acid 1 to amino acid 1255. The transmembrane region of the protein is noted with a black box labeled “Tm”. The two heptad repeat regions are indicated with striped boxes and labels HR1 and HR2 respectively. A vertical line demarcates the approximate location of the division between the S1 and S2 subunits of the protein. The two regions containing the heptad repeats (amino acids 879 to 980) of the SARS-CoV S glycoprotein is shown enlarged below and is aligned with the same region of the S glycoprotein from BCoV. Residues conserced between the two viruses represented are overlayed by shaded boxes .....................................170
Figure 4.5. Schematic diagram of the SARS-CoV and BCoV S glycoprotein endodomains. Amino acid sequences of the carboxyl termini are shown for the wild-type S glycoproteins as well as truncated proteins. Each SARS-CoV S truncation is coupled with its corresponding BCoV S truncation. The cysteine cluster and the charged rich regions of the S wild-type proteins are encompassed in brackets and appropriately labeled. The shaded residues represent the transmembrane portion of the endodomain. Amino acids that serve as predicted phosphorylation sites are denoted by asterisks.......................................................................................173
Figure 4.6. Immunohistochemical detection of total expression of the BCoV S wild-type and truncated proteins. Vero cells were transfected with T1359 (Panel A), T1355 (Panel B), T1346 (Panel C), and T1333 (Panel D), and wild-type respiratory (BCoV-Lun So 3xF) (Panel E) and enteric (BCoV-End So 3xF) (Panel F) strains of the BCoV optimized S) labeled with an amino terminus 3xFLAG tag. At 48 hours post-transfection, cells were immunohistochemically processed wither under permeabilized conditions to show total expression with anti-FLAG antibody...........................175
Figure 4.7. Quantitation of the extent of S-mediated cell fusion. The average size of syncytia for each mutant was determined by digitally analyzing the area of approximately 300 syncytia stained by immunohistochemistry for S glycoprotein expression using the Image Pro Plus 5.0 software package (see Materials and Methods). The fusion levels quantitated for the VERO-E6 cell line are represented by black bars while the levels for VERO cells are represented by grey bars. Error bars shown represent the standard deviation calculated through comparison of the data from each of three experiments .........................................................................................................177
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ABSTRACT
The SARS-coronavirus (SARS-CoV) is the etiological agent of the severe acute
respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates
membrane fusion events during virus entry and virus-induced cell-to-cell fusion.
Investigations, described herein, have focused on the genetic manipulation of the SARS-
CoV S glycoprotein in order to delineate functional domains within the protein. This was
accomplished by incorporating single point mutations, cluster-to-lysine and cluster-to-
alanine mutations, as well as carboxyl terminal truncations into the protein and
investigating these mutants in transient expression experiments. Mutagenesis of either the
coiled-coil domain of the S glycoprotein amino terminal heptad repeat, the predicted
fusion peptide, or adjacent but distinct regions, severely compromised S-mediated cell-to-
cell fusion, while intracellular transport and cell-surface expression were not adversely
affected. Surprisingly, a carboxyl terminal truncation of 17 amino acids substantially
increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17
amino acids regulate the S fusogenic properties. In contrast, truncation of 26 or 39 amino
acids eliminating either one or both of the two endodomain cysteine-rich motifs,
respectively, inhibited cell fusion in comparison to the wild-type S. The cysteine rich
domains were further studied by constructing cysteine cluster to alanine mutants in order
to ascertain their importance in the function of the protein. Results showed that the two
cysteine clusters proximal to the transmembrane region were vital in the functioning of
the spike protein in mediating cell-to-cell fusion. Mutagenesis of the acidic amino acid
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cluster in the carboxyl terminus of the S glycoprotein as well as modification of a
predicted phosphorylation site within the acidic cluster revealed that this amino acid
motif may play a functional role in the retention of S at cell-surfaces. A panel of
truncations for Bovine Coronavirus (BCoV) S was also constructed and compared to
truncations made for the SARS-CoV S glycoprotein. It was found that the two sets of
truncations had very little comparable effects on protein function when compared to one
another. This genetic analysis reveals that the SARS-CoV S glycoprotein contains
extracellular domains that regulate cell fusion as well as distinct endodomains that
function in intracellular transport, cell-surface expression and cell fusion.
1
CHAPTER I
INTRODUCTION
STATEMENT OF RESEARCH PROBLEM AND HYPOTHESIS
The severe acute respiratory syndrome coronavirus (SARS-CoV) is the most
recently discovered coronavirus. It first appeared in the Guangdong Province of southern
China in November of 2002. Unlike other human coronaviruses whose infections are
usually very mild, the SARS-CoV produced mortality rates as high as 15% in some age
groups (Anand et al., 2003). Due to this high mortality rate, intense research interest has
been generated to elucidate certain aspects of the virus life cycle as well as characteristics
of viral proteins that could possibly allude to why this virus is so lethal when compared to
the other serotypes of human coronavirus.
The SARS-CoV encodes for the typical genes found in all coronaviruses which
include the nonstructural replicase gene as well as the structural proteins nucleocapsid
(N), membrane protein (M), envelope protein (E), and spike glycoprotein (S), which are
assembled into virus particles. The S glycoprotein is primarily responsible for entry of
all coronaviruses into susceptible cells through binding to specific receptors on cells and
mediating subsequent virus-cell fusion (Cavanagh, 1995). Because of the roles it plays in
virus attachment, virus entry, and cell-cell fusion, the S glycoprotein seems to potentially
be the protein most likely responsible for the increase in virulence of the virus.
Recently the SARS-CoV S glycoprotein has been codon optimized for
mammalian cells in order to allow for more efficient expression of the protein (Li et al.,
2003). This optimization of the protein allows the protein to be studied using a transient
transfection system. The overall experimental approach was to introduce site specific
2
mutations and truncations into the codon optimized SARS-CoV S glycoprotein in order
to ascertain the functional impact of certain motifs found in the S glycoprotein. The
delineation of functional domains in the endodomain as well as the role of hydrophobic
amino acids found in and around the heptad repeat regions were the focus of Chapter II.
A second aspect of the work focused on cysteine rich clusters in the endodomain of the S
protein that are conserved throughout all coronaviruses. Cysteine cluster to alanine
mutations were introduced into the SARS-CoV S glycoprotein in order to determine the
importance of several conserved cysteines. These mutants were then tested for their
ability to mediate cell-cell fusion as compared to the wild-type proteins as well as their
ability to be properly transported to the cellular surface; this work is described in Chapter
III. A codon optimized version of the bovine coronavirus (BCoV) S glycoprotein was
constructed in order to do a comparison of the motifs found in the endodomain of the
BCoV S protein versus the SARS-CoV S protein. Several truncations of the BCoV S
protein were made in order to compare them with homologous truncations made in the
SARS-CoV S (described in Chapter IV).
STATEMENT OF RESEARCH OBJECTIVES
The overall goal of this research was to gain a more thorough understanding of
the functional domains found in the SARS-CoV S glycoprotein. The specific objectives
were as follows: 1) To delineate functional domains in the SARS-CoV S glycoprotein; 2)
To ascertain the importance of the conserved cysteine residues found in the endodomain
of the SARS-CoV S glycoprotein; 3) To compare the impact on protein function of
homologous truncations made in both the SARS-CoV and BCoV S glycoproteins.
3
LITERATURE REVIEW
Taxonomy of Coronaviridae
Nidovirales is the taxonomic order in which enveloped, positive-sense RNA
viruses that synthesize a 3' co-terminal set of subgenomic mRNAs during infection of
host cells are grouped in (Cavanagh, 1997). Members of this order include
coronaviruses, toroviruses, and arteriviruses. The toroviruses and coronaviruses are
further grouped into the family Coronaviridae that contain variable length genomes and
nucleocapsid structure.
There are three groups of coronaviruses that have little to no cross-reactivity
among antigens found on virions of the different groups (Table 1.1). Two of the groups
are composed of mammalian coronaviruses, while avian coronaviruses such as the
Infectious Bronchitis Virus (IBV) and Turkey Coronavirus (TCoV) form a third group
(Guy et al., 1997; Pedersen, Ward, and Mengeling, 1978). The model virus for the first
group of coronaviruses is the HCoV-229E. Coronaviruses of pigs (transmissible
gastroenteritis virus [TGEV] and porcine respiratory coronavirus [PRCoV]), cats (feline
coronavirus [FCoV], and dogs (canine coronavirus [CCoV]) (Pedersen, Ward, and
Mengeling, 1978) belong to group I. Murine Hepatitis Virus (MHV) is the prototype of
group II coronaviruses. Some viruses found in this group are the coronaviruses of
humans (HCoV-OC43), rats (rat coronavirus [RCoV]), cattle (bovine coronavirus
[BCoV]), pigs (hemagglutinating encephalomyelitis virus [HEV]), and other species
(Pedersen, Ward, and Mengeling, 1978). Analysis of the severe acute respiratory
4
5
syndrome associated coronavirus (SARS-CoV) viral genome has demonstrated that it is
phylogenetically divergent from the three known antigenic groups of coronaviruses
(Drosten et al., 2003; Ksiazek et al., 2003). Analysis of the polymerase gene alone,
however, indicates that the SARS-CoV may be an early off-shoot from the group II
coronaviruses (Snijder et al., 2003).
Coronavirus Architecture
Virion Classification and Morphology
The basic structure of coronavirus virions is depicted in Figure 1.1. Coronavirus
virions are spherical enveloped particles that have a diameter of 100 to 120 nm. Interior
to the virion is a single-stranded, positive-sense RNA genome that ranges from 27 to 32
kb in length, the largest of all known RNA viral genomes (Boursnell et al., 1987; Eleouet
et al., 1995; Herold et al., 1993; Lai and Cavanagh, 1997; Lee et al., 1991). The SARS-
CoV genome, specifically, has a length of 29,727 nucleotides (Rota et al., 2003). The
RNA genome is associated with the nucleocapsid (N) phosphoprotein which allows it to
form a long, flexible, helical nucleocapsid (Macneughton and Davies, 1978; Sturman,
Holmes, and Behnke, 1980). When not associated with virion particles, the
nucleocapsids appear as extended tubular strands of 14 to 16 nm in diameter (Risco et al.,
1996; Sturman, Holmes, and Behnke, 1980). It has been shown that for at least two
coronaviruses (TGEV and MHV), the helical nucleocapsid is enclosed within a 65 nm in
diameter, spherical, possibly icosahedral “internal core structure” that can be released
from the virion particle by NP-40 treatment (Risco et al., 1996). This virus core is
encapsidated by a lipoprotein envelope that is formed during virus budding from
6
Figure 1.1. A schematic diagram of a typical coronavirion structure. S, spike glycoprotein; HE hemagglutinin-esterase glycoprotein (found only in a subset of coronaviruses); M, membrane glycoprotein; E, small envelope protein; N nucleocapsid phosphoprotein.
7
8
intracellular membranes (Griffiths and Rottier, 1992; Oshiro, 1973; Tooze and Tooze,
1985). The most prominent feature of the coronavirus virion is the “halo of crowns”
found to line the outside of the virion. These long proteins (20 nm in length) consist of
the spike (S) glycoprotein and are present on all coronaviruses. Some other
coronaviruses have shorter spike proteins that also line the outside of the virion which
consist of the hemagglutinin-esterase (HE) glycoprotein. The matrix (M) glycoprotein,
because it spans the lipid bilayer three times (Machamer et al., 1993; Machamer et al.,
1990; Machamer and Rose, 1987), is thought to be a component of both the internal core
structure and the envelope. The envelope protein (E) also makes up part of the viral
envelope, although it is present in much smaller quantities than the other viral envelope
proteins (Godet et al., 1992; Vennema et al., 1996; Yu et al., 1994).
Structural Proteins
Nucleocapsid Protein
The coronavirus nucleocapsid protein, N, is a 50 to 60 kDa highly basic protein
that interacts with the viral genome in order to form the viral nucleocapsid. More
specifically, the SARS-CoV N protein is 422 amino acids that shares only a 20-30%
homology with other coronaviruses (Marra et al., 2003; Rota et al., 2003). Translation of
the protein occurs on free polysomes and is rapidly phosphorylated on serine residues in
the cytosol (Stern and Sefton, 1982); the extent and physiological relevance of
phosphorylation, however, remains unclear (Stohlman and Lai, 1979; Wilbur et al.,
1986). The synthesized protein consists of three highly conserved domains that are
separated by spacer regions of variable length (Parker and Masters, 1990).
9
The most prominent feature of the N protein is its ability to bind to RNA. Several
studies have pinpointed the RNA binding region of the N protein to the second of the
three conserved domains found in the N protein (Masters, 1992; Nelson and Stohlman,
1993; Nelson, Stohlman, and Tahara, 2000; Peng et al., 1995). The complexes formed
between the N protein and the viral RNA of coronaviruses and orthomyxoviruses are
more easily disrupted at high salt concentrations and offer little protection against RNase
when compared to the complexes formed by N proteins and viral RNA of rhabdoviruses
and paramyxoviruses (Masters and Sturman, 1990).
The second type of interaction is N protein interaction with itself. Disulfide
linked multimeric forms of the N protein have been shown to exist (Robbins et al., 1986).
It has also been shown for the SARS-CoV that the carboxyl terminal conserved domain
functions as a dimerization domain (He et al., 2004). The third type of interaction is the
N protein’s interaction with the matrix glycoprotein (Sturman, Holmes, and Behnke,
1980) which leads to the formation of virus particles.
In addition to the structural role of N, it is hypothesized that it may function in
viral RNA synthesis, transcription, translation, and virus budding (He et al., 2003; Lai
and Cavanagh, 1997; Tahara et al., 1998). Specifically, it has been shown that the N
protein participates in RNA synthesis. It was found that RNA synthesis was inhibited by
greater than 90% when antibodies to N were introduced into an in vitro synthesizing
system prepared from MHV-infected cells (Compton et al., 1987). This inhibition
implies a critical role for the N protein in transcription and replication of the viral RNA.
Although N plays a critical role in RNA synthesis, the relative amounts of free N protein
in infected cells are quite large and do not appear to be the rate limiting factor for MHV
10
RNA synthesis (Perlman et al., 1986). Also, some N protein may be complexed with
cellular membranes (An, Maeda, and Makino, 1998; Stern and Sefton, 1982) where it
functions in budding of the virus. The SARS-CoV N protein has not been extensively
studied, except that the SARS-CoV N protein may selectively activate the AP-1 signal
transduction pathway (He et al., 2003).
Membrane Protein
The membrane glycoprotein, the most abundant envelope component, is a triple-
membrane spanning protein with a short amino-terminus on the virion exterior surface,
an α-helical domain, and a large carboxyl terminal domain inside the virion envelope
(Armstrong et al., 1984; Locker et al., 1992; Machamer et al., 1993; Routledge et al.,
1991). For some coronaviruses, however such as TGEV, the carboxyl terminus of the M
protein is exposed on the surface of the virion (Risco et al., 1995). The protein is
inserted into the ER membrane through the action of a signal sequence (Rottier,
Armstrong, and Meyer, 1985) after being synthesized on membrane bound polysomes.
After being synthesized, the protein undergoes posttranslational modification in the form
of glycosylation. Interestingly, for group I and group III coronaviruses, such as TGEV
and IBV, the matrix protein undergoes N-linked glycosylation (Stern and Sefton, 1982),
whereas for group II coronaviruses, such as MHV, the matrix protein undergoes O-linked
glycosylation (Holmes, Doller, and Sturman, 1981; Niemann et al., 1982; Niemann et al.,
1984). Through the use of a virus like particle system it has been demonstrated that
neither glycosylation of the M protein nor its interaction with the S glycoprotein is
necessary for virus assembly (de Haan et al., 1998; de Haan et al., 1999). These
observations are consistent with studies using the glycosylation inhibitors tunicamycin
11
(Rossen et al., 1998; Stern and Sefton, 1982) and monensin (Niemann et al., 1982) in
infected cells. Mature matrix protein accumulates in the Golgi apparatus and is not
transported to the plasma membrane (Machamer et al., 1990; Machamer and Rose, 1987;
Swift and Machamer, 1991). The nature of the membrane-targeting sequence that causes
this accumulation in the Golgi varies for each coronavirus (Locker et al., 1992;
Machamer et al., 1993).
One of the main functions of the M protein is to direct the incorporation of the S
glycoprotein (Nguyen and Hogue, 1997; Opstelten et al., 1995) and the N protein
(Narayanan et al., 2000) into the budding virion particle. The M protein itself does not,
however, determine the actual budding site, since when expressed by itself it migrates
beyond the budding compartment and localizes in the late-Golgi complex (Klumperman
et al., 1994). Interactions with other viral proteins have been shown to be mediated
through the carboxyl terminus of the protein (Corse and Machamer, 2003; de Haan et al.,
1998; Kuo and Masters, 2002). For several coronaviruses, cells expressing the M and E
proteins alone have been shown to produce virion like particles which are exported from
the cell (Baudoux et al., 1998; Bos et al., 1996; Corse and Machamer, 2000; Corse and
Machamer, 2003; de Haan et al., 1998; Vennema et al., 1996). For the SARS-CoV,
however, this is not the case. Expression of M and E alone is not sufficient for capsid
formation, however the expression of M and N alone is able to produce virus capsids
(Huang et al., 2004).
Envelope Protein
The small envelope protein, E, is typically a 9 to 12 kDa protein that is a
component of the virion envelope (Godet et al., 1992; Liu et al., 1991; Yu et al., 1994).
12
When compared to the other structural proteins, however, the E protein is in much lower
abundance relative to the M, N, and S proteins (Godet et al., 1992; Liu and Inglis, 1991;
Yu et al., 1994). Within the three groups of coronaviruses, the E proteins are well
conserved, but between the three groups, they show limited homology. All of the
proteins do share a general structure: a short hydrophilic region on the amino terminus,
followed by a large hydrophobic region, preceding a large hydrophilic carboxyl terminus
(Liu and Inglis, 1991). Recent studies of the topology of the protein have found for
MHV and IBV that the E protein is an integral membrane protein presenting its carboxyl
terminus on the cytoplasmic face of the endoplasmic reticulum or Golgi which
corresponds to the interior of the assembled virion (Corse and Machamer, 2000;
Raamsman et al., 2000; Vennema et al., 1996). For IBV, this Golgi targeting of the
molecule has been shown to be associated with the carboxyl tail, in the absence of the
membrane-bound domain (Corse and Machamer, 2002). The orientation of the amino
terminus of the protein has not been well established; one study shows a luminal
orientation for the IBV E protein (Corse and Machamer, 2000) and another study shows
the amino terminus being buried within the membrane near the cytoplasmic face for the
MHV protein (Maeda et al., 2001). E protein is localized primarily in the perinuclear
space of infected cells, although it also has been detected on the cell surface (Godet et al.,
1992; Yu et al., 1994). The main function of the E protein is its role in the formation of
the coronavirus envelope. For several coronaviruses, expression of MHV M and E alone
has been shown to be sufficient for the formation of virion like particles which are
exported from the cell (Baudoux et al., 1998; Bos et al., 1996; Corse and Machamer,
2000; Corse and Machamer, 2003; de Haan et al., 1998; Vennema et al., 1996). For the
13
SARS-CoV, however, expression of M and E alone is not sufficient for capsid formation,
although expression of M and N is sufficient for capsid formation (Huang et al., 2004).
Another function of the E protein is its ability to induce apoptosis in the infected cell (An
et al., 1999).
The SARS-CoV envelope protein is approximately 76 amino acids long with an
approximate molecular weight of 10-15 kDa. Several studies of the SARS-CoV E
protein have shown additional properties that may be specific to the SARS-CoV. The
SARS-CoV E protein has the ability to form cation-selective ion channels (Wilson et al.,
2004) similar to those of the influenza virus protein, M2 (Duff and Ashley, 1992; Duff et
al., 1994; Pinto, Holsinger, and Lamb, 1992), the HIV-1 proteins VPu (Ewart et al., 1996)
and VPr (Piller et al., 1996), and the hepatitis C virus protein, p7 (Griffin et al., 2003;
Pavlovic et al., 2003; Premkumar et al., 2004). In addition, a segment of the SARS-CoV
E protein appears to form three disulfide bonds with another segment of corresponding
cysteines in the carboxyl-terminus of the S glycoprotein (Wu et al., 2003). The SARS-
CoV E protein appears to have a unique structural feature in that it forms a highly
unusually short, palindromic transmembrane helical hairpin around a previously
unidentified pseudo-center of symmetry (Arbely et al., 2004). It is through the action of
this hairpin, by way of deforming the lipid bilayer and increasing membrane curvature,
that there finally may be a molecular explanation of the E proteins pivotal role in virus
budding (Arbely et al., 2004).
Hemagglutinin-Esterase Glycoprotein
Although not found in the SARS-CoV, another protein found on some
coronaviruses is the hemagglutinin-esterase (HE) glycoprotein (Rota et al., 2003). The
14
protein exists as a homodimer of 65 to 70 kDa proteins that form short spikes on the
surface of some group II coronavirus virions as well as turkey coronavirus virions
(Hogue, Kienzle, and Brian, 1989; Kienzle et al., 1990; Kunkel and Herrler, 1993;
Schultze et al., 1991). Cotranslational N-linked glycosylation occurs in the rough
endoplasmic reticulum after which the dimerization of proteins occurs by disulfide
interactions to form a 165 to 170 kDa protein complex (Hogue, Kienzle, and Brian, 1989;
Kienzle et al., 1990; Yokomori et al., 1989). This protein complex is then incorporated
into the envelope of budding virus particles after being transported to the Golgi where the
N-linked glycans are converted to the complex form (Yokomori et al., 1989). HE that
does not incorporate into the virion envelope is expressed on cellular surfaces (Kienzle et
al., 1990). Some studies have hypothesized that the HE protein is dispensable for viral
replication since the presence or absence is highly inconsistent even among the group II
coronaviruses. In addition, the HE gene is frequently mutated or completely deleted
during serial virus passaging in cell culture (Yokomori, Banner, and Lai, 1991). The HE
protein of various coronaviruses binds 9-O-acetylated neuraminic acid residues which is
comparable to the binding activity of S in BCoV and HCoV-OC43 (Schultze and Herrler,
1992; Schultze et al., 1991; Vlasak et al., 1988). This activity is expected to contribute to
the hemagglutination and hemadsorption activities of coronaviruses. The HE protein also
has an acetylesterase activity that cleaves acetyl groups from 9-O-acetylated neuraminic
acid (Vlasak et al., 1988; Yokomori et al., 1989) which reverses or prevents the
hemagglutination and hemadsorption activity induced by S and HE. This acetylesterase
activity, along with the inhibition of BCoV infectivity through neutralization of HE with
15
monoclonal antibodies (Deregt et al., 1989), points to a role for HE in virus entry or virus
release from infected cells.
Spike Glycoprotein
The SARS spike glycoprotein, a 1,255-amino-acid type I membrane glycoprotein
(Rota et al., 2003), is the major protein present in the viral membrane forming the typical
spike structure found on all coronavirions. Mature proteins form oligomers in the form
of homotrimers (Delmas and Laude, 1990; Xiao et al., 2004) and are known to be
glycosylated. Posttranslational glycosylation occurs on at least four different sites of the
spike protein (Krokhin et al., 2003; Ying et al., 2004). Once fully processed the protein
exists as a 180 kDa single protein species with the homotrimer species of approximately
500 kDa. Although some S oligomers can be found on the infected cell surface where it
may mediate cell-cell fusion, most newly synthesized S accumulates in the Golgi of
infected cells where it participates in virus particle assembly (Griffiths and Rottier, 1992;
Vennema et al., 1990). The S glycoprotein of mouse hepatitis virus (MHV) is cleaved
into (Cavanagh, 1995) S1 and S2 subunits, although cleavage is not necessarily required
for virus-cell fusion (Bos, Luytjes, and Spaan, 1997; Gombold, Hingley, and Weiss,
1993; Stauber, Pfleiderera, and Siddell, 1993). Similarly, the SARS-CoV S glycoprotein
seems to be cleaved into S1 and S2 subunits in Vero-E6 infected cells (Wu et al., 2004),
but it is not known whether this cleavage affects S-mediated cell fusion. This cleavage of
the SARS-CoV S protein, however, may be attributed to overexpression of the protein
and may not occur in the context of the virus (personal communication). The S protein in
group I coronaviruses appears not to be cleaved although some of the viruses can induce
cell-cell fusion (De Groot et al., 1989). The S1 subunit contains the receptor binding
16
domain which causes it to be the main determinate of viral tropism (Hingley et al., 1994;
Sanchez et al., 1999; Suzuki and Taguchi, 1996). Angiotensin converting enzyme 2
(ACE2) (Li et al., 2003) and CD209L (L-SIGN) (Jeffers et al., 2004) have been identified
to be two receptors for the SARS-CoV. The S2 subunit contains internal hydrophobic
sequences which are thought to be responsible for the membrane fusion activity of the
protein (Luo and Weiss, 1998). It is worth noting, however, that sequences in multiple
sites of S2 as well as in S1 can affect fusion activity (Gallagher, Escarmis, and
Buchmeier, 1991; Routledge et al., 1991). Expression of the S protein alone has been
shown to induce membrane fusion on cells that are susceptible to coronavirus infection
(De Groot et al., 1989; Taguchi, 1993; Yoo, Parker, and Babiuk, 1991).
The internal hydrophobic sequences that are found in the S2 portion of the SARS
S glycoprotein are known as heptad repeats (HR). These regions contain a sequence motif
characteristic of coiled-coils which appear to be a common motif in many viral and
cellular fusion proteins (Skehel and Wiley, 1998). These coiled-coil regions allow the
protein to fold back upon itself as a prerequisite step to initiating the membrane fusion
event. There are usually two HR regions: an N terminal HR region adjacent to the fusion
peptide and a C-terminal HR region close to the transmembrane region of the protein.
Within the HR segments, the first amino acid (a) and fourth amino acid (d) are typically
hydrophobic amino acids that play a vital role in maintaining coiled-coil interactions.
Based on structural similarities, two classes of viral fusion proteins have been
established. Class I viral fusion proteins contain two heptad repeat regions and an N-
terminal or N-proximal fusion peptide. Class II viral fusion proteins lack heptad repeat
regions and contain an internal fusion peptide (Lescar et al., 2001). The MHV S
17
glycoprotein, which is similar to other coronavirus S glycoproteins, is a class I membrane
protein that is transported to the plasma membrane after being synthesized in the
endoplasmic reticulum (Bosch et al., 2003). Typically, the ectodomains of the S2
subunits of coronaviruses contain two regions with a 4, 3 hydrophobic (heptad) repeat the
first being adjacent to the fusion peptide and the other being in close proximity to the
transmembrane region (de Groot et al., 1987).
The S glycoprotein is primarily responsible for entry of all coronaviruses into
susceptible cells through binding to specific receptors on cells and mediating subsequent
virus-cell fusion (Cavanagh, 1995). Although the exact mechanism by which the SARS-
CoV enters the host cell has not been elucidated, it is most likely similar to other
coronaviruses. Upon receptor binding at the cell membrane, the S glycoprotein is thought
to undergo a dramatic conformational change causing exposure of a hydrophobic fusion
peptide, which is subsequently inserted into cellular membranes. This conformational
change of the S glycoprotein causes close apposition followed by fusion of the viral and
cellular membranes resulting in entry of the virion nucleocapsids into cells (Eckert and
Kim, 2001; Tsai et al., 2003; Zelus et al., 2003). This series of S-mediated virus entry
events is similar to other class I virus fusion proteins (Baker et al., 1999; Melikyan et al.,
2000; Russell, Jardetzky, and Lamb, 2001).
Nonstructural Proteins
The Replicase Protein
Two replicase polyproteins, termed pp1a and pp1ab, are produced from two open
reading frames (ORF1a and ORF1b) contained within the first 21 kb of the
approximately 29.7 kb SARS-CoV genome (Marra et al., 2003; Rota et al., 2003; Thiel et
18
al., 2003). Pp1a is an approximately 486 kDa polyprotein that has been predicted to
include a papain-like protease (PLpro), two putative membrane proteins, MP1 (nsp4) and
MP2 (nsp6), a picornavirus 3C-like protease (3CLpro), and several other products of
unknown function. Pp1ab is approximately 790 kDa. It is generated by ribosomal
frameshifting so that both ORF1a and ORF1b are included in the translation. It is
predicted that ORF1b contains a helicase domain (nsp13) (Ivanov et al., 2004) as well as
the predicted core RNA polymerase (nsp12), exonuclease (nsp14), endoribonuclease
(nsp15), and methyltransferase (nsp16) activities (Schmidt-Mende et al., 2001; Snijder et
al., 2003). A total of 16 protein products (nonstructural proteins nsp1 to nsp16),
produced from the processed pp1a and pp1ab polyproteins, are predicted to assemble into
a membrane-associated viral replication complex (Snijder et al., 2003; Stadler et al.,
2003; Thiel et al., 2003). This processing of the polyproteins has been hypothesized to be
coordinated by two virus-encoded proteinases, the picornavirus 3C-like proteinase
(3CLpro) and a papain-like proteinase (PLP) (Denison et al., 1992; Snijder et al., 2003;
Thiel et al., 2003). The precursor proteins and mature processed replicase proteins likely
mediate the progression from replication complex formation, followed by subgenomic
mRNA transcription, and finally genome replication. Essential functions carried out by
the replication complex are as follows: 1) transcription of genome-length negative and
positive stranded RNAs, 2) transcription of a 3´-coterminal nested set of subgenomic
mRNAs that have a common 5´ “leader” sequence derived from the 5´ end of the
genome, and 3) transcription of the subgenomic derived negative-stranded RNAs with
common 5´ ends and complementary leader sequences at their 3´ ends (Lai and Holmes,
2001; Thiel et al., 2003). Replicase complex activity has been shown to take place at
19
double-membrane vesicles in the host cell cytoplasm (Gosert et al., 2002; Pedersen et al.,
1999; Prentice et al., 2004).
Novel SARS-CoV Specific Proteins
In addition to the four main structural proteins (S, E, M, and N) and the viral
encoded RNA dependent RNA polymerase, there are nine other potential ORFs found in
the SARS-CoV genome that vary from 39 to 274 amino acids in length (Marra et al.,
2003; Rota et al., 2003; Thiel et al., 2003). The functions of these additional ORFs are
largely unknown and further characterization of these proteins is necessary to be able to
assess any impact on the virus life cycle.
U274
The largest of the additional ORFs (ORF 3a) and the second largest subgenomic
mRNA encode a protein that has been termed U274 because of its length of 274 amino
acids (Tan et al., 2004c). The subgenomic mRNA encoding U274 has been found in high
quantities in infected cells and also has a strong match to the transcription regulating
consensus sequence close to and upstream of its first ORF (Marra et al., 2003; Rota et al.,
2003; Thiel et al., 2003). The exact function of the protein is unknown but a region in its
carboxyl terminus is similar to calcium-transporting adenosine triphosphatases (Marra et
al., 2003; Rota et al., 2003; Snijder et al., 2003; Thiel et al., 2003). Analysis of sera taken
from patients infected with the SARS-CoV has shown that the sera contains antibodies
against the U274 protein, which indicates that the protein may play a role in the
biogenesis of SARS-CoV (Tan et al., 2004b). In cells infected with SARS-CoV and cells
transiently transfected with U274, the protein localized to the plasma membrane as well
as to the perinuclear region (Tan et al., 2004c). This surface expression may explain the
20
presence of U274 antibodies found in sera taken from infected patients mentioned above
(Tan et al., 2004b). Surfaced expressed U274 is able to undergo endocytosis and its
cytoplasmic domains contain sorting signals that allow proper transport of the protein
(Tan et al., 2004c). Interactions were observed between the U274 protein and other
structural proteins (M, E, and S) as well as the uncharacterized SARS-CoV protein U122.
This suggests that the U274 protein may play a role in virus assembly (Tan et al., 2004c).
U122
Another group specific gene product encoded by ORF7a (also known as ORFX4
or ORF8) (Marra et al., 2003; Rota et al., 2003; Snijder et al., 2003) has been partially
characterized (Fielding et al., 2004). Like the U274 protein, U122 derives its name from
its amino acid length. The U122 appears to localize to the perinuclear region and is
associated with the endoplasmic reticulum in both cells infected with the SARS-CoV and
in cells transiently transfected with the U122 protein (Fielding et al., 2004). From
sequence analysis, it appears that the protein is a type I membrane protein because of its
probable cleaved signal peptide located on its amino terminal and a carboxyl terminal
transmembrane helix (Fielding et al., 2004). Another study has indicated that over-
expression of U122 is able to induce apoptosis via a caspase-dependent pathway (Tan et
al., 2004a).
Protein Antigenicity
The induction of the host immunological response against coronavirus infection is
largely due to the spike glycoprotein (Collins et al., 1982; Koolen et al., 1990; Spaan,
Cavanagh, and Horzinek, 1988). Analysis of its antigenic structure has been useful
particularly in vaccine design (Posthumus et al., 1990). Particular emphasis has been
21
placed on locating potential neutralization epitopes on the spike glycoprotein which
include linear and conformational epitopes (Daniel et al., 1993; Kubo, Yamada, and
Taguchi, 1994; Moore, Jackwood, and Hilt, 1997; Takase-Yoden et al., 1991; Talbot and
Buchmeier, 1985; Vautherot, Laporte, and Boireau, 1992; Vautherot et al., 1992).
Induction of a protective immune response was able to be accomplished through
immunization of animals with a synthetic peptide from a particular immunodominant
region of the MHV spike protein (Daniel, Lacroix, and Talbot, 1994; Koo et al., 1999;
Koolen et al., 1990; Yu et al., 2000). This immunodominant region of the MHV spike
was also shown to be recognized by viral neutralizing antibodies (Daniel et al., 1993;
Luytjes et al., 1989; Talbot and Buchmeier, 1985; Talbot et al., 1984). As with other
coronaviruses, there have been regions of the SARS-CoV spike that have been found that
are able to elicit a neutralizing antibody response from animals injected with the synthetic
peptides from these regions (Zhang et al., 2004). Expression of the other structural
proteins (M, E, or N), in the absence of SARS-CoV spike expression, is unable to provide
protective immunity to SARS-CoV infection (Buchholz et al., 2004).
Organization of the Viral Genome
The genomes of several coronaviruses are compared and shown in Figure 1.2.
Coronaviral genomes are capped, polyadenylated, positive stranded RNAs of 27 to 32 kb
in length. Since they are positive strand genomes, they are able to serve as mRNAs and it
has been shown that the purified genomic RNA is infectious (Lomniczi, 1977;
Schochetman, Stevens, and Simpson, 1977). The leader RNA is a sequence of 65 to 98
nucleotides that is present at the 5' end of the genome as well as at the 5' ends of all
22
Figure 1.2. Genomic Organization of several serotypes of coronavirus. The genome structure for five previously sequenced coronavirus RNAs are shown. All genes, except for gene 1 (pp1ab) are drawn approximately to scale. The severe acute respiratory syndrome (SARS-CoV), mouse hepatitis virus (MHV), avian infectious bronchitis virus (IBV), porcine transmissible gastroenteritis virus (TGEV), and human respiratory coronavirus (HCoV-229E) are composed of positive stranded RNA genomes of approximately 29.7, 31.2, 27.6, 28.5, and 27.2 kb respectively. The 5' end of RNA genome is capped and contains a 65- to 98-base long leader sequence (L). Structural proteins represented by shaded boxes while nonstructural proteins are shown as unshaded boxes. Vertical lines shown between open reading frames (ORFs) represent intergenic sequences. The area found bracketed by two intergenic sequences represents a single gene. Each separate ORF within that single gene is translated from a single mRNA species. Several variations exist between the serotypes of coronaviruses in the number, location, and sequence of ORFs encoding nonstructural proteins. The positive stranded RNAs encoding the coronavirus genomes are polyadenylated at their 3' end.
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24
subgenomic mRNAs (Lai et al., 1983; Shieh et al., 1987; Spaan et al., 1983). This leader
sequence is followed by an untranslated region of approximately 200 to 400 nucleotides.
There is another untranslated region of approximately 200 to 500 nucleotides at the 3' end
of the genome which is followed by a poly(A) tail that varies in length. The sequences
that make up both the 5' and 3' untranslated region are essential for RNA transcription
and replication. The remaining genome is made up of 7 to 10 open reading frames that
encode the genes necessary for virus replication. Specifically for SARS, the genome
contains 10 open reading frames encoding the replicase gene, 4 structural proteins, and 5
potential nonstructural genes that are more than 50 amino acids in length (Rota et al.,
2003). The first open reading frame makes up two thirds of the viral genome
(approximately 20 to 22 kb in length) and encodes a polyprotein that is the precursor of
the viral polymerase. This gene actually consists of two over lapping open reading
frames that is effectively combined into a single open reading frame through ribosomal
frame shifting. The order in which the polymerase polyprotein and the four structural
proteins found in all coronaviruses is 5'-Pol-S-E-M-N-3'. Several other open reading
frames that encode a variety of nonstructural proteins can be found interspersed between
the known structural proteins. Some coronaviruses also contain a gene that encodes for a
hemagglutinin-esterase protein. The number of nonstructural genes present as well as
their order in the viral genome, sequence, and method of expression vary widely among
coronaviruses (Lai and Cavanagh, 1997). The functions of most of these nonstructural
proteins are widely unknown; some are even absent in genomes of some coronaviruses
(Schwarz, Routledge, and Siddell, 1990; Yokomori and Lai, 1991).
25
The Coronavirus Lifecycle
A general schematic of the major occurrences in the coronavirus lifecycle are
depicted in Figure 1.3.
Viral Attachment and Entry
The initial step in any virus life cycle is the binding of virions to the plasma
membrane of the host cell. The viral protein that is primarily responsible for the binding
of the virion to the plasma membrane of the host cell is the spike glycoprotein. It is the
spike glycoprotein that drives infection of the host cell by facilitating viral attachment to
the target cell and promoting fusion between the host cell plasma membrane and the viral
membrane. This fusion of the two membranes allows the insertion of the viral genome
into the cellular cytoplasm. Viral attachment is accomplished through the interaction of
the spike protein and specific-receptor glycoproteins on the cell surface. Given its role in
viral attachment, the spike protein is the main determinant of viral tropism.
After binding to the specific cellular receptor, the virus enters the cell through the
fusion event between the viral envelope and the plasma membrane or the endosomal
membrane of the host cell. The pH of the environment has been shown to affect the
efficiency of membrane fusion for some coronaviruses such as BCoV, MHV, and IBV
whose optimum pH for cell-cell fusion is either neutral or slightly alkaline (Li and
Cavanagh, 1992; Payne and Storz, 1988; Sturman, Ricard, and Holmes, 1990; Weismiller
et al., 1990). Given that these viruses may cause fusion of the viral envelop with the
plasma membrane at the typical physiological pH of the extracellular environment, it is
likely that they probably enter cells by virus-cell fusion at the plasma membrane. For
26
Fig. 1.3. Schematic diagram of a typical coronavirus replication cycle. Interactions between the spike protein on the virion particle and specific receptor glycoproteins or glycans found on the surface of the target host cell facilitate virion binding to the plasma membrane of the host cell. Spike protein mediated fusion between plasma membrane or endosomal membrane with the viral envelop allows penetration of the cell by the virus. Once inside the cell, gene one encoded by the positive sensed RNA genome is translated into a large polyprotein. Cotranslational or posttranslational processing of this large polyprotein produces an RNA-dependent RNA polymerase as well as several other proteins that are involved in viral RNA synthesis. The genomic RNA is then used as a template by the RNA-dependent RNA polymerase and the other polyprotein products to synthesize negative stranded RNAs. These negative stranded RNAs are subsequently used to produce genomic and subgenomic mRNAs. These mRNAs have an overlapping nested set of 3' coterminal RNAs that possess a common leader sequence at the 5' end. Each of the subgenomic mRNAs, with few exceptions, only have their 5' most ORF translated into viral proteins effectively making them monocistronic. Proteins produced include the S, spike glycoprotein; M, membrane glycoprotein; E, small envelope protein; and N nucleocapsid phosphoprotein as well as several nonstructural proteins. Also the HE (hemagglutinin-esterase glycoprotein) is produced in a small subset of coronaviruses. Once translated, the N protein interacts in the cytoplasm with newly synthesized genomic RNA in order to form helical nucleocapsids. The M and E proteins are inserted in the endoplasmic reticulum and anchored in the Golgi apparatus. The helical nucleocapsid, produced through N protein and RNA interactions, most likely first joins with M at the budding compartment which is located between the rough endoplasmic reticulum and the Golgi apparatus. Interactions between the M and E proteins elicit the budding of virions which encloses the helical nucleocapsid. Also associated in the Golgi, the S and HE structural proteins are translated on membrane-bound polysomes, inserted into the rough endoplasmic reticulum and then transported to the Golgi complex. During protein transport, some of the S and HE proteins interact with the M protein and are incorporated into maturing virion particles. S and HE proteins not incorporated into virions are transported to the cellular surface where they may play a role in mediating cell-cell fusion or hemaadsorption, respectively. Virions seem to be released by exocytosis-like fusion of smooth-walled vesicles that contain the virion particles with the plasma membrane. Virions also may remain attached to the plasma membranes of infected cells. The entire coronavirus replication cycle takes place solely in the cytoplasm of the host cell.
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MHV, however, the efficiency of virus infectivity has been shown to be reduced through
the use of lysosomotropic drugs which suggests that the virus utilizes the endosomal
pathway for entry (Gallagher, Escarmis, and Buchmeier, 1991; Krzystyniak and Dupuy,
1984; Mizzen et al., 1985). Further experimental evidence indicates that different
coronaviruses can enter cells either by the acidic pH-dependent endocytosis or by pH
independent fusion at the plasma membrane (Kooi, Cervin, and Anderson, 1991). Once
the virus has entered the host cell, the next step in life cycle is the uncoating of the virus
and the release of the genomic RNA into the cytoplasm. The mechanism for this
uncoating and release is not currently clear and may require specific cellular proteins in
addition to the incorporated viral elements. Some murine cells, despite having the
presence of a functional MHV receptor, are known to block MHV infections at
penetration, uncoating, or other steps in entry (Asanaka and Lai, 1993; Flintoff, 1984;
Yokomori et al., 1993). These cell types can by grouped into three complementation
groups which suggests that at least three cellular genes are involved in the entry process
(Asanaka and Lai, 1993)
Viral Receptors
Receptors have been elucidated for several coronaviruses. For mouse hepatitis
virus, the receptor is a murine biliary glycoprotein which belongs to the
carcinoembryonic antigen (CEA) family in the Ig superfamily (Dveksler et al., 1991;
Williams, Jiang, and Holmes, 1991). The spike glycoprotein binds to the N-terminal Ig-
like domain of the mouse hepatitis virus receptor in order to facilitate the fusion of the
two membranes (Dveksler et al., 1993b). MHV attachment to host cells can be blocked
by monoclonal antibodies to the viral receptor (Parker, Gallagher, and Buchmeier, 1989;
29
Shieh et al., 1987). Further studies have shown that the MHV is able to use other
members of the CEA family and several human CEA-related glycoproteins as receptors
for viral entry (Chen et al., 1997; Chen et al., 1995; Dveksler et al., 1993a; Nedellec et
al., 1994; Yokomori and Lai, 1992a; Yokomori and Lai, 1992b). Other coronaviruses
such as HCoV-229E, TGEV, FIPV, and most likely canine coronavirus, use the cell
membrane-bound metalloprotease, aminopeptidase N (APN) that is specific for their host
species (Benbacer et al., 1997; Delmas et al., 1992; Yeager et al., 1992) as their entry
receptor. Unlike the CEA molecules which are normally expressed in cells of the liver,
gastrointestinal tract, macrophages, and B cells, but not thymic T cells (Coutelier et al.,
1994; Godfraind et al., 1995), APN is widely dispersed on many cell types which include
respiratory and enteric epithelial cells as well as neuronal and glial cells (Kusters et al.,
1989). Some monoclonals against APN have been shown to block binding of TGEV or
HCoV-229E virions to the host cell receptor (Delmas et al., 1992; Yeager et al., 1992);
however APN protease activity is not a requirement for viral infection (Delmas et al.,
1992; Williams, Jiang, and Holmes, 1991). The cellular receptor for SARS-CoV was
originally found to be the metallopeptidase angiotensin-converting enzyme 2 (ACE2) (Li
et al., 2003; Wang et al., 2004). Recently, however, an additional receptor, CD209L (L-
SIGN), has been shown to function as a receptor for the SARS-CoV (Jeffers et al., 2004).
Angiotensin Converting Enzyme 2
Angiotensin-converting enzyme 2 is a type I transmembrane protein that is made
up of 805 amino acids. It contains a single metalloprotease active site with an HEXXH
zinc binding domain (Kuhn et al., 2004). The ACE2 protein is synthesized in the human
heart muscle, kidneys, testis (Donoghue et al., 2000), gastrointestinal tract, and the lungs
30
(Hamming et al., 2004). In particular, high levels of ACE2 expression have been found
by immunohistochemical examinations in the endothelium of intramyocardial and
intrarenal vessels and in the renal tubular epithelium (Donoghue et al., 2000). The
enzyme appears be the physiological counterweight of the related angiotensin-converting
enzyme (ACE) which is known to cleave the inactive peptide angiotensin I in order to
produce the highly potent vasoconstrictor angiotensin II (Kuhn et al., 2004). Specifically,
ACE2 has been shown to cleave angiotensin I to the metabolite angiotensin (1-9), which
in turn is cleaved to angiotensin (1-7) (Donoghue et al., 2000; Harmer et al., 2002). It
also cleaves des-arg-bradykinin, neurotensin, and kinetensin (Donoghue et al., 2000).
There is significant overlap between the tissues that express the ACE2 protein and those
tissues that are most correlated with SARS-CoV replication and symptomatic
manifestation (Hamming et al., 2004). An obvious example of this overlap would be the
lungs. The lungs are the primary site of SARS-CoV infection (Kuiken et al., 2003) ,and
the lungs express the ACE2 protein (Donoghue et al., 2000; Hamming et al., 2004;
Harmer et al., 2002; Li et al., 2003). A less obvious example is the gastrointestinal tract
and kidneys. These areas have high levels of ACE2 expression and have also been
shown to be an active site of SARS-CoV infection (Donoghue et al., 2000; Hamming et
al., 2004; Harmer et al., 2002). SARS-CoV has not, however, been found to replicate in
the human heart which is a site a high expression of ACE2 (Donoghue et al., 2000).
CD209L (L-SIGN)
CD209L, a homologue of CD209, is a type II transmembrane glycoprotein in the
C-type lectin family that serve as adhesion receptors for ICAM-2 and ICAM-3
(Bashirova et al., 2001; Geijtenbeek et al., 2000; Mummidi et al., 2001). The isoform
31
that has been identified as the receptor for the SARS-CoV is composed of 376 amino
acids. Structurally, the protein has a short cytoplasmic tail, a transmembrane domain, an
extracellular stalk that contains seven repeats of a 23 amino acid sequence
(KAAVGELXEKSKXQEIYQELTXL), and a large carboxyl terminal carbohydrate
recognition domain (Jeffers et al., 2004). This carbohydrate recognition domain has been
shown to bind specifically to high mannose glycans on glycoproteins (Geijtenbeek,
Engering, and Van Kooyk, 2002). It has not yet been determined if it is this carbohydrate
recognition domain that participates in viral interactions with the SARS-CoV spike
glycoprotein, or if the SARS-CoV spike glycoprotein recognizes the protein’s specific
amino acid sequence.
Genome Expression
Once released into the cytoplasm, the viral RNA serves as a template for the
synthesis of the viral RNA dependent RNA polymerase. This RNA dependent RNA
polymerase first synthesizes negative stranded RNAs which are used as templates for the
synthesis of multiple subgenomic RNAs as well as full length copies of the genome. It
has been shown that there are comparable levels of negative polarity genomic and
subgenomic RNA to levels of corresponding positive sense genomic and subgenomic
mRNA in infected cells (Sawicki and Sawicki, 1990; Sethna, Hofmann, and Brian, 1991;
Sethna, Hung, and Brian, 1989). Although levels were comparable, there was no single-
stranded negative sense RNA found in the infected cell, only double-stranded (Perlman et
al., 1986; Sawicki and Sawicki, 1986; Sawicki and Sawicki, 1990). These subgenomic
RNAs account for all of the viral proteins except for the ORF 1ab polyprotein.
Depending on the strain of the virus, coronaviruses produce five to seven subgenomic
32
mRNAs. All of the mRNAs form a nested set that have common 3′ ends followed by a
poly(A) tail. Each of these mRNAs, except for the smallest, contain two or more ORFs
in which only the 5′ most ORF, with few exceptions, is translated. This effectively makes
each subgenomic mRNA functionally monocistronic. The coronavirus subgenomic
RNAs and the genomic RNA share an identical 5′ end leader sequence that is 65 to 98
bases long (Lai et al., 1983; Shieh et al., 1987; Spaan et al., 1983). The leader sequence
is a unique part of the viral genome and only shares partial homology with sequences
found between each gene that have been termed both the transcription-associated
sequence (Hiscox et al., 1995) and the intergenic sequence (Baric et al., 1987). The latter
term will be used for the purposes of this literature review. The core intergenic sequence
shares a common homology with the seven to eighteen nucleotides found at the 3′ end of
the leader (Shieh et al., 1987). Depending on which cell type is used, coronavirus RNA
synthesis occurs at membranous structures associated with the endoplasmic reticulum,
late endosomes, or Golgi complex (Denison et al., 1999; Shi et al., 1999; van der Meer et
al., 1999). Studies have shown that in addition to the RNA dependent RNA polymerase,
the N protein as well as other host components may be involved in viral RNA synthesis
(Compton et al., 1987; Li et al., 1999; Li et al., 1997).
Several models have been proposed to clarify how the subgenomic mRNAs are
synthesized so that the leader sequence RNA is fused to the subgenomic mRNAs are
produced (Figure 1.4). Proposed models of transcription include a discontinuous
transcription process by which the leader and mRNA sequences come from two different
RNA molecules (Jeong and Makino, 1994; Zhang, Liao, and Lai, 1994).
33
Figure 1.4 Schematic diagram of the three proposed models of coronavirus mRNA transcription. Positive stranded RNA is represented by solid black lines while negative stranded RNA is represented by dashed grey lines. Leader RNA and antileader RNA is represented by black and grey boxes respectively. Arrows are used to indicate direction of transcription.
34
35
One model uses a leader-primed transcription (Figure 1.4A) in which a discontinous
transcription occurs throughout positive-stranded RNA synthesis (Lai, 1986). The first
step in this model is the genomic RNA being transcribed into a full-length negative-
stranded copy of the RNA genome. This is followed by the transcription of the leader
RNA beginning at the 3’ end of the negative-stranded RNA. Termination of transcription
occurs at the end of the leader sequence which allows the leader to dissociate from the
template. The dissociated leader, possibly with the polymerase still bound to it, then
reanneals to any of the intergenic sequences on the negative-stranded RNA allowing the
priming of mRNA transcription. In this model, the intergenic sequences function as
promoters for mRNA transcription. These intergenic sequences, at least in the case of
MHV, have a minimal core promoter sequence of seven nucleotides (UCUAAAC) (Joo
and Makino, 1992; Makino and Joo, 1993). A novel subgenomic RNA is produced from
this seven nucleotide sequence when it is incorporated into a defective interfering (DI)
RNA which is allowed to be expressed in MHV-infected cells (Makino, Joo, and Makino,
1991). This shows that the seven nucleotide sequence is capable of priming the synthesis
of a subgenomic RNA that is normally not synthesized. The mRNAs produced in this
model include a leader sequence from the 5’ end of the same RNA molecule or a
different RNA molecule (Zhang, Liao, and Lai, 1994). Fusion of the leader RNA occurs
within the core promoter sequence (UCUAAAC), although adjacent sequences may
possibly contribute to the joining of the leader RNA and the subsequent subgenomic
RNA (van der Most, de Groot, and Spaan, 1994). Mutagenesis or deletion of the leader
RNA eradicates or severely compromises mRNA transcription (Liao and Lai, 1994;
Zhang, Liao, and Lai, 1994). Several studies have been concluded whose data support
36
this model of transcription. Cells infected with MHV contain dissociated free leader
RNA ranging in size from 50 to 90 nucleotides (Baric et al., 1987). Although in the case
of bovine coronavirus, such free leader RNAs were not found in infected cells (Chang,
Krishnan, and Brian, 1996). Another experiment in which two distinct strains of MHV
are used to infect the same cell shows that leader RNAs transcribed can be
indiscriminately joined to the subgenomic RNAs from either strain (Makino, Stohlman,
and Lai, 1986). Leader RNAs can also be incorporated into subgenomic mRNAs when
exogenously added to an in vitro transcription system using lysates obtained from MHV-
infected cells (Baker and Lai, 1990).
A second model is based on discontinous transcription during the negative-
stranded RNA synthesis (Figure 1.4B) from the full-length genomic RNA template
(Sawicki and Sawicki, 1990). In this model, the polymerase pauses transcription at one
of the intergenic sequences and then “jumps” to the 3’ end of the leader sequence in the
genomic RNA template. This jumping mechanism generates a negative-stranded
subgenomic RNA with an antisensed leader sequence at its 3’ end which serves as a
template for the synthesis of mRNAs. Intergenic sequences on the positive-stranded
strand may serve as transcriptional termination sites. It is also possible that these
sequences interact with the leader RNA to promote polymerase jumping during negative-
strand RNA synthesis. It is during this phase of negative-strand synthesis in which the
intergenic sequences facilitate leader-mRNA fusion. As with the first model proposed,
there have been several studies that support this model of transcription. One such study
shows that there are equal amounts of subgenomic RNAs and their negative-stranded
counterparts found in infected cells (Sethna, Hung, and Brian, 1989). It has also been
37
shown that the negative-stranded RNAs are exact complementary copies of the viral
subgenomic RNAs complete with 5’ poly(U) sequence and an antisensed leader sequence
at the 3’ end (Hofmann and Brain, 1991; Sethna, Hofmann, and Brian, 1991). Also, it
appears that each subgenomic mRNA is possibly transcribed from a corresponding
subgenomic-sized, negative-stranded template (Sawicki and Sawicki, 1990). These
negative-stranded RNAs have also been found in membrane associated replication
complexes (Sethna and Brian, 1997).
The third and final model proposed for the synthesis of subgenomic mRNAs are
based on the findings of incorporated subgenomic mRNAs (Figure 1.4C), along with the
full length viral genome, within the virions of some coronaviruses such as BCoV, TGEV,
and IBV (Hofmann, Sethna, and Brian, 1990; Sethna, Hung, and Brian, 1989; Zhao,
Shaw, and Cavanagh, 1993). It is postulated by this model that subgenomic RNAs
brought in with the infecting virion are used directly as templates for the synthesis of
negative-sensed subgenomic RNAs. These negative-sensed RNAs then serve as
templates for additional copies of subgenomic mRNAs (Schwarz, Routledge, and Siddell,
1990; Senanayake et al., 1992). This model does not correspond with the discontinous
nature of coronaviral RNA synthesis. Also, not all coronaviruses have been shown to
have subgenomic mRNAs incorporated in the virion. Studies have also tried to promote
mRNA amplification by transfecting subgenomic RNAs into infected cells but were
unsuccessful (Chang et al., 1994; Liao and Lai, 1994). Currently, it cannot be
unequivocally said which of these proposed models is the correct explanation for
coronavirus mRNA synthesis. It is very possible that various aspects of these proposed
models are active at different points in the virus lifecycle.
38
The leader RNA and the intergenic sequences are not the only regulators of
coronavirus mRNA transcription. Several other facets of the RNA have been found to
play a role in transcriptional regulation. One such regulator is the 3’ end of the viral
genome (Lin, Liao, and Lai, 1994; Lin et al., 1996). A significant portion of the 3’ end,
approximately 55 nucleotides at the 3’ end of the genomic RNA as well as the poly(A)
sequence, is required for the initiation of negative-stranded RNA synthesis (Lin, Liao,
and Lai, 1994). Another example of regulation is the need for the complete 3’-UTR to be
present in order for the transcription of the subgenomic mRNAs to occur (Lin et al.,
1996). These two findings seem to indicate that the 3’ end (or the 5’ end in the case of
the negative-stranded RNA) cis-acting sequence is involved in the synthesis of positive-
stranded RNA which in turn indicates that it probably interacts with the leader or
intergenic sequences of the genome to regulate mRNA transcription.
Replication of Genomic RNA The majority (95%) of the genome sized RNA in the infected cell is packaged into
nucleocapsids and virions while the other 5% is used to synthesize mRNA encoding the
polymerase polyprotein (Perlman et al., 1986; Spaan et al., 1981). The replication of the
full length RNA would seemingly require a different mechanism than transcription of the
subgenomic mRNAs since replication of the full length RNA relies on continuous
transcription rather than the discontinuous transcription of subgenomic mRNA synthesis.
This presumption, however, may not be the case. Some studies suggest that some
genome length replication may actually be performed by discontinuous transcription. It
has been shown that some genome sized MHV genomic RNA is in fact a fusion of the
leader sequence to an intergenic sequence that immediately follows the leader RNA
39
which creates a genome sized RNA with a small deletion (Zhang and Lai, 1996). It has
also been found that the leader sequence of MHV DI RNA can be replaced with that of
the helper virus leader sequence (Makino and Lai, 1989b). This substitution of the leader
sequence by the helper virus implies that the transcription of the full length genome
utilizes a step in which the leader sequence dissociates from the template and then
rebinds are some point downstream to begin transcription. This mechanism is also
utilized in subgenomic mRNA synthesis. The quantity of UCUAA repeats at the 3’ end
of the leader have been shown to undergo rapid evolution (Makino and Lai, 1989a) and it
is at these regions that high levels of RNA recombination occur (Keck et al., 1987).
These studies suggest that genomic RNA replication uses the leader dissociation
discontinous synthesis involved in mRNA transcription.
Defective Interfering (DI) RNAs have been used to delineate which cis-acting
sequences are required for the replication of the genomic RNA (Brian and Spaan, 1997;
Makino, Fujioka, and Fujiwara, 1985; Makino et al., 1988a; Makino et al., 1988b; van
der Most, Bredenbeek, and Spaan, 1991). For MHV, replication of the viral genome
requires a 135 nucleotide internal replication sequence along with approximately 400 to
800 nucleotides at both the 3’ and 5’ ends of the viral genome (Kim, Lai, and Makino,
1993; Lin and Lai, 1993). A 57 nucleotide portion of the replication sequence in the
MHV DI RNAs, which is required for DI replication, has been found to form a secondary
structure in the positive strand (Kim and Makino, 1995; Lin and Lai, 1993). Both the
higher order structure and the sequence itself have been shown to be vital for the function
of the replication signal (Repass and Makino, 1998). The length of required cis-acting
40
regions for viral genome replication and subgenomic mRNA synthesis differ slightly
from one another (Liao and Lai, 1994; Lin and Lai, 1993; Lin et al., 1996).
Translation of Viral Proteins As previously noted, transcription of the viral genome results in multiple
subgenomic mRNAs which, with the exception of the smallest mRNA, contain two or
more ORFs in which only the 5’ most ORF, with few exceptions, is translated. Structural
proteins are translated by a cap-dependent ribosomal scanning mechanism from separate
mRNAs. Translation in virus infected lysates is enhanced by a 5’ leader sequence found
in all of the subgenomic mRNAs (Tahara et al., 1994). This enhancement of translation
may give viral mRNAs an advantage as host cell translation is being shut off by the viral
infection. Besides the structural proteins, the virus encoded polymerase is encoded
within two large overlapping ORFs (ORF1AB) that utilize a ribosomal frameshifting
mechanism in order to synthesize the entire polyprotein (Boursnell et al., 1987; Brierley,
Jenner, and Inglis, 1992; Eleouet et al., 1995; Herold et al., 1993; Lee et al., 1991). This
translation is initiated by the conventional cap-dependent translation mechanism. For
several coronaviruses, the second or third ORF has the highest efficiency of translation.
This is due to the presence of an internal ribosomal entry site just before the ORF that
allows the ribosome to bypass the preceding ORFs. This allows the translation of the
targeted ORF through a cap-independent translation mechanism (Liu and Inglis, 1992b;
Thiel and Siddell, 1994). Other than the virus encoded polymerase, there are other
proteins in the genome that contain more than one ORF. These proteins are translated by
unknown mechanisms. Examples of this include an internal ORF within the N genes of
41
MHV and BCoV (Fischer et al., 1997; Senanayake et al., 1992) and the two ORFs found
on the fifth subgenomic mRNA of IBV (Liu and Inglis, 1992a).
Assembly and Release of Virions
The initial step in virion assembly is the formation of helical nucleocapsids by the
nucleocapsid proteins binding to the viral RNA. This binding of viral RNA to
nucleocapsid protein is facilitated by a sequence of nucleotides found in ORF 1B which
is only present in the genomic-length RNA. This sequence has been shown to bind the
nucleocapsid protein (Masters et al., 1994), and is thought to facilitate packaging of viral
genomes into virions. Small DI RNAs composed only of terminal sequences are able to
be packaged into virions, which indicate that there is some form of packaging signal
contained within these terminal sequences. This packaging has been observed in several
coronaviruses such as TGEV, BCoV, and IBV (Hofmann, Sethna, and Brian, 1990;
Sethna, Hung, and Brian, 1989; Zhao, Shaw, and Cavanagh, 1993). Although the
terminal signals allow packaging of the DI particle, it may not be responsible for
packaging the viral genome. A 69 nucleotide packaging signal has been identified
through the creation of artificial chimeric DI RNAs of MHV (Fosmire, Hwang, and
Makino, 1992; Makino, Yokomori, and Lai, 1990; van der Most, Bredenbeek, and Spaan,
1991). This putative packaging signal maps to a region within ORF 1b and has been
shown to function by maintaining secondary structure (Fosmire, Hwang, and Makino,
1992), which allows interaction between the packaging signal and the RNA being
packaged (Bos et al., 1997; Woo et al., 1997). Packaging of reporter RNAs has also been
accomplished by fusing a homologous region of the bovine coronavirus genome to the
nonviral RNAs (Cologna and Hogue, 2000). Packaging efficiency for large TGEV DI
42
RNAs is affected by the presence of other portions of the ORF 1B suggesting that the
packaging of viral RNA may use additional packaging signals found in this region (Izeta
et al., 1999).
Once the nucleocapsid forms, it interacts with the matrix protein at cellular
membranes (Sturman, Holmes, and Behnke, 1980) of the ER or the Golgi complex. The
nucleocapsid protein can only be incorporated into virions when complexed with the viral
RNA; no unbound protein is able to be packaged into virions (Bos et al., 1996; Vennema
et al., 1996). This suggests that the M protein must interact with the viral genome
directly. Alternatively, a conformational change may occur when the N protein interacts
with the viral genome promoting the interaction between the N and M proteins. This
interaction may enable the nucleocapsid to be packaged into budding virus particles
formed on the membranes of the ER and the Golgi while simultaneously allowing the
formation of the spherical internal core shell surrounding the nucleocapsid (Risco et al.,
1996).
Virus like particles are able to be formed by the expression of the M and E
proteins without the presence of any other additional viral proteins (Bos et al., 1996;
Vennema et al., 1996). This suggests, for most coronaviruses, that the interactions
between the M and E proteins facilitate the formation of virion particles. Mutations
introduced into the E and M proteins have shown altered virus morphology (Fischer et
al., 1998) and abrogated virus like particle formation (de Haan et al., 1998), respectively.
Unlike other coronaviruses, the SARS coronavirus can produce virus like particles
through expression of the M and N proteins alone but particles are not formed with
expression of the M and E proteins (Huang et al., 2004). Regardless of the coronavirus
43
type, these experiments show that the spike protein is dispensable in virus particle
formation. This conclusion is also supported by the fact that tunicamycin-treated cells
infected with MHV produce noninfectious particles that do not contain the spike protein
(Holmes, Doller, and Sturman, 1981). It should be noted, however, that when the spike
protein is expressed along with M and E (M and N for the SARS-CoV), virus like
particles are produced with the spike protein incorporated in them (Bos et al., 1996;
Huang et al., 2004; Vennema et al., 1996).
The budding compartment, located between the ER and Golgi, is the site at which
virus budding is first detected (Dubois-Dalcq et al., 1982; Klumperman et al., 1994;
Tooze, Tooze, and Warren, 1984; Tooze and Tooze, 1985). Interaction between the M
protein, which accumulates at the budding compartment (Klumperman et al., 1994) and
the E protein is thought to trigger virus budding. Because virus budding has only been
shown to occur at the budding compartment and the E protein has been detected at sites
other than that of virus budding (Godet et al., 1994; Yokomori and Lai, 1992a), it is
thought that the M protein dictates the location of virus budding. Although the E protein
is required for particle formation, it may only serve as a scaffolding protein which is not
essential part of virus maturation. This is because the E protein is present in such low
quantities compared to that of the M protein (Vennema et al., 1996). It may function in
the pinching off of the budding virions at the budding compartment because it has been
shown to induce curvature of intracellular membranes containing M (Vennema et al.,
1996).
Incorporation of the spike protein and the HE protein into virions is directed by
interactions with the M protein which occurs in the pre-Golgi complex (Nguyen and
44
Hogue, 1997; Opstelten et al., 1995). The glycoproteins S and HE are processed as the
virions pass through the Golgi where they may undergo additional morphological
changes resulting in the compact, electron dense internal core typical of the mature virus
particle (Risco et al., 1998; Salanueva, Carrascosa, and Risco, 1999). After the Golgi,
mature virions accrue in large, smooth-walled vesicles that fuse with plasma membrane
in order to release the virions into the extracellular space (Griffiths and Rottier, 1992).
Although virus release is restricted to certain areas of the cell, the exact mechanism
dictating this site restriction is poorly understood.
Cellular Proteins Involved in Coronavirus Replication Coronaviruses require cellular proteins in addition to its viral proteins in order to
replicate. These cellular proteins are hijacked from their normal functions in the cell to
assist the virus in its replication. Crosslinking experiments failed to show any
interactions, with the exception of the N protein, between the viral RNA and any of the
coronavirus proteins which suggest that viral proteins interact with the viral RNA
indirectly through cellular proteins. Several cellular proteins have been shown to bind to
MHV viral RNA. These portions of viral RNA are known to play regulatory functions in
other aspects of the virus lifecycle. Such regulatory elements include the 5’ and 3’ ends
of the genomic RNA, intergenic regions of the RNA genome, and the 3’ end of the
negative-strand RNA.
HNRNP A1
Several different cellular proteins have been identified that bind to the intergenic
regions of the template coronavirus RNA (Zhang and Lai, 1995a). Deletion analysis and
mutagenesis of the intergenic sites have correlated transcription efficiency with RNA
45
protein binding. This suggests that these cellular RNA binding proteins play an
important role in the regulation of coronavirus mRNA transcription. One of the proteins
was identified by partial peptide mapping to be hnRNP A1 (Li et al., 1997). This cellular
protein is known to be an RNA-binding protein that contains two RNA-binding domains
along with a glycine-rich domain responsible for protein-protein interactions. hnRNP A1
primarily exists as a nuclear protein, but it also has a shuttling function that cycles it
between the nucleus and the cytoplasm (Pinol-Roma and Dreyfuss, 1992). Its primary
function depends on its location. When in the cytoplasm, hnRNP A1 modulates mRNA
turnover and translation by binding to AU-rich elements on cytoplasmic mRNA
(Hamilton et al., 1997; Hamilton et al., 1993; Henics et al., 1994). While in the nucleus,
it is involved in pre-mRNA splicing and transport of cellular RNAs (Dreyfuss et al.,
1993). Specifically for MHV, hnRNP A1 binds the negative-strand leader along with
intergenic sequences (Furuya and Lai, 1993; Li et al., 1997). These sequences are known
to be critical elements for discontinuous viral RNA transcription. Mutagenesis of the
intergenic sequences disrupts hnRNP A1 binding which hinders efficiency of
transcription from the altered intergenic site (Furuya and Lai, 1993; Li et al., 1997; Zhang
and Lai, 1995b). Another function of hnRNP A1 may be in its interaction with the
nucleocapsid protein (Wang and Zhang, 1999) which is known to bind to the viral RNA
directly (Baric et al., 1988; Stohlman et al., 1988).
PTB
PTB, also known as hnRNP I, is another cellular protein found to be involved in
coronavirus replication. It is known to bind to the UC-rich RNA sequences found near
the 3’ end of introns, to play a role in the regulation of translation of viral and cellular
46
RNAs, as well as alternative splicing of pre-mRNAs, and is similar in function to hnRNP
A1 in that it also shuttles between the nucleus and the cytoplasm (Kaminski et al., 1995;
Svitkin et al., 1996; Valcarcel and Gebauer, 1997). Immunoprecipitation and
crosslinking studies have established PTB binds to the MHV positive-strand leader RNA
(Li et al., 1999). This region of the MHV RNA has been shown previously to be needed
for MHV RNA synthesis and to regulate transcription (Kim, Jeong, and Makino, 1993;
Liao and Lai, 1994; Tahara et al., 1994). Deletion of the mentioned binding sites causes
significant inhibition of RNA transcription (Li et al., 1999). This suggests that PTB may
play a role in coronavirus mRNA translation although it does not have a direct effect on
the cap-dependent MHV RNA translation (Choi and Lai, unpublished data).
PABP
Poly(A)-binding protein (PABP) binds to the 3’ UTR region of coronaviral RNA
which is necessary for the synthesis of negative-stranded viral RNA and both genomic
and subgenomic positive-strand RNA synthesis (Kim, Jeong, and Makino, 1993; Lin and
Lai, 1993; Lin, Liao, and Lai, 1994; Lin et al., 1996). These 3’ UTR regions contain
structures that are conserved among several different strains of coronaviruses (Hsue,
Hartshorne, and Masters, 2000; Hsue and Masters, 1997; Liu, Johnson, and Leibowitz,
2001). It is known that PABP is a highly abundant cytoplasmic protein that binds to the
3’ poly(A) tail on eukaryotic mRNAs (Gorlach, Burd, and Dreyfuss, 1994). Binding of
PABP to the 3’ UTR of DI RNA replicons correlates with the ability of the RNA to
replicate which suggests that the PABP interaction with the poly(A) tail may have an
effect on coronavirus RNA replication (Huang and Lai, 2001; Liu, Yu, and Leibowitz,
1997; Spagnolo and Hogue, 2000; Yu and Leibowitz, 1995a; Yu and Leibowitz, 1995b).
47
Mitochondrial Aconitase
Despite the absence of a consensus RNA-binding domain, crosslinking
experiments have indicated that mitochondrial aconitase binds to the MHV 3’ protein-
binding element (Burd and Dreyfuss, 1994). This binding increases the stability on the
viral mRNA enhancing the translation of viral proteins. In addition to binding to the
MHV genome, mitochondrial aconitase also colocalizes with the MHV nucleocapsid
protein suggesting a potential interaction with the MHV replication complex (Nanda and
Leibowitz, 2001).
Pathology and Disease
Coronaviruses are known to cause a wide array of pathologies including acute
respiratory disease, hepatitis, chronic demyelination in the central nervous system (CNS),
encephalitis, and enteritis (Holmes, 1996). Generally the virus infects the respiratory and
enteric mucosal surfaces (Navas-Martin and Weiss, 2003) and is able to cause both self-
limiting acute and chronic persistent infections. Some coronaviruses, such as MHV,
target other cells such as hepatocytes, endothelial cells, neurons, macrophages,
astrocytes, and oligodendrocytes (Haring and Perlman, 2001).
Before the discovery of the SARS-CoV only two coronaviruses were known to
infect humans, HCoV-229E and HCoV-OC43. The infections caused by theses viruses
were self-limiting upper respiratory tract infections (Myint, 1994) that account for
approximately 30% of all common colds. These viruses, however, have never been
reported to cause severe illness. Other coronaviruses such as TGEV (porcine), BCoV
(bovine), and IBV (avian), cause respiratory and enteric diseases that can result in severe
economic loss in the farming industry. The SARS-CoV originated in Guangdong
48
Province, China in late 2002 (Parry, 2003). From there it spread throughout Asia and by
the end of the epidemic caused more than 8000 cases of infection worldwide, according
to the Centers for Disease Control (CDC) and the World Health Organization (WHO)
(Navas-Martin and Weiss, 2003). Out of the more than 8000 cases reported, more than
800 of these resulted in death with mortality rates, depending on the age of the victim, as
high as 15% (Anand et al., 2003).
The SARS-CoV infection exhibits a wide clinical course characterized by fever,
dyspnea, lymphopenia and lower tract respiratory infection (Nie et al., 2003; Tsui et al.,
2003) along with gastrointestinal symptoms and diarrhea (Booth et al., 2003; Lee et al.,
2003; Leung et al., 2003; Peiris et al., 2003). A three stage disease model that has been
proposed consists of viral replication, immune hyperactivity, and pulmonary destruction
(Tsui et al., 2003). SARS pathology of the lung has been correlated with diffuse alveolar
damage, epithelial cell proliferation, and an increase of macrophages (Navas-Martin and
Weiss, 2004). Similar to many coronavirus infections, multinucleate giant-cell infiltrates
of macrophage and epithelial origin have been associated with putative syncytium-like
formation (Nicholls et al., 2003). As found in the fatal influenza subtype H5N1 disease
in 1997, lymphopenia, hemophagocytosis in the lung and white-pulp atrophy of the
spleen are also observed during the SARS-CoV infection (To et al., 2001). This
hemophagocytosis found in the lung supports a cytokine dysregulation (Fisman, 2000)
which may have a role in the pathogenesis of SARS due to the proinflammatory
cytokines being released by stimulatory macrophages in the alveoli.
Based on the known pathology of the SARS-CoV, several treatments have been
tried including the administration of steroids to try to control the exacerbated cytokine
49
response (Lai et al., 2003). However, treatments of the infection have been largely
ineffective (Koren et al., 2003; Lee et al., 2003; Tsui et al., 2003). SARS-CoV infection
is also resistant to ribavirin, a nucleoside analog that normally has a broad antiviral
activity and is presently being used for the treatment of respiratory syncytial virus (RSV)
(Everard et al., 2001) and hepatitis C (Lipman and Cotler, 2003; Martin et al., 1998).
Interferons α, β, and γ have also been tested for their antiviral properties against the
SARS-CoV in vitro with interferon β showing the most promise (Cinatl et al., 2003).
The exact mechanism that allowed the emergence of the SARS-CoV possible is
unknown. Although the coronavirus biological vector is not known, it is speculated that
the virus jumped from an animal species to humans (Holmes, 2003). Several domestic
and wild animals from the Guangdong Province have been examined in order to
determine if any of them were carrying the SARS-CoV. Viruses similar to SARS have
been isolated from Himalayan palm civets, raccoon dogs, and Chinese ferret badgers
found in a retail market in China (Guan et al., 2003). It is speculated that the SARS-CoV
does indeed have an animal reservoir (Holmes, 2003).
In addition to the recent impact that the SARS-CoV has had on public health,
other coronaviruses have an economic impact in the cattle industry. Primarily, bovine
coronaviruses were known to be enteropathogenic viruses that caused severe diarrhea in
neonatal calves. Coronavirus virions were able to be isolated from diarrhea fluid and
intestinal samples from animals infected with the disease (Doughri et al., 1976; Mebus et
al., 1973). Theses enteric viruses were also able to be isolated from the feces of adult
cattle with winter dysentery (Saif et al., 1988). It was not until recently that
coronaviruses were able to be isolated from the nasal secretions and lung tissues of cattle
50
infected with fatal cases of shipping fever pneumonia. These isolated viruses were
termed respiratory coronaviruses (Storz et al., 2000a; Storz et al., 2000b) in order to
differentiate them from the commonly found enteric strains known as enteropathogenic
bovine coronaviruses. There two related viruses are separated by phenotypic variation,
antigenic differences, and genetic divergence (Chouljenko et al., 1998; Chouljenko et al.,
2001; Lin et al., 2000; Storz et al., 2000a). A comparison study between respiratory and
enteropathogenic coronavirus isolated from the same animal with fatal shipping
pneumonia suggests that the difference in viral tropism is due to slight variations in the
S1 subunit of the Spike glycoprotein (Chouljenko et al., 1998; Gelinas et al., 2001;
Hasoksuz et al., 2002; Rekik and Dea, 1994).
It is the continuing research on all coronaviruses as well as the SARS-CoV
specifically that will allow a better understanding of the SARS virus and its implications
on public health.
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CHAPTER II
GENETIC ANALYSIS OF THE SARS-CORONAVIRUS SPIKE GLYCOPROTEIN FUNCTIONAL DOMAINS INVOLVED IN CELL-SURFACE
EXPRESSION AND CELL-TO-CELL FUSION*
INTRODUCTION
An outbreak of atypical pneumonia, termed severe acute respiratory syndrome
(SARS), appeared in the Guangdong Province of southern China in November, 2002. The
mortality rates of the disease reached as high as 15% in some age groups (Anand et al.,
2003). The etiological agent of the disease was found to be a novel coronavirus (SARS-
CoV), which was first isolated from infected individuals by propagation of the virus on
Vero E6 cells (Drosten et al., 2003; Ksiazek et al., 2003; Peiris et al., 2003). Analysis of
the viral genome has demonstrated that the SARS-CoV is phylogenetically divergent
from the three known antigenic groups of coronaviruses (Drosten et al., 2003; Ksiazek et
al., 2003). Analysis of the polymerase gene alone, however, has indicated that the SARS-
CoV may be an early off-shoot from the group 2 coronaviruses (Snijder et al., 2003).
The coronaviruses are the largest of the enveloped RNA viruses with a positive-
stranded RNA genome of 28 to 32 kb (Holmes, 2003). Coronaviruses possess a wide host
range, capable of infecting mammalian and avian species. All identified coronaviruses
have a common group of indispensable genes that encode nonstructural proteins
including the RNA replicase gene open reading frame (ORF) 1ab and the structural
proteins nucleocapsid (N), membrane protein (M), envelope protein (E), and spike
glycoprotein (S), which are assembled into virus particles. A hemagglutinin-esterase
* Reprinted from Virology , Vol 341 (2), C. M. Petit, J. M. Melancon, V. N. Chouljenko, R. Colgrove, M. Farzan, D. M. Knipe, and K. G. Kousoulas, Genetic Analysis of the SARS-Coronavirus Spike Glycoprotein Functional Domains Involved in Cell-surface Expression and Cell-to-Cell Fusion, Pages 215-308, Copyright 2005, with permission from the Elsevier.
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(HE) protein is also encoded by some coronaviruses. Distributed among the major viral
genes are a series of ORFs that are specific to the different coronavirus groups. Functions
of the majority these ORFs have not been determined.
The SARS spike glycoprotein, a 1,255-amino-acid type I membrane glycoprotein
(Rota et al., 2003), is the major protein present in the viral membrane forming the typical
spike structure found on all coronavirions. The S glycoprotein is primarily responsible
for entry of all coronaviruses into susceptible cells through binding to specific receptors
on cells and mediating subsequent virus-cell fusion (Cavanagh, 1995). The S
glycoprotein specified by mouse hepatitis virus (MHV), is cleaved into S1 and S2
subunits, although cleavage is not necessarily required for virus-cell fusion (Bos, Luytjes,
and Spaan, 1997; Gombold, Hingley, and Weiss, 1993; Stauber, Pfleiderera, and Siddell,
1993). Similarly, the SARS-CoV S glycoprotein seems to be cleaved into S1 and S2
subunits in Vero-E6 infected cells (Wu et al., 2004), while it is not known whether this
cleavage affects S-mediated cell fusion. The SARS-CoV receptor has been recently
identified as the angiotensin converting enzyme 2 (ACE2) (Li et al., 2003). Although the
exact mechanism by which the SARS-CoV enters the host cell has not been elucidated, it
is most likely similar to other coronaviruses. Upon receptor binding at the cell
membrane, the S glycoprotein is thought to undergo a dramatic conformational change
causing exposure of a hydrophobic fusion peptide, which is subsequently inserted into
cellular membranes. This conformational change of the S glycoprotein causes close
apposition followed by fusion of the viral and cellular membranes resulting in entry of
the virion nucleocapsids into cells (Eckert and Kim, 2001; Tsai et al., 2003; Zelus et al.,
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2003). This series of S-mediated virus entry events is similar to other class I virus fusion
proteins (Baker et al., 1999; Melikyan et al., 2000; Russell, Jardetzky, and Lamb, 2001).
Heptad repeat (HR) regions, a sequence motif characteristic of coiled-coils,
appear to be a common motif in many viral and cellular fusion proteins (Skehel and
Wiley, 1998). These coiled-coil regions allow the protein to fold back upon itself as a
prerequisite step to initiating the membrane fusion event. There are usually two HR
regions: an N terminal HR region adjacent to the fusion peptide and a C-terminal HR
region close to the transmembrane region of the protein. Within the HR segments, the
first amino acid (a) and fourth amino acid (d) are typically hydrophobic amino acids that
play a vital role in maintaining coiled-coil interactions. Based on structural similarities,
two classes of viral fusion proteins have been established. Class I viral fusion proteins
contain two heptad repeat regions and an N-terminal or N-proximal fusion peptide. Class
II viral fusion proteins lack heptad repeat regions and contain an internal fusion peptide
(Lescar et al., 2001). The MHV S glycoprotein, which is similar to other coronavirus S
glycoproteins, is a class I membrane protein that is transported to the plasma membrane
after being synthesized in the endoplasmic reticulum (Bosch et al., 2003). Typically, the
ectodomains of the S2 subunits of coronaviruses contain two regions with a 4, 3
hydrophobic (heptad) repeat the first being adjacent to the fusion peptide and the other
being in close proximity to the transmembrane region (de Groot et al., 1987).
In the present study, we investigated the role of several predicted structural and
functional domains of the SARS spike glycoprotein by introducing specific alterations
within selected S glycoprotein regions. The results show that the SARS-CoV S
glycoprotein conforms to the general structure and function relationships that have been
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elucidated for other coronaviruses, most notably the MHV (Chang, Sheng, and Gombold,
2000; Ye, Montalto-Morrison, and Masters, 2004) . However, in contrast to the MHV
endodomain, the carboxyl terminus of the SARS-CoV S glycoprotein contains multiple
non-overlapping domains that function in intracellular transport, cell-surface expression,
and endocytosis as well as in S glycoprotein-mediated cell-to-cell fusion.
RESULTS
Genetic Analysis of S Glycoprotein Functional Domains
To delineate domains of the S glycoprotein that function in membrane fusion,
intracellular transport, and cell-surface expression, two types of mutations were
introduced within the S gene: a) mutations were introduced within and adjacent to the
predicted amino terminal heptad repeat (HR1) core and the predicted fusion peptide,
which are known to play important roles in membrane fusion (Bosch et al., 2004; Bosch
et al., 2003; Ingallinella et al., 2004; Tripet et al., 2004); b) mutations and carboxyl
terminal truncations of the S glycoprotein were engineered to delineate S cytoplasmic
domains that function in glycoprotein synthesis, intracellular transport, and membrane
fusion (Fig. 2.1). Specifically, to investigate the amino acid requirements of the HR1 of
the SARS-CoV S glycoprotein, the a and d amino acid positions L(898) and N(901) were
both replaced by lysine residues in the cluster mutation CL2, effectively collapsing the
predicted α-helical structure at the amino terminal terminus of the HR. This amino acid
sequence is thought to align with the L(1184) of HR2 in the formation of the HR1/HR2
core complex (Xu et al., 2004). In addition, cluster-to-lysine mutations CL3 and CL4
replaced the a and d positions within the HR1 region (Fig. 2.1B). The CL5 cluster
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Figure 2.1. Schematic diagram of the SARS-CoV S glycoprotein. (A) Graphical representation of the S glycoprotein showing the approximate location of the cluster to lysine mutations CL1-CL5 relative to known and indicated functional domains. (B) Shown on the top of the diagram is a graphical representation of the SARS-CoV S glycoprotein. The predicted fusion peptide and the HR1 region are enlarged below to show the sets of amino acids replaced by lysines in the cluster mutations. The heptad repeat a and d positions are labeled above the corresponding amino acid. Amino acids changed to lysine are demarcated by arrows with the name of that particular mutation shown in brackets. (C) Amino acid sequences of the carboxyl termini of the truncation and acidic cluster associated mutations. Cysteine clusters (CRM1 and CRM2) are denoted by underlined italicized text as well as a bracket encompassing their respective regions. The charged cluster is bracketed over the region. Amino acids mutated to alanines for the CL6 and CL7 cluster mutations are in bold.
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mutation was placed adjacent to the HR1 region to investigate whether regions proximal
to HR1 had any effect on S mediated cell fusion. Similarly, the role of the a and d
positions within the predicted fusion peptide, located immediately proximal to the N
terminus of HR1, was investigated by constructing the CL1 cluster mutation (Fig. 2.1B).
It has been shown for other viral class I fusion proteins that the carboxyl terminus
plays a regulatory role in membrane fusion (Bagai and Lamb, 1996; Sergel and Morrison,
1995; Seth, Vincent, and Compans, 2003; Tong et al., 2002; Yao and Compans, 1995).
Specifically for coronaviruses, the MHV S glycoprotein endodomain has been shown to
contain charged-rich and cysteine-rich regions, which are critical for fusion of infected
cells (Bos et al., 1995; Chang, Sheng, and Gombold, 2000; Ye, Montalto-Morrison, and
Masters, 2004). The carboxyl terminal portion of the S glycoprotein contains a consensus
acidic amino acid cluster containing a motif, which is predicted by the NetPhos 2.0
software to be phosphorylated (Blom, Gammeltoft, and Brunak, 1999). To investigate the
potential role of the acidic amino acid cluster in synthesis, transport and cell fusion, serial
truncations of S were constructed. The acidic cluster was specifically targeted by
mutagenizing the predicted phosphorylation site embedded within the acidic cluster as
well as by replacing acidic residues of the acidic cluster with alanine residues. In
addition, carboxyl-terminal truncations of 8, 17, 26, and 41 amino acids were engineered
by insertion of stop codons within the S glycoprotein gene. The 8 aa truncation (T1247)
was designed to bring the predicted charged cluster DEDDSE proximal to the carboxyl
terminus of the mutated S glycoprotein (Fig. 2.1C). Similarly, the 17 aa truncation
(T1238) was designed to delete the DEDDSE acidic cluster. The SARS-CoV S
glycoprotein endodomain contains two cysteine residue clusters, a CCMTSCCSC
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(CRM1) cluster immediately adjacent to the membrane and a CSCGSCC (CRM2)
downstream of the first cluster. To address the role of these domains in S glycoprotein-
mediated cell-to-cell fusion, the 26 aa truncation (T1229) was designed to delete the
CRM2 domain (T1229), while the 41 aa truncation (T1214) deleted both the CRM1 and
CRM2 domains (Fig. 2.1C).
Effect of Mutations on S Synthesis
To investigate the effect of the different mutations on S synthesis, western
immunoblot analysis was used to detect and visualize all of the constructed mutant
glycoproteins as well as the wild type S (Fig. 2.2). Cellular lysates prepared from
transfected cells at 48 hours post transfection were electrophoretically separated by SDS-
PAGE and the S glycoproteins were detected via chemiluminescence using a monoclonal
antibody specific for the SARS-CoV S glycoprotein. Carbohydrate addition was shown to
occur in at least four different locations of the SARS-CoV S glycoprotein (Krokhin et al.,
2003; Ying et al., 2004). Furthermore, transiently expressed S glycoprotein in Vero E6
cells was proteolytically cleaved into S1 and S2 components (Wu et al., 2004). The anti-S
monoclonal antibody SW-111 detected a protein species in cellular extracts from
transfected cells, which migrated with an apparent molecular mass of approximately 180
kDa, as reported previously (Song et al., 2004). All mutated S glycoproteins produced
similar S-related protein species to that of the wild-type S indicating that none of the
engineered mutations adversely affect S synthesis and intracellular processing (Fig.
2.2A). The SARS S glycoprotein is known to form homotrimers in its native state (Song
et al., 2004). To investigate the effect of the mutations on S oligomerization, cellular
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Figure 2.2. Western blot analysis of the expressed mutant SARS-CoV S mutant glycoproteins. (A,B) Immunoblots of wild-type (3xFLAG So (WT)), cluster to lysine, cluster to alanine, and carboxyl truncation mutant S glycoproteins probed with monoclonal anti-SARS S antiserum. "Cells only" represents a negative control in which Vero cells with no protein transfected into them were probed with the monoclonal antibody to SARS S glycoprotein. (B) In order to detect trimer formation more efficiently, the protein extracts of the mutants were neither boiled nor subject to treatment with beta mercaptoethanol.
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lysates from transfected cells were electrophoretically separated without prior boiling of
the samples and in the absence of reducing agents (Song et al., 2004). The different S
species were detected via chemiluminescence using the monoclonal SW-111 to the SARS
S glycoprotein. A S protein species was detected that had an approximate apparent
molecular mass of 500 kDa, which was consistent with previously published data (Song
et al., 2004) (Fig 2.2B). Although levels of oligomer expression seemed to vary slightly
between mutant forms, all mutated S glycoproteins produced similar species to the wild
type, indicating that none of the mutations blocked oligomerization from occurring.
Ability of Mutant S Glycoproteins to be Expressed on the Cell Surface
To determine if the mutant S glycoproteins were expressed on the surface of cells,
immunohistochemical analysis was used to label cell-surface expressed S under live cell
conditions that restrict antibody binding to cell surfaces. In addition,
immunohistochemistry was used to detect the total amount of S expressed in cells by
fixing and permeabilizing the cells prior to reaction with the antibody. A recombinant S
protein having the 3xFLAG added in-frame to the carboxyl terminus of S was used as a
negative control; since it would not be stained by the live cell reaction conditions (see
Materials and Methods). Both wild-type versions of S having the 3xFLAG, either at the
amino or carboxyl terminus of S, caused similar amounts of fusion (Fig. 2.3), which also
was similar to that obtained with the untagged wild-type S (not shown). The relative
amounts of cell-surface versus total cellular expression of S were obtained through the
use of an ELISA. A ratio between the cell-surface localized S and total cellular S
expression was then calculated and normalized to the corresponding ratio obtained with
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Figure 2.3. Immunohistochemical detection of cell-surface and total expression of the SARS-CoV S wild-type and mutant proteins. Vero cells were transfected with the wild-type SARS-CoV optimized S (3xFLAG So (WT)) (F1, F2), CL1 (A1, A2), CL2 (B1, B2), CL3 (C1, C2), CL4 (D1, D2), CL5 (E1, E2), CL6 (L1, L2), CL7 (M1, M2), T1214 (K1, K2), T1229 (J1, J2), T1238 (I1, I2), T1247(H1, H2) and a wild-type SARS-CoV optimized S labeled with a 3xFLAG carboxyl tag (G1, G2), which served as a negative control. At 48 hours post-transfection, cells were immunhistochemically processed either under live conditions to show surface expression. (A2, B2, C2, D2, E2, F2, G2, H2, I2, J2, K2, L2, and M2) or fixed and permeabilized conditions to show total expression(A1, B1, C1, D1, E1, F1, G1, H1, I1, J1, K1, L1, and M1) with anti-FLAG antibody.
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the wild-type S glycoprotein (see Materials and Methods) (Fig. 2.4). The CL1 (95%),
CL2 (78%), CL3 (86%), CL4 (80%), CL5 (92%), CL6 (86%) mutants as well as the
T1229 (91%), T1238 (94%), and T1247 (79%) truncations were expressed on the cellular
surface by the percentages indicated when compared to the cell surface expression of the
wild type protein. In contrast, the 1214T mutant expressed 77% less S on cell surfaces in
comparison to the wild-type S (Fig. 2.4).
Effect of Mutations on S-mediated Cell-to-Cell Fusion
Transiently expressed wild type S causes extensive cell-to-cell fusion (syncytial
formation), especially in the presence of the SARS-CoV ACE2 receptor (Li et al., 2003).
To determine the ability of each mutant S glycoprotein to cause cell-to-cell fusion and the
formation of syncytia, fused cells were labeled by immunohistochemistry using the anti-
FLAG antibody (Fig. 2.5), and the extent of cell-to-cell fusion caused by each mutant
glycoprotein was calculated by obtaining the average size of approximately 300 syncytia.
The average syncytium size for each mutant was then normalized to that found in wild
type S transfected cells (see Materials and Methods). The CL1 (73%), CL2 (75%), CL3
(68%), CL4 (71%), CL5 (76%), CL6 (51%) as well as the T1214 (86%) and T1247
(66%) mutants inhibited the formation of syncytia by the percentages indicated. The
T1229 truncation and the cluster mutant CL7 produced syncytia, which were on the
average 22% and 15% smaller, respectively, than that of the wild-type S. In contrast, the
T1238 mutant produced on the average 43% larger syncytia than that of the wild-type S
(Fig. 2.5).
Comparison of the membrane fusion and cell-surface expression results allowed the
grouping of the different mutant S phenotypes into four distinct groups (Table 2.1): 1) S
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Figure 2.4. Ratios of cell-surface to total cellular expression of mutant SARS-CoV S glycoproteins. Detection of cell surface and total glycoprotein distribution was determined by immunohistochemistry and ELISA (see Materials and Methods). Cell-surface expression of the S glycoprotein was measured by incubating the transfected cell monolayers with anti-FLAG antibody at room temperature before permeabilization. For total S glycoprotein detection, cells were fixed and permeabilized prior to incubation with the anti-FLAG antibody. A ratio between the surface localization and the total expression was calculated and normalized to the wild type protein, then set to a percentage of the wild-type. The error bars represent the maximum and minimum surface to total ratios obtained from three independent experiments, and the bar height represents the average surface to total ratio.
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Figure 2.5. Quantitation of the extent of S-mediated cell fusion. The average size of syncytia for each mutant was determined by digitally analyzing the area of approximately 300 syncytia stained by immunohistochemistry for S glycoprotein expression using the Image Pro Plus 5.0 software package (see Materials and Methods). Error bars shown represent the standard deviations calculated through comparison of the data from each of three experiments.
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mutant forms in group I (CL1-CL6 and T1247) resulted in high levels of cell-surface
expression (78-95% of the wild-type S); however, the average size of syncytial formed
by these mutated S glycoproteins was reduced substantially in comparison to the wild-
type S (23-48% of the wild-type). CL1 affects the predicted fusion peptide, CL2-CL4
affect the HR1 domain, and CL5 affects a region downstream of the HR1 domain. The
CL6 mutation is located within the S carboxyl terminal acidic cluster. The T1247
mutation truncates the S carboxyl terminus by 8 amino acids; 2) S mutant forms in group
II produced high levels of S cell-surface expression and an average size of syncytia
slightly smaller than that of the wild-type S. These mutations included CL7, which
modified the acidic cluster and the T1229 truncations that deleted the cysteine-rich motif
CRM2; 3) The single S mutant in group III, T1214, produced significantly less cell-
surface expression and concomitantly the average size of syncytia was substantially
reduced in comparison to the wild type S; 4) The T1238 truncation in group IV produced
high levels of cell surface expression equivalent to that of the wild-type (94% of the wild-
type S), while the average syncytium size was 43% larger than that the syncytial
produced by the wild-type S (Table 2.1).
Detection of the Intracellular Distribution of S Mutant Glycoproteins Via Confocal Microscopy To visualize the intracellular distribution of S mutant glycoproteins, cells were
transfected with plasmids encoding the wild type or S mutants and examined by confocal
microscopy at 48 h post transfection (Fig. 2.6). The wild type and all the S mutants were
detected throughout the cytoplasm of transfected cells and exhibited similar intracellular
distribution patterns (Fig. 2.6, panels B, D, F, H, J, L). To determine and compare the
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Figure 2.6. Confocal microscopic visualization of endocytosed and intracellular distribution of SARS-CoV S glycoprotein mutants. Vero cells expressing wild-type SARS-CoV S glycoprotein (3xFLAG So (WT)) (A and B), CL6 (C and D), CL7 (E and F), T1229 (G and H), T1238 (I and J) and T1247 (K and L) were processed for confocal microscopy using two different methods in order to assess different properties of the mutants. Endocytosis patterns (A,C,E,G,I, and K) were visualized by adding anti-FLAG (green) antibody into the media 12 h prior to processing, enabling detection of the mutant protein after endocytosis from cellular surfaces. Early endosomes were also detected for these panels using a polyclonal anti-early endosomal antigen I antibody (red). For total glycoprotein detection (B, D, F, H, J, and L), cells were fixed and permeabilized prior to labeling with anti-FLAG (green).
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endocytotic profiles of wild-type and S mutant forms, transfected cells were reacted with
the anti-FLAG antibody under live conditions for 12 hours at 37ºC and visualized by
confocal microscopy. The majority of the wild-type S detected by the anti- FLAG
antibody appeared to remain on cell surfaces (Fig. 2.6, panel A). In contrast, a significant
fraction of cell-surface expressed CL6 and CL7 as well as the T1229, T1238 and T1247 S
mutants appeared to partially endocytose to cytoplasmic compartments (Fig. 2.6, panels
C, E, G, I, K). The CL7 mutant, but not the other S mutants, appeared to colocalize with
the early endosomal marker EEA-1 (Fig. 2.6, panel E). The S mutants CL1, CL2, CL3,
CL4, and CL5 remained in plasma membranes exhibiting profiles similar to that of the S
wild-type glycoprotein (data not shown).
Time-dependent Endocytotic Profiles of Wild-type and Mutant S Proteins
A time-dependent endocytosis assay was utilized to better visualize the endocytotic
patterns of the wild-type and mutant S glycoproteins as well as to exclude the possibility
that the observed plasma membrane accumulation of the wild-type S and some of the S
mutants was due to recirculation of endocytosed S to cell-surfaces. In this assay, cell-
surface expressed S was reacted with anti-FLAG antibody at 4°C and subsequently, cells
were incubated at 37°C for different time periods before processing for confocal
microscopy (see Materials and Methods). Generally, these time-dependent endocytosis
studies were in agreement with the results shown in Figure 2.6. Specifically, in cells that
were not shifted to 37ºC, referred to as time zero cells, wild type and mutant spike were
detected exclusively at the surface of the cells (Fig. 2.7, panels A1, B1, C1, D1, E1, F1).
At five and fifteen minutes after the shift to 37ºC, the wild-type S remained exclusively at
the surface while the other S mutants were detected in numerous intracellular vesicles
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Figure 2.7. Analysis of the endocytotic kinetic profile of the truncation mutants and the acidic cluster mutants using confocal microscopy. After transfection, SARS-CoV S glycoprotein wild-type (3xFLAG So (WT)) (A1-A4), T1229 (B1-B4), T1238 (C1-C4), T1247 (D1-D4), CL6 (E1-E4), and CL7 (F1-F4) expressing cells were incubated with an anti-FLAG monoclonal antibody (green) for 1 hr and then returned to 37ºC for different times. Cell nuclei were labeled with To-Pro-3 Iodide (blue). Panels A1-A4, B1-B4, C1-C4, D1-D4, E1-E4, and F1-F4 correspond to 0-, 5-, 15-, and 60-min incubation times at 37ºC, respectively.
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dispersed inside the cell (Fig. 2.7, panels A2 and A3 compared to panels B2 and B3, C2
and C3, D2 and D3, E2 and E3, F2 and F3). By sixty minutes after the shift to 37ºC, the
wild type S was still localized exclusively to the surface of the cell (Fig. 2.7, panel A4),
while the T1229, T1238, CL6 and CL7 S mutants appeared to be present throughout the
cytoplasm of the cell (Fig. 2.7, panels B4, C4, E4, F4). The T1247 S mutant seemed to
undergo rapid and complete endocytosis during the 60 min observation and appeared to
localize into punctuate structures in the cytoplasm of cells unlike the fairly even cellular
distribution of all other S mutants (Fig. 2.7, panels D1-D4 compared A4, B4, C4, D4, E4,
F4).
DISCUSSION
The mechanism by which class I fusion proteins such as the coronavirus S
glycoprotein, the hemagglutinin protein (HA) of influenza virus, the gp41 of human
immunodeficiency virus (HIV), the Ebola virus surface glycoprotein (GP), and the fusion
protein (F) of paramyxovirus facilitate membrane fusion during viral entry into cells has
been extensively investigated (Eckert and Kim, 2001; Hernandez et al., 1996; Tsai et al.,
2003; White, 1992; Zelus et al., 2003). Currently, specific membrane fusion models have
been proposed all of which include the following general steps: a) binding of a receptor
through a receptor specific domain located within the ectodomain of the viral
glycoprotein; b) induction of a conformational change via low pH or binding to the
receptor that exposes a fusion peptide, typically a hydrophobic region in the membrane
anchored subunit, which inserts into the cellular lipid membrane; c) formation of a
trimer-of-hairpins like structure by α-helical peptides, termed heptad repeat segments, via
a transient pre-hairpin intermediate that facilitates the juxtaposition of the viral and
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cellular membranes which then leads to fusion of the viral envelope with cellular
membranes (reviewed in (Eckert and Kim, 2001; Hernandez et al., 1996)). Although the
most important domains of the class I fusion proteins are naturally located in their
ectodomains, it has been reported that intracytoplasmic endodomains play an important
role in intracellular transport and virus-induced cell fusion (Bagai and Lamb, 1996; Bos
et al., 1995; Chang, Sheng, and Gombold, 2000; Lontok, Corse, and Machamer, 2004;
Schwegmann-Wessels et al., 2004; Sergel and Morrison, 1995; Seth, Vincent, and
Compans, 2003; Tong et al., 2002; Waning et al., 2004; Yao and Compans, 1995).
In this paper, we show that mutations that alter the HR1 and predicted fusion
peptide domains of the SARS-CoV S glycoprotein as well as mutations located within α-
helical regions well separated from the HR1, HR2 and predicted fusion peptide domains
drastically affected S-mediated cell fusion. Importantly, mutagenesis of the S cytoplasmic
domains suggests that the carboxyl terminus of the S glycoprotein contains multiple but
distinct regulatory domains that may function in virus-induced cell fusion through
different mechanisms.
Functional Domains of the S Ectodomain
The CL1 cluster mutation is located within the predicted fusion peptide. The
constructed cluster mutations replaced the amino terminal a and d positions of the
predicted fusion peptide resulting in shortening the predicted α-helical portion of the
fusion peptide. The S mutant glycoprotein carrying the CL1 mutation was apparently
synthesized in comparable levels to the wild type S glycoprotein. As expected, although
this mutant S form was able to be expressed on cell surfaces (95% of wild-type S levels),
its ability to cause cell fusion was inhibited by more than 70% (Table 1; Group I
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mutants). This result confirms that the predicted fusion peptide it absolutely essential for
S mediated cell fusion, although the engineered collapse of the predicted region does not
significantly effect glycoprotein synthesis, processing, and cell-surface expression.
Recent studies have shown that interactions between HR1 and HR2 of SARS-CoV
are critical in producing the necessary conformation changes that result in exposure of the
fusion peptide and its insertion into apposed membranes (Ingallinella et al., 2004; Tripet
et al., 2004; Xu et al., 2004). Biochemical and x-ray crystallography studies have shown
that the HR1 and HR2 form a stable six-helix bundle, in which the HR1 helices form a
central coiled-coil surrounded by three HR2 helices in an oblique, antiparallel manner
termed the fusion core (Ingallinella et al., 2004; Tripet et al., 2004; Xu et al., 2004),
which is consistent with other class I fusion proteins (Baker et al., 1999; Bullough et al.,
1994; Caffrey et al., 1998; Chan et al., 1997; Lu, Blacklow, and Kim, 1995; Tan et al.,
1997; Weissenhorn et al., 1998a; Weissenhorn et al., 1998b; Weissenhorn et al., 1997).
Amino acid residues 902-947 in the SARS-CoV S HR1 domain fold into a predicted 12-
turn α-helix (entire length of the fusion core) with hydrophobic amino acids
predominantly occupying the a and d positions. The CL3 and CL4 mutations were
designed to change the a and d hydrophobic residues to hydrophilic (lysine) residues.
Both mutant glycoproteins were expressed on cell surfaces at reduced levels in
comparison to the wild-type S glycoprotein (14% and 20% reduction, respectively).
However, these mutations inhibited S-mediated fusion by 68% and 71%, respectively
(Table 1; Group I mutants). Therefore, the inability of the CL3 and CL4 mutants to cause
fusion is most likely due to ectodomain structural changes involving the HR1 domain.
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Inhibition of proper HR1 interaction with HR2 may be the primary cause of the observed
inhibition of S-mediated cell fusion.
The CL2 and CL5 cluster mutations are located upstream and downstream of HR1
within a predicted α-helical portion of the S ectodomain extending from residues 875 to
1014. Both CL2 and CL5 S mutant forms exhibited reduced S-mediated cell fusion (75%
and 76% reduction in comparison to the S wild-type, respectively), while they were
synthesized and expressed on cell-surfaces at levels similar to the S wild-type (Table 1;
Group I mutants). These data suggest that the inability of these S mutants to cause
extensive cell fusion was mostly due to structural alterations of the extracellular portion
of the S glycoprotein. Furthermore, these results suggest that α-helical portions of the S
ectodomain that are well-separated from HR1 and HR2 or the predicted fusion peptide
are important for S-mediated cell fusion. It is possible that these mutations affect HR1
interactions with HR2 by inhibiting ectodomain conformational changes required for
their optimal interactions. Specifically, the CL2 mutation is only 16 amino acids
downstream of the predicted fusion peptide. Therefore, this mutation may interfere with
fusion peptide-associated functions
Functional Domains of the Endodomain of S
The T1247 S mutation deletes 8 amino acids from the carboxyl terminus of S. This
S mutant exhibited a 65% reduction in cell fusion in comparison to the wild-type S, while
cell-surface expression was reduced by 20% in comparison to the wild type S (Table 1;
Group I mutants). Kinetic endocytosis experiments revealed that the T1247 S mutant
also endocytosed much faster than the wild-type S glycoprotein. Acidic cluster motifs are
known to serve as endocytotic signals (Brideau et al., 1999; Zhu et al., 1996). The rapid
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endocytosis of the T1247 S mutant form may be due to the relocation of the acidic cluster
KFDEDDSE proximal to the carboxyl terminus of the S glycoprotein after removal of the
terminal 8 amino acids resulting in more efficient endocytosis. Therefore, the inability of
the S mutant form to cause extensive cell fusion may be due primarily to its rapid
endocytosis from cell surfaces. It is worth noting that a similar deletion of 8 amino acids
from the carboxyl terminus of the MHV S glycoprotein resulted in enhanced S-mediated
cell fusion (Chang, Sheng, and Gombold, 2000). A comparison of the carboxyl terminal
amino acid sequences of these two glycoproteins reveals that the MHV S does not have a
well-defined acidic cluster in the SARS-COV S homologous location; however, the last
three amino acids of the MHV S are charged residues (HED). Therefore, the ability of the
truncated MHV S to cause more cell fusion may be due to increased surface retention
resulting from a reduction in endocytosis mediated by these charged residues.
Alternatively, the MHV deletion may cause structural changes that enhance the MHV S
fusogenicity by destabilizing the overall structure of the glycoprotein. Stabilization of the
carboxyl terminus has been shown to decrease the fusion activity of the vesicular
stomatitis virus G glycoprotein (Waning et al., 2004), therefore, conversely, it is possible
that destabilization of the carboxyl terminus may cause an increase in fusion.
Of particular interest is the T1238 truncation which removed the S carboxyl
terminal acidic domain. This S mutant glycoprotein exhibited a more than 40% increase
in cell fusion relative to the S wild type, while there was only a slight decrease in cell-
surface expression (Table 1; Group IV mutants). The fact that the overall levels of S
glycoprotein detected on cell surfaces as well as the endocytosis profile were not altered
suggests that this deletion may enhance fusion via a structural destabilization of the
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glycoprotein. In contrast, changing acidic amino acids of the acidic motif to alanine
residues inhibited cell fusion indicating that the acidic motif was important for S-
mediated cell fusion. Specifically, the CL6 cluster mutation changed the acidic residues
(DEDDSE) to alanine residues (AAAASA). This mutant protein, while being efficiently
expressed at cellular surfaces (87% of the wild type protein), exhibited a 51% reduction
in fusion activity in comparison to the wild type (Table 1; group I mutant). The observed
reduction in S-mediated cell fusion suggests that the acidic cluster plays an important
regulatory role in S-mediated cell fusion without appreciably affecting intracellular
transport and cell-surface expression. This result is in sharp contrast to the T1238
truncation that deleted the acidic cluster and caused enhanced S-mediated cell fusion. A
possible explanation for these seemingly disparate results is that modifications of the
carboxyl terminus produce differential effects on the structure and function of the protein
by rendering the S glycoprotein more or less prone to S-mediated fusion. Alternatively, it
is possible that the acidic cluster plays important roles only in the context of the entire S
glycoprotein by regulating binding to other viral or cellular proteins that may modify the
S fusogenic properties.
The acidic cluster located in the cytoplasmic portion of the SARS CoV S contains a
predicted phosphorylation site (DEDDSE). To address the role of this predicted
phosphorylation site within the acidic cluster, the CL7 cluster mutation was constructed.
The CL7 replaces the serine residue with an alanine residue within the acidic cluster.
This mutant S protein fused cells extensively (85% of the wild type) and was expressed
on cell-surfaces at levels similar to that of the wild type S (88% of the wild type) (Table
1; Group II mutants), suggesting that the potential phosphorylation site does not play an
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important role in S-mediated cell fusion and cell-surface expression. However, the CL7
mutant appeared to recycle to early endosomes in contrast to the wild type S, which
remained mostly on cell surfaces. Therefore, it is possible that the altered putative
phosphorylation site within the acidic cluster may play a yet unknown role in S retention
at cell surfaces. Conversely, lack of this signal may cause aberrant endocytosis to early
endosomes. The overall charge of the carboxyl terminus may also play some role in the
structure and function of S. In this regard, there are additional charged amino acids
dispersed upstream and downstream of the mutated charged cluster that may play some
role in S transport and S-mediated fusion through electrostatic interactions with other
viral and cellular proteins. Additional alanine scanning mutations would be needed to
resolve their potential contribution to S functions.
In contrast to the MHV S glycoprotein, in which the domains overlap, the charged
region of the SARS-CoV S glycoprotein and the two cysteine-rich motifs CRM1 and
CRM2 are separate distinct regions (Fig 2.8). The T1229 mutation deleted the CRM2
domain, while the T1214 truncation deleted both CRM1 and CRM2. Deletion of the
CRM2 slightly inhibited surface expression (91% of the wild type) while reducing fusion
activity by 22% (Table 1; Group II mutants). A similar truncation of the MHV S
glycoprotein produced a comparable effect on cell fusion (Chang, Sheng, and Gombold,
2000). The 1214 truncation severely inhibited cell surface expression by 77%, when
compared to the wild type, while cell-to-cell fusion activity was reduced by 84% (Table
1; Group III mutant). These results differ from previously published data on similar
truncations of the MHV S glycoprotein. Specifically, it was found that the replacement
of the entire cysteine rich domain with amino acid sequences derived from the
111
Figure 2.8. Cluster alignment of the carboxyl terminus of the SARS-CoV S and the MHV S glycoproteins. The shaded residues indicate the position of the cysteine rich motif in their respective protein. The cysteine residues are bolded. The charged clusters are indicated by a bracket over the corresponding regions.
112
113
cytoplasmic terminus of the herpes simplex virus 1 (HSV-1) glycoprotein D (gD)
severely inhibited MHV S glycoprotein function without necessarily affecting cell-
surface expression (Chang, Sheng, and Gombold, 2000; Ye, Montalto-Morrison, and
Masters, 2004). In contrast, the SARS-CoV S T1214 mutant glycoprotein failed to be
expressed on cell surfaces explaining the inability of this glycoprotein to cause cell
fusion. These results suggest that the proximal cysteine residues of the SARS CoV S play
crucial roles in intracellular transport and cell-surface expression. The discrepancy with
the MHV S carboxyl terminal replacements may be due to additional gD sequences that
facilitated intracellular transport and cell-surface expression. It is worth noting that
depending on the algorithm used to predict the membrane spanning domain of S, a few of
the cysteine residues may be included in the membrane spanning region (Chang, Sheng,
and Gombold, 2000; Ye, Montalto-Morrison, and Masters, 2004). Therefore, it is
possible that deletion of these cysteine residues may lead to S misincorporation into
membranes, resulting in the apparent transport defects.
Overall, these results suggest that the S-mediated cell fusion is regulated by both
the ecto- and endodomains, which play important roles in cell-surface expression.
Furthermore, the data suggest that the 17 carboxyl terminal amino acid residues of S
exert a negative regulatory (repressor) effect on S-mediated cell fusion, while both the
carboxyl terminal acidic cluster and CRM2 domains exert secondary regulatory roles in
S-mediated cell fusion. Additional studies are required to elucidate the specific amino
acid requirements of the S endodomain that can affect S-mediated cell fusion potentially
via a transmembrane signal transduction process that leads to destabilization of the S
ectodomain.
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MATERIALS AND METHODS
Cells
African green monkey kidney (Vero) cells were obtained from the American Type
Culture Collection (Rockville, Md.). Cells were propagated and maintained in Dulbecco
modified Eagle medium (Sigma Chemical Co., St. Louis, Mo.) containing sodium
bicarbonate and 15 mM HEPES supplemented with 10% heat-inactivated fetal bovine
serum.
Plasmids
The parental plasmid used in the present study, SARS-S-Optimized, has been
previously described (Li et al., 2003). The Spike-3XFLAG-N gene construct was
generated by cloning the codon-optimized S gene, without the DNA sequence coding for
the signal peptide, into the p3XFLAG-CMV-9 plasmid vector (Sigma). PCR overlap
extension (Aiyar, Xiang, and Leis, 1996) was used to construct the alanine mutants,
cluster mutants, and the single point mutants. In order to construct the truncation
mutants, primers were designed that incorporated a stop codon and a BamHI restriction
site at the appropriate gene site. Restriction endonuclease sites HindIII and BamHI were
then used to clone the gene construct into the Spike-3XFLAG-N plasmid.
The constructed cluster mutants targeting the S ectodomain changed the following
sets of amino acids to lysine residues: CL:1 Y(855), L(859), G(862); CL2: L(898), N
(901); CL3: L(927), L(930), V(934); CL4: L(941), L(944); CL5: L(983), L(986). The
cluster mutants targeting the S endodomain changed the following amino acids to alanine
residues: CL6: D(1239), E(1240), D(1241), D(1242), E(1244); CL7: S(1243) (Figs. 2.1B
and 2.1C).
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Production of SARS-CoV S Monoclonal Antibodies
The monoclonal antibodies SW-111 was raised against the Spike envelope
glycoprotein of the SARS virus. A synthetic, codon-optimized gene corresponding to
SAR-CoV S glycoprotein coding sequences (Li et al., 2003) was engineered to produce
truncated, secreted proteins containing C-terminal His-tags which encode either the entire
ectodomain or just the receptor binding domain of S1. These genes were cloned into
baculoviral vectors, and the resulting virus was used to infect insect cell lines (High Five)
(Invitrogen, Carlsbad, CA). The supernatants were then tested by western blotting using
anti-6-His antibodies. Protein was partially purified from supernatants by passage
through a nickel column and then concentrated by ultrafiltration. Additional protein
derived in an analogous fashion was provided by the laboratory of Stephen Harrison.
Standard protocols for mouse immunization were used. The animals were maintained in
the animal facility of Dana-Farber Cancer Institute, and hybridomas and monoclonal
antibodies were produced in the Dana Farber/Harvard Cancer Center Monoclonal
Antibody Core. Spleenocytes from immunogenized mice were fused with NS-1 myeloma
cells (ATCC) using standard protocols. Antibody-producing clones were identified by
Western blot analysis, using purified SARS-CoV S. The positive cells were then sub-
cloned and re-tested against the purified SARS-CoV S glycoprotein. Isotype analysis
revealed that the antibody belonged to the IgG-1 class.
Western Blot and S Oligomerization Analysis
Vero cell monolayers in six-well plates were transfected with the indicated plasmids
utilizing the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s
directions. At 48 hours (h) post transfection, cells were collected by low-speed
116
centrifugation, washed with Tris-buffered saline (TBS), and lysed at on ice for 15 min in
mammalian protein extraction reagent supplemented with a cocktail of protease inhibitors
(Invitrogen/Life Technologies). Insoluble cell debris was pelleted, samples were
electrophoretically separated by SDS-PAGE, transferred to nitrocellulose membranes,
and probed with anti-SARS CoV monoclonal antibody at a 1:10 dilution. Samples being
analyzed for transport and processing were boiled for 5 min and treated with beta
mercaptoethanol, while samples being analyzed for trimer formation were not.
Subsequently, blots were incubated for 1 h with a peroxidase-conjugated secondary
antibody at a 1:50,000 dilution and then visualized on X-ray film by chemiluminescence
(Pierce Chemicals, Rockford, Ill.). All antibody dilutions and buffer washes were
performed in TBS supplemented with 0.135 M CaCl2 and 0.11 M MgCl2 (TBS-Ca/Mg).
Cell Surface Immunohistochemistry
Vero cell monolayers in six-well plates were transfected with the indicated plasmids
utilizing the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s
directions. At 48 h post transfection, the cells were washed with TBS-Ca/Mg and either
fixed with iced cold methanol or left unfixed (live). Immunohistochemistry was
performed by utilizing the Vector Laboratories Vectastain Elite ABC kit (Vector
Laboratories, Burlingame, CA) essentially as described in the manufacturer’s directions.
Briefly, cells were washed with TBS-Ca/Mg and incubated in TBS supplemented with
5% normal horse serum and 5% bovine serum albumin at room temperature for 1 h. After
blocking, cells were reacted with anti-FLAG antibody (1:500) in TBS blocking buffer for
3 h, washed four times with TBS-Ca/Mg, and incubated with biotinylated horse anti-
mouse antibody. Excess antibody was removed by four washes with TBS-Ca/Mg and
117
subsequently incubated with Vectastain Elite ABC reagent for 30 min. Finally, cells were
washed three times with TBS-Ca/Mg, and reactions were developed with NovaRed
substrate (Vector Laboratories) according to the manufacturer’s directions.
Determination of Cell-surface to Total Cell S Glycoprotein Expression
Vero cell monolayers in six-well plates were transfected with the indicated plasmids
and processed for immunohistochemistry as described above with the exception that the
ABTS Substrate Kit , 2, 2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Vector
Laboratories) was used instead of the NovaRed substrate. After the substrate was allowed
to develop for 30 min, 100 μl of the developed substrate was transferred, in triplicate, to a
96 well plate. The samples were then analyzed for color change at a wavelength of 405
nm. The absorbance reading from cell-surface labeling experiments obtained from live
cells were divided by the total labeled absorbance readings obtained from fixed cells
which was then normalized to the wild type protein values. The measurements were then
converted to percentages reflecting the ratio of S present on cell-surfaces versus the total
S expressed in the transfected cells.
Confocal Microscopy
Vero cell monolayers grown on coverslips in six-well plates were transfected with
the indicated plasmids utilizing the Lipofectamine 2000 reagent (Invitrogen) according to
the manufacturer’s directions. For the endocytosis analysis of the mutants, anti-FLAG
diluted 1:500 in TBS supplemented with 5% normal goat serum and 5% bovine serum
albumin (TBS blocking buffer) was added to the cell culture media for 12 hours before
processing. At 48 h post transfection, cells were washed with TBS and fixed with
electron microscopy-grade 3% paraformaldehyde (Electron Microscopy Sciences, Fort
118
Washington, Pa.) for 15 min, washed twice with TBS-Ca/Mg, and permeabilized with
0.1% Triton X-100. Monolayers were blocked for 1 hour with TBS blocking buffer
before incubation for 3 h with anti-FLAG antibody (sigma) diluted 1:500 in TBS
blocking buffer. Cells were then washed extensively and subsequently incubated for 1 h
with Alexafluor 488-conjugated anti-immunoglobulin G (IgG) (Molecular Probes)
diluted 1:750 in TBS blocking buffer. To visualize the early endosomes, cells were
stained with a 1:500 dilution of anti-early endosomal antigen I antibody (Affinity
Bioreagents Inc). Cells were examined by using a Leica TCS SP2 laser-scanning
microscope (Leica Microsystems, Exton, Pa.) fitted with a 63x Leica objective lens
(Planachromatic; 1.4 numerical aperture). Individual optical sections in the z axis,
averaged eight times, were collected simultaneously in the different channels at a 512 x
512 pixel resolution as described previously (Foster et al., 2004; Lee et al., 2000). Images
were compiled and rendered in Adobe Photoshop.
S Glycoprotein Endocytosis Assay
Vero cell monolayers grown on coverslips in six-well plates were transfected with
the indicated plasmids utilizing the Lipofectamine 2000 reagent (Invitrogen) according to
the manufacturer’s directions. At 48 hours posttransfection, the cells were washed once
with room temperature TBS-Ca/Mg. The plates were then moved to a 4ºC cold room and
washed with 4ºC TBS-Ca/Mg. The cells were labeled for 1 hour at 4°C with anti-FLAG
antibody diluted 1:500 in TBS blocking buffer. The cells were washed with 4ºC TBS
three times and then brought back to 37°C and allowed to incubate for their respective
time points. Then, the cells were immediately fixed and permeabilized with ice cold
methanol. Monolayers were blocked for 2 hrs in TBS blocking buffer before incubation
119
for 1 h with Alexafluor 488-conjugated anti-immunoglobulin G (IgG) (Molecular Probes)
diluted 1:750 in TBS-Ca/Mg blocking buffer. After incubation, excess antibody was
removed by washing five times with TBS-Ca/Mg. The nuclei was counterstained for 15
min with TO-PRO-3 iodide (1:5,000 dilution) and visualized at 647 nm. Cells were
examined by using a Leica TCS SP2 laser-scanning microscope (Leica Microsystems,
Exton, Pa.) fitted with a 63x Leica objective lens (Planachromatic; 1.4 numerical
aperature). Individual optical sections in the z axis, averaged eight times, were collected
simultaneously in the different channels at 512 x 512 pixel resolution as described
previously (Foster et al., 2004; Lee et al., 2000). Images were compiled and rendered in
Adobe Photoshop.
Quantitation of the Extent of S-mediated Cell Fusion
Vero cell monolayers in six-well plates were transfected in triplicate with the indicated
plasmids utilizing the Lipofectamine 2000 reagent (Invitrogen) according to the
manufacturer’s directions. Concurrently, Vero cell monolayers in six-well plates were
transfected with the plasmid encoding the ACE2 receptor protein utilizing the
Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s directions. At
24 h post transfection, cells containing the mutant plasmids, the ACE2 receptor, and
normal untransfected cells were washed with TBS-Ca/Mg, trypsinized, and overlayed in
a single well of a six-well plate at a ratio of 2 ml (cells transfected with the ACE2
receptor) : 0.5 ml (cells transfected with the mutant) : 1.5 ml (untransfected cells). All of
the cells transfected with ACE2 were pooled to ensure that every well had an equal
amount of cells with receptor expressed on their surface. After incubation for 24 h, the
cells were washed with TBS-Ca/Mg and fixed with ice cold methanol.
120
Immunohistochemistry was performed by utilizing the Vector Laboratories Vectastain
Elite ABC kit essentially as described in the manufacturer’s directions. Briefly, cells were
washed with TBS-Ca/Mg and incubated in TBS blocking buffer supplemented with
normal horse serum at room temperature for 1 h. After blocking, cells were reacted with
anti-FLAG antibody (1:500) in TBS blocking buffer for 3 h, washed four times with
TBS-Ca/Mg, and incubated with biotinylated horse anti-mouse antibody. Excess antibody
was removed by four washes with TBS-Ca/Mg and subsequently incubated with
Vectastain Elite ABC reagent for 30 min. Finally, cells were washed three times with
TBS-Ca/Mg, and reactions were developed with NovaRed substrate (Vector
Laboratories) according to the manufacturer’s directions. The average size of syncytia for
each mutant was determined by analyzing the area of approximately 300 syncytia, from
digital images, using the Image Pro Plus 5.0 software package. The averages were then
converted to percentages of the average syncytia size of the wild type SARS-CoV S.
Error bars shown represent the standard deviations calculated through comparison of the
data from each of three experiments.
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CHAPTER III
GENETIC ANALYSIS OF THE CYSTEINE RICH DOMAINS IN THE CARBOXYL TERMINUS OF THE SARS-COV SPIKE GLYCOPROTEIN
INTRODUCTION
An outbreak of atypical pneumonia, termed severe acute respiratory syndrome
(SARS), appeared in the Guangdong Province of southern China in November, 2002. The
mortality rates of the disease reached as high as 15% in some age groups (Anand et al.,
2003). The etiological agent of the disease was found to be a novel coronavirus (SARS-
CoV), which was first isolated from infected individuals by propagation of the virus on
Vero E6 cells (Drosten et al., 2003; Ksiazek et al., 2003; Peiris et al., 2003). Analysis of
the viral genome has demonstrated that the SARS-CoV is phylogenetically divergent
from the three known antigenic groups of coronaviruses (Drosten et al., 2003; Ksiazek et
al., 2003). Analysis of the polymerase gene alone, however, has indicated that the SARS-
CoV may be an early off-shoot from the group 2 coronaviruses (Snijder et al., 2003).
The coronaviruses are the largest of the enveloped RNA viruses with a positive-
stranded RNA genome of 28 to 32 kb (Holmes, 2003). Coronaviruses possess a wide host
range, capable of infecting mammalian and avian species. All identified coronaviruses
have a common group of indispensable genes that encode nonstructural proteins
including the RNA replicase gene open reading frame (ORF) 1ab and the structural
proteins nucleocapsid (N), membrane protein (M), envelope protein (E), and spike
glycoprotein (S), which are assembled into virus particles. A hemagglutinin-esterase
(HE) protein is also encoded by some coronaviruses. Distributed among the major viral
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genes are a series of ORFs that are specific to the different coronavirus groups. Functions
of the majority of these ORFs have not been determined.
The SARS spike glycoprotein, a 1255-amino-acid type I membrane glycoprotein
(Rota et al., 2003), is the major protein present in the viral membrane forming the typical
spike structure found on all coronavirions. The S glycoprotein facilitates virion entry of
all coronaviruses into susceptible cells by binding to specific receptors on cells and
mediating virus-cell fusion (Cavanagh, 1995). The S glycoprotein specified by mouse
hepatitis virus (MHV) is cleaved into S1 and S2 subunits, although cleavage is not
necessary for virus-cell fusion (Bos, Luytjes, and Spaan, 1997; Gombold, Hingley, and
Weiss, 1993; Stauber, Pfleiderera, and Siddell, 1993). Similarly, the SARS-CoV S
glycoprotein seems to be cleaved into S1 and S2 subunits in VeroE6-infected cells (Wu et
al., 2004), while it is not known whether this cleavage affects S-mediated cell fusion. The
SARS-CoV receptor has been recently identified as the angiotensin-converting enzyme 2
(ACE2) (Li et al., 2003). Although the exact mechanism by which the SARS-CoV enters
the host cell has not been elucidated, it is most likely similar to other coronaviruses.
Upon receptor binding at the cell membrane, the S glycoprotein is thought to undergo a
dramatic conformational change causing exposure of a hydrophobic fusion peptide,
which is subsequently inserted into cellular membranes. This conformational change of
the S glycoprotein causes close apposition followed by fusion of the viral and cellular
membranes resulting in entry of the virion nucleocapsids into cells (Eckert and Kim,
2001; Tsai et al., 2003; Zelus et al., 2003). This series of S-mediated virus entry events is
similar to other class I virus fusion proteins (Baker et al., 1999; Melikyan et al., 2000;
Russell, Jardetzky, and Lamb, 2001).
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Heptad repeat (HR) regions, a sequence motif characteristic of coiled-coils,
appear to be a common motif in many viral and cellular fusion proteins (Skehel and
Wiley, 1998). These coiled-coil regions allow the protein to fold back upon itself as a
prerequisite step to initiating the membrane fusion event. There are usually two HR
regions: an N terminal HR region adjacent to the fusion peptide and a C-terminal HR
region close to the transmembrane region of the protein. Within the HR segments, the
first amino acid (a) and fourth amino acid (d) are typically hydrophobic amino acids that
play a vital role in maintaining coiled-coil interactions. Based on structural similarities,
two classes of viral fusion proteins have been established. Class I viral fusion proteins
contain two heptad repeat regions and an N- terminal or N-proximal fusion peptide. Class
II viral fusion proteins lack heptad repeat regions and contain an internal fusion peptide
(Lescar et al., 2001). The MHV S glycoprotein, which is similar to other coronavirus S
glycoproteins, is a class I membrane protein that is transported to the plasma membrane
after being synthesized in the endoplasmic reticulum (Bosch et al., 2003). Typically, the
ectodomains of the S2 subunits of coronaviruses contain two regions with a 4, 3
hydrophobic (heptad) repeat the first being adjacent to the fusion peptide and the other
being in close proximity to the transmembrane region (de Groot et al., 1987).
In addition, the SARS-CoV S glycoprotein has a high (3%) cysteine content (39
residues. Nine of the cysteine resides are concentrated in a domain that spans the
transmembrane region and the cytoplasmic domain, with six of these residues extremely
well conserved throughout all coronaviruses (Fig 3.1). This unusual concentration of
cysteine residues along with the conservation of the residues among all coronaviruses
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Figure 3.1. Alignment of the membrane spanning domain and endodomain of the spike glycoprotein from ten different coronaviruses (Abraham et al., 1990; Binns et al., 1985; Delmas et al., 1992; Kunkel and Herrler, 1993; Luytjes et al., 1987; Marra et al., 2003; Mounir and Talbot, 1993; Parker, Gallagher, and Buchmeier, 1989; Raabe, Schelle-Prinz, and Siddell, 1990; Rasschaert and Laude, 1987). A schematic diagram of the SARS-CoV S protein is shown on top from amino acid 1 to amino acid 1255. A vertical line demarcates the approximate location of the division between the S1 and S2 subunits of the protein. The carboxyl terminus (amino acids 1193 to 1255) of the SARS-CoV S glycoprotein is shown enlarged below and is aligned with the same region of the S glycoprotein from nine other coronaviruses. Viruses from antigenic group I (feline infectious peritonitis virus [FIPV], transmissible gastroenteritis virus [TGEV], human coronavirus 229E [HCoV-229E]), antigenic group II (three different mouse hepatitis virus strains [A59, JHM, and MHV2], bovine coronavirus [BCoV], and human coronavirus OC43 [HCoV-OC43]), and antigenic group III (infectious bronchitis virus [IBV]) are represented in the alignment. The membrane spanning domain and the cytoplasmic tail are denoted with arrows above the alignment. Residues conserved in at least eight of the ten coronaviruses represented are indicated by the shaded residues. Cysteines that are highly conserved throughout all of the S proteins are noted by asterisks.
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suggest that these residues play an important role in S glycoprotein function although this
exact role is largely unknown. For MHV, studies have shown that the cysteine rich
domain is required for coronavirus-induced membrane fusion. Substituting the
cytoplasmic portion of this cysteine rich region and the cytoplasmic tail with the
cytoplasmic tail of the VSV-G protein abolished MHV S glycoprotein mediated cell-cell
fusion (Bos et al., 1995). Also, it was shown that while not being the sole functional
domain of the transmembrane anchor required for fusion activity, it was necessary for
fusion activity (Chang, Sheng, and Gombold, 2000).
In this study, cluster to alanine mutagenesis was used to elucidate which cysteine
clusters were dispensable for protein transport and SARS-CoV S mediated cell-cell
fusion. The results indicate that the SARS-CoV S glycoprotein conforms to the general
structure and function relationships that have been elucidated for the cysteine rich
domains of other coronaviruses, most notably the MHV (Bos et al., 1995; Chang, Sheng,
and Gombold, 2000). Specifically, the results show that the cysteine residues proximal to
the membrane are required for S-mediated fusion.
RESULTS
Genetic Analysis of S Glycoprotein Cysteine Rich Domain
It has been shown for other viral class I fusion proteins that the carboxyl terminus
plays a regulatory role in membrane fusion (Bagai and Lamb, 1996; Sergel and Morrison,
1995; Seth, Vincent, and Compans, 2003; Tong et al., 2002; Yao and Compans, 1995).
Specifically for coronaviruses, the MHV S glycoprotein endodomain has been shown to
contain cysteine rich regions, which are critical for fusion of infected cells (Bos et al.,
1995; Chang, Sheng, and Gombold, 2000; Ye, Montalto-Morrison, and Masters, 2004).
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To elucidate the role of the cytoplasmic cysteine rich domain in membrane fusion,
intracellular transport, and cell surface expression, cysteine cluster to alanine mutations
were made in the four cysteine clusters in the cytoplasmic domain of the S glycoprotein
(Fig. 3.2) (as described in materials and methods).
Effects of Mutations on S Synthesis
In order to investigate the effect of the different cluster to alanine mutations,
western immunoblot analysis was used to detect and visualize all of the mutant
glycoproteins as well as the wild type glycoprotein (Fig. 3.3). Cellular lysates prepared
from transfected cells at 48 hours post transfection were electrophoretically separated by
SDS-PAGE and the S glycoproteins were detected via chemiluminescence using a
monoclonal antibody specific for the SARS-CoV S glycoprotein. Carbohydrate addition
was shown to occur in at least four different locations of the SARS-CoV S glycoprotein
(Krokhin et al., 2003; Ying et al., 2004). Furthermore, transiently expressed S
glycoprotein in Vero E6 cells was proteolytically cleaved into S1 and S2 components
(Wu et al., 2004). The anti-S monoclonal antibody SW-111 detected a protein species in
cellular extracts from transfected cells, which migrated with an apparent molecular mass
of approximately 180 kDa, as reported previously (Song et al., 2004). All mutated S
glycoproteins produced similar S-related protein species to that of wild-type S indicating
that none of the engineered mutations adversely affected S synthesis and intracellular
processing (Fig. 3.3).
Ability of Mutant S Glycoproteins to Be Expressed on the Cell Surface
To determine if the mutant S glycoproteins were expressed on the surface of cells
efficiently, immunohistochemical analysis was used to label cell-surface expressed S
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Figure 3.2. Schematic diagram of the SARS-CoV S glycoprotein endodomain and the cysteine cluster to alanine mutations. Amino acid sequences of the carboxyl termini and the cysteine cluster to alanine mutations are shown for the wild-type as well as the mutant proteins. The cysteine cluster and the charged rich regions of the S proteins are encompassed in brackets and appropriately labeled. The transmembrane portion of the endodomain is italicized and underlined. Amino acids mutated to alanines for the C1217A, C1223A, C1230A, and C1235A cluster mutations are in bold.
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Figure 3.3 Western blot analysis of the expressed mutant SARS-CoV mutant glycoproteins. Immunoblots of wild-type [So-3xF(WT)] and cysteine to alanine mutant S glycoproteins probed with monoclonal anti-SARS S antiserum. “Cells only” represents a negative control for which Vero were mock transfected and probed with the monoclonal antibody to the SARS-CoV S glycoprotein.
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under live cell conditions that restrict antibody binding to cell surfaces. In addition,
immunohistochemistry was used to detect the total amount of S expressed in cells by
fixing and permeabilizing the cells prior to reaction with the antibody (see Materials and
Methods) (Fig 3.4). An ELISA was used to quantitatively determine the relative amounts
of cell-surface and total cellular expressed S glycoprotein. A ratio between the cell-
surface localized S and total cellular S expressed was then calculated and normalized to
the corresponding ratio obtained with the wild-type S glycoprotein (see Materials and
Methods) (Fig 3.5). All cysteine cluster to alanine mutants were expressed on the cell
surface at levels comparable to that of the wild-type. Specifically, the CL2-C1217A
(91%), CL3-C1223A (87%), CL2-C1230A (101%), and CL2-C1235A (110%) were
expressed on the cellular surface by the percentages indicated when compared to the cell
surface expression of the wild-type protein.
Effect of Mutations of S-mediated Cell-to-Cell Fusion
Transiently expressed wild-type S causes extensive cell-to-cell fusion (syncytial
formation), especially in the presence of the SARS-CoV ACE2 receptor (Li et al., 2003).
To determine the ability of each cysteine cluster to alanine mutant S glycoprotein to
cause cell-to-cell fusion and the formation of syncytia, fused cells were labeled by
immunohistochemistry using the anti-FLAG antibody, and the extent of cell-to-cell
fusion caused by each mutant glycoprotein was calculated by obtaining the average size
of approximately 300 syncytia (Fig 3.6). The average syncytium size for each mutant
was then normalized to that found in wild type S transfected cells (see Materials and
Methods). The C1217A (54%), C1223A (62%), C1230A (15%), and C1235A (14%)
mutants inhibited the formation of syncytia by the percentages indicated.
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Figure 3.4 Immunohistochemical detection of cell-surface and total expression of the SARS-CoV S wild-type and mutant proteins. Vero cells were transfected with the wild-type SARS-CoV optimized S (SARS So 3xF) (E1, E2), C1217A (A1, A2), C1223A (B1,B2), C1230A (C1,C2), C1235A (D1,D2), and a wild-type SARS-CoV optimized S labeled with a 3xFLAG carboxyl tag (F1, F2), which served as a negative control. At 48 hours post-transfection, cells were immunohistochemically processed wither under live conditions to show surface expression (A1, B1, C1, D1, E1, and F1) and permeablilized conditions to show total expression (A2, B2, C2, D2, E2, and F2) with anti-FLAG antibody.
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Figure 3.5. Ratios of cell-surface to total cellular expression of mutant SARS-CoV S glycoproteins. Detection of cell surface and total glycoprotein distribution was determined by immunohistochemistry and ELISA (see materials and methods). Cell-surface expression of the S glycoprotein was measured by incubating the transfected cell monolayers with anti-FLAG antibody at room temperature before permeabilization. For total S glycoprotein detection, cells were fixed and permeabilized prior to incubation with the anti-FLAG antibody. A ratio between the surface localization and the total expression was calculated and normalized to the wild-type protein, then set to a percentage of the wild-type. The error bars represent the maximum and minimum surface to total ratios obtained from three independent experiments, and the bar height represents the average surface to total ratio as a percentage of the wild-type.
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Figure 3.6 Quantitation of the extent of S-mediated cell fusion. The average size of syncytia for each mutant was determined by digitally analyzing the area of approximately 300 syncytia stained by immunohistochemistry for S glycoprotein expression using the Image Pro Plus 5.0 software package (see Materials and Methods). Error bars shown represent the standard deviation calculated through comparison of the data from each of three experiments.
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DISCUSSION
Although the most important domains of the class I fusion are naturally located in
their ectodomains, it has been reported that intracytoplasmic endodomains play an
important role in intracellular transport and virus-induced cell-to-cell fusion (Bagai and
Lamb, 1996; Bos et al., 1995; Chang, Sheng, and Gombold, 2000; Lontok, Corse, and
Machamer, 2004; Petit et al., 2005; Schwegmann-Wessels et al., 2004; Sergel and
Morrison, 1995; Tong et al., 2002; Waning et al., 2004; Yao and Compans, 1995). This
study shows that the two cysteine clusters closest to the transmembrane regions were
vital for the protein to be able to induce cell-to-cell fusion while the two cysteine clusters
closest to the carboxyl end of the protein did not drastically affect cell-to-cell fusion. Two
similar studies performed on the MHV S glycoprotein produced similar results found for
the SARS-CoV S glycoprotein.
The C1217A and C1223A are cysteine to alanine cluster mutations that target the
two cysteine rich clusters most proximal to the transmembrane region of the S
glycoprotein. Both of these proteins were expressed on cell surfaces at levels comparable
to that of the wild-type (91% and 87% of the of the wild-type protein, respectively).
However, these mutant forms had a significant impact on S glycoprotein induced cell-to-
cell fusion. Similar mutations of the cysteine clusters in the MHV S glycoprotein
produced a comparable effect on cell fusion (Chang, Sheng, and Gombold, 2000). The
C1217A cluster mutant reduced fusion activity by 55 % while the equivalent cysteine to
serine mutation in MHV reduced fusion by 56% (Chang, Sheng, and Gombold, 2000). In
addition, the C1223A cluster to alanine mutation had a substantial effect on fusion (60%
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reduction) in a similar manner to the corresponding MHV S mutant which caused a 94%
inhibition of S-mediated cell fusion. (Chang, Sheng, and Gombold, 2000).
The S2 fragment of the S glycoprotein is palmitylated on its carboxyl-terminal
region (Niemann and Klenk, 1981; Sturman, Holmes, and Behnke, 1980; van Berlo et al.,
1987). In general, cysteines proximal to the inner leaflet of the plasma membrane in viral
proteins often undergo palmitate attachment through a thioester bond (Ponimaskin and
Schmidt, 1995; Rose, Adams, and Gallione, 1984; Schlesinger, Veit, and Schmidt, 1993;
Schmidt, 1989; Sefton and Buss, 1987). Both MHV mutants corresponding to the SARS-
CoV C1217A and C1223 mutants have been shown to be acylation sites for the MHV S
glycoprotein (Bos et al., 1995). Covalent palmitic acid attachment to these cysteines may
have an impact on the conformation of the transmembrane and subsequently fusion.
Since palmytilation is able to affect an assortment of processes either mediated by or
involving fusion proteins including membrane fusion, infection of cells, and virus
assembly (Glick and Rothman, 1987; Jin et al., 1996; Melikyan et al., 2000; Naim et al.,
1992; Schroth-Diez et al., 1998; Zurcher, Luo, and Palese, 1994), it is plausible that
mutation of these cysteine clusters prevented palmytilation which in turn adversely
affected S-mediated fusion.
The C1230A and C1235A cluster mutations are the two cysteine clusters that are
closest to the carboxyl end of the S glycoprotein. Both C1230A and C1235A mutant
forms exhibited levels of S-mediated cell fusion that were similar to that of the wild-type
(85% and 88% of the wild-type protein, respectively, while they were synthesized and
expressed on cell-surfaces at levels similar to that of the wild-type S. These data suggests
that the two cysteine clusters are not vital for the proper synthesis, transport, or function
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of the S glycoprotein. Instead of participating in protein function and trafficking, these
two cysteine clusters may play a role in virion assembly or virus-protein interactions.
MATERIALS AND METHODS Cells
African green monkey kidney (Vero) cells were obtained from the American Type
Culture Collection (Rockville, Md.). Cells were propagated and maintained in Dulbecco
modified Eagle medium (Sigma Chemical Co., St. Louis, Mo.) containing sodium
bicarbonate and 15 mM HEPES supplemented with 10% heat-inactivated fetal bovine
serum.
Plasmids
The parental plasmid used in the present study, SARS-S-Optimized, has been
previously described (Li et al., 2003). The Spike-3XFLAG-N gene construct was
generated by cloning the codon-optimized S gene, without the DNA sequence coding for
the signal peptide, into the p3XFLAG-CMV-9 plasmid vector (Sigma). PCR overlap
extension (Aiyar, Xiang, and Leis, 1996) was used to construct the cluster to alanine
mutants. To construct the truncation mutants, primers were designed that incorporated a
stop codon and a BamHI restriction site at the appropriate gene site. Restriction
endonuclease sites BamHI and Pml-I were then used to clone the gene construct into the
Spike-3XFLAG-N plasmid.
The constructed cluster mutants targeting the S cysteine rich region changed the
following sets of amino acids to alanine residues: C1217A: C(1217), C(1218); C1223A:
C(1223), C (1224), C(1226); C1230A: C(1230), C(1232); C1235A: C(1235), C(1236).
(Fig 3.2)
148
Production of SARS-CoV S Monoclonal Antibodies
The monoclonal antibodies SW-111 was raised against the Spike envelope
glycoprotein of the SARS virus. A synthetic, codon-optimized gene corresponding to
SAR-CoV S glycoprotein coding sequences (Li et al., 2003) was engineered to produce
truncated, secreted proteins containing C-terminal His-tags which encode either the entire
ectodomain or just the receptor binding domain of S1. These genes were cloned into
baculoviral vectors, and the resulting virus was used to infect insect cell lines (High Five)
(Invitrogen, Carlsbad, CA). The supernatants were then tested by western blotting using
anti-6-His antibodies. Protein was partially purified from supernatants by passage
through a nickel column and then concentrated by ultrafiltration. Additional protein
derived in an analogous fashion was provided by the laboratory of Stephen Harrison.
Standard protocols for mouse immunization were used. The animals were maintained in
the animal facility of Dana-Farber Cancer Institute, and hybridomas and monoclonal
antibodies were produced in the Dana Farber/Harvard Cancer Center Monoclonal
Antibody Core. Spleenocytes from immunogenized mice were fused with NS-1 myeloma
cells (ATCC) using standard protocols. Antibody-producing clones were identified by
Western blot analysis, using purified SARS-CoV S. The positive cells were then sub-
cloned and re-tested against the purified SARS-CoV S glycoprotein. Isotype analysis
revealed that the antibody belonged to the IgG-1 class.
Western Blot Analysis
Vero cell monolayers in six-well plates were transfected with the indicated plasmids
utilizing the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s
directions. At 48 hours (h) post transfection, cells were collected by low-speed
149
centrifugation, washed with Tris-buffered saline (TBS), and lysed at on ice for 15 min in
mammalian protein extraction reagent supplemented with a cocktail of protease inhibitors
(Invitrogen/Life Technologies). Insoluble cell debris was pelleted, samples were
electrophoretically separated by SDS-PAGE, transferred to nitrocellulose membranes,
and probed with anti-SARS CoV monoclonal antibody at a 1:10 dilution. Samples were
boiled for 5 min and treated with beta mercaptoethanol. Subsequently, blots were
incubated for 1 h with a peroxidase-conjugated secondary antibody at a 1:50,000 dilution
and then visualized on X-ray film by chemiluminescence (Pierce Chemicals, Rockford,
Ill.). All antibody dilutions and buffer washes were performed in TBS supplemented with
0.135 M CaCl2 and 0.11 M MgCl2 (TBS-Ca/Mg).
Cell Surface Immunohistochemistry
Vero cell monolayers in six-well plates were transfected with the indicated plasmids
utilizing the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s
directions. At 48 h post transfection, the cells were washed with TBS-Ca/Mg and either
fixed with iced cold methanol or left unfixed (live). Immunohistochemistry was
performed by utilizing the Vector Laboratories Vectastain Elite ABC kit (Vector
Laboratories, Burlingame, CA) essentially as described in the manufacturer’s directions.
Briefly, cells were washed with TBS-Ca/Mg and incubated in TBS supplemented with
5% normal horse serum and 5% bovine serum albumin at room temperature for 1 h. After
blocking, cells were reacted with anti-FLAG antibody (1:500) in TBS blocking buffer for
3 h, washed four times with TBS-Ca/Mg, and incubated with biotinylated horse anti-
mouse antibody. Excess antibody was removed by four washes with TBS-Ca/Mg and
subsequently incubated with Vectastain Elite ABC reagent for 30 min. Finally, cells were
150
washed three times with TBS-Ca/Mg, and reactions were developed with NovaRed
substrate (Vector Laboratories) according to the manufacturer’s directions.
Determination of Cell-surface to Total Cell S Glycoprotein Expression
Vero cell monolayers in six-well plates were transfected with the indicated plasmids
and processed for immunohistochemistry as described above with the exception that the
ABTS Substrate Kit , 2, 2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Vector
Laboratories) was used instead of the NovaRed substrate. After the substrate was allowed
to develop for 30 min, 100 μl of the developed substrate was transferred, in triplicate, to a
96 well plate. The samples were then analyzed for color change at a wavelength of 405
nm. The absorbance reading from cell-surface labeling experiments obtained from live
cells were divided by the total labeled absorbance readings obtained from fixed cells
which was then normalized to the wild type protein values. The measurements were then
converted to percentages reflecting the ratio of S present on cell-surfaces versus the total
S expressed in the transfected cells.
Quantitation of the Extent of S-mediated Cell Fusion
Vero cell monolayers in six-well plates were transfected in triplicate with the
indicated plasmids utilizing the Lipofectamine 2000 reagent (Invitrogen) according to the
manufacturer’s directions. Concurrently, Vero cell monolayers in six-well plates were
transfected with the plasmid encoding the ACE2 receptor protein utilizing the
Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s directions. At
24 h post transfection, cells containing the mutant plasmids, the ACE2 receptor, and
normal untransfected cells were washed with TBS-Ca/Mg, trypsinized, and overlayed in
a single well of a six-well plate at a ratio of 2 ml (cells transfected with the ACE2
151
receptor) : 0.5 ml (cells transfected with the mutant) : 1.5 ml (untransfected cells). All of
the cells transfected with ACE2 were pooled to ensure that every well had an equal
amount of cells with receptor expressed on their surface. After incubation for 24 h, the
cells were washed with TBS-Ca/Mg and fixed with ice cold methanol.
Immunohistochemistry was performed by utilizing the Vector Laboratories Vectastain
Elite ABC kit essentially as described in the manufacturer’s directions. Briefly, cells were
washed with TBS-Ca/Mg and incubated in TBS blocking buffer supplemented with
normal horse serum at room temperature for 1 h. After blocking, cells were reacted with
anti-FLAG antibody (1:500) in TBS blocking buffer for 3 h, washed four times with
TBS-Ca/Mg, and incubated with biotinylated horse anti-mouse antibody. Excess antibody
was removed by four washes with TBS-Ca/Mg and subsequently incubated with
Vectastain Elite ABC reagent for 30 min. Finally, cells were washed three times with
TBS-Ca/Mg, and reactions were developed with NovaRed substrate (Vector
Laboratories) according to the manufacturer’s directions. The average size of syncytia for
each mutant was determined by analyzing the area of approximately 300 syncytia, from
digital images, using the Image Pro Plus 5.0 software package. The averages were then
converted to percentages of the average syncytia size of the wild type SARS-CoV S.
Error bars shown represent the standard deviations calculated through comparison of the
data from each of three experiments.
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CHAPTER IV
GENETIC COMPARISON OF THE SARS-COV AND BCOV S GLYCOPROTEINS
INTRODUCTION
In November 2002, an outbreak of atypical pneumonia, termed severe acute
respiratory syndrome (SARS), appeared in the Guangdong Province of southern China.
The etiological agent of the disease was found to be a novel coronavirus (SARS-CoV),
which was first isolated from infected individuals by propagation of the virus on Vero E6
cells (Drosten et al., 2003; Ksiazek et al., 2003; Peiris et al., 2003). For some age groups
the mortality rates of the disease reached as high as 15% (Anand et al., 2003).
In addition to the recent impact that coronaviruses, in the form of the SARS-CoV,
have had on public health, other coronaviruses produce significant diseases and economic
impact in various animal species. These viral infections include transmissible
gastroenteritis virus (TGEV), infectious bronchitis virus (IBV) ,and murine hepatitis virus
(MHV). Of particular interest are bovine coronaviruses since their disease spectrum seem
to be similar to that of the SARS-CoV. Primarily, bovine coronaviruses were known to
be enteropathogenic viruses that caused severe diarrhea in neonatal calves. The enteric
strains of the virus were known as enteric bovine coronavirus (EBCoV). Recently,
however, respiratory bovine coronaviruses were identified as the primary cause of acute
respiratory disease “shipping fever” as well as deep lung pneumonia in two Texas-based
epizootics (Storz et al., 2000a; Storz et al., 2000b). These isolated viruses were termed
respiratory coronaviruses (RBCoV). In a similar scenario to SARS-CoV, bovine
coronavirus virions were isolated from both diarrhea fluid and intestinal samples as well
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as from nasal secretions and lung tissues of animals experiencing both respiratory and
enteric disease (Doughri et al., 1976; Mebus et al., 1973; Storz et al., 2000a; Storz et al.,
2000b). These RBCoV and EBCoV strains can be differentiated on the basis of
phenotypic variation, antigenic differences, and genetic divergence (Chouljenko et al.,
1998; Chouljenko et al., 2001; Lin et al., 2000; Storz et al., 2000a). It is not known
however, whether respiratory and enteric disease are caused by different viruses or
alternatively, whether viruses are selected from a parental strain for their ability to
replicate in specific tissues. A comparison study between respiratory and
enteropathogenic coronavirus spikes isolated from the same animal with fatal shipping
pneumonia indicated at least 7 amino acid differences including amino acid changes
within the S1 portion that may account for a potential alteration in tissue specificity
(Chouljenko et al., 1998; Gelinas et al., 2001; Hasoksuz et al., 2002; Rekik and Dea,
1994). A comparison of the RBCoV and EBCoV S glycoproteins is shown in Figure 4.1.
The coronaviruses are the largest of the enveloped RNA viruses with a positive-
stranded RNA genome of 28 to 32 kb (Holmes, 2003). Coronaviruses possess a wide host
range, capable of infecting mammalian and avian species. All identified coronaviruses
have a common group of indispensable genes that encode nonstructural proteins
including the RNA replicase gene open reading frame (ORF 1ab) and the structural
proteins: nucleocapsid (N), membrane protein (M), envelope protein (E), and spike
glycoprotein (S), which are assembled into virus particles. A hemagglutinin-esterase
(HE) protein is also encoded by some coronaviruses. The SARS-CoV does not contain
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Figure 4.1. Schematic diagram of the BCoV S glycoprotein denoting the differences between RBCoV and EBCoV (Chouljenko et al., 2001). A schematic diagram of the BCoV S protein is shown on top from amino acid 1 to amino acid 1363. The transmembrane region of the protein is noted with a grey box labeled “Tm”. The two heptad repeat regions are indicated with grey boxes labeled HR1 and HR2, respectively. A vertical line demarcates the location of the division between the S1 and S2 subunits of the protein with the amino acid number between S1 and S2 shown. Amino acid differences are denoted with the amino acid number shown and the corresponding residues listed below.
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the HE protein while it is found in the BCoV (Lai and Cavanagh, 1997; Rota et al.,
2003).
The SARS S glycoprotein is composed of 1255-amino-acid with an estimated
post-translational mass of approximately 180 kDa. S is a type I membrane glycoprotein
(Rota et al., 2003) that is the major protein present in the viral membrane forming the
typical spike structure found on all coronavirions. After posttranslational modifications
are completed, the SARS-CoV S glycoprotein may be cleaved into S1 and S2 subunits in
VeroE6-infected cells (Wu et al., 2004). The receptor for SARS-CoV S has been
recently identified as the angiotensin-converting enzyme 2 (ACE2) (Li et al., 2003) and
CD209L (L-SIGN) (Jeffers et al., 2004).
The bovine coronavirus spike glycoprotein is made up of 1363 amino acids with
an estimated preglycosylated mass of 151 kDa (Abraham et al., 1990; Boireau, Cruciere,
and Laporte, 1990; Jeffers et al., 2004; Parker et al., 1990; Zhang, Kousoulas, and Storz,
1991). Once fully glycosylated, the BCoV S glycoprotein has an estimated mass of 190
kDa with 19 potential sites of glycosylation (Abraham et al., 1990; Cavanagh, 1995).
After posttranslational modification, the S glycoprotein is cleaved into two subunits, the
N-terminal S1 (110 kDa) and the C-terminal S2 (100 kDa) (Cyr-Coats and Storz, 1988).
This cleavage into the two subunits occurs between amino acid 768 and 769 of the spike
glycoprotein and is thought to be mediated by cellular trypsin-like proteases (Storz, Rott,
and Kaluza, 1981).
The S glycoprotein is primarily responsible for entry of all coronaviruses into
susceptible cells through binding to specific receptors on cells and mediating subsequent
virus-cell fusion (Cavanagh, 1995). Although the exact mechanism by which the SARS-
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CoV enters the host cell has not been elucidated, it is most likely similar to other
coronaviruses. Upon receptor binding at the cell membrane, the S glycoprotein is
thought to undergo a dramatic conformational change causing exposure of a hydrophobic
fusion peptide, which is subsequently inserted into cellular membranes. This
conformational change of the S glycoprotein causes close apposition followed by fusion
of the viral and cellular membranes resulting in entry of the virion nucleocapsids into
cells (Eckert and Kim, 2001; Tsai et al., 2003; Zelus et al., 2003). This series of S-
mediated virus entry events is similar to other class I virus fusion proteins (Baker et al.,
1999; Melikyan et al., 2000; Russell, Jardetzky, and Lamb, 2001).
Although the most important domains of the class I fusion proteins are naturally
located in their ectodomains, it has been reported that their cytoplasmic endodomains
play an important role in intracellular transport and virus-induced cell fusion (Bagai and
Lamb, 1996; Bos et al., 1995; Chang, Sheng, and Gombold, 2000; Lontok, Corse, and
Machamer, 2004; Schwegmann-Wessels et al., 2004; Sergel and Morrison, 1995; Seth,
Vincent, and Compans, 2003; Tong et al., 2002; Waning et al., 2004; Yao and Compans,
1995). Both the BCoV and SARS-CoV S glycoprotein have similar motifs in their
intracytoplasmic tails that could possibly play a role in the proper functioning of the
protein (Fig 4.2). In this study BCoV and SARS Spikes were compared with regard to
their ability to cause S-mediated cell fusion paying particular attention to comparing and
contrasting the functional domains contained within their carboxyl termini. The salient
features of this study are: 1) Transient expression of the RBCoV in Vero cells produced
significantly less cell fusion than the SARS-CoV; 2) Surprisingly, both RBCoV and
EBCoV spike proteins induced similar amounts of cell fusion; 3) The carboxyl
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Figure 4.2. Schematic diagram of the SARS-CoV and BCoV S glycoprotein endodomains. Amino acid sequences of the carboxyl termini are shown for the wild-type S glycoproteins. The cysteine cluster and the charged rich regions of the S proteins are encompassed in brackets and appropriately labeled. The shaded residues represent the transmembrane portion of the endodomain. Amino acids that serve as predicted phosphorylation sites are denoted by asterisks.
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terminal domains of RBCoV S function substantially differently than their homologous
domains of the SARS-CoV.
RESULTS
Comparison of the Ectodomain of the SARS-CoV and BCoV Spike Glycoproteins
Analysis of the viral genome has demonstrated that the SARS-CoV is
phylogenetically divergent from the three known antigenic groups of coronaviruses
(Drosten et al., 2003; Ksiazek et al., 2003). Analysis of the polymerase gene alone,
however, has indicated that the SARS-CoV may be an early off-shoot from the group 2
coronaviruses (Snijder et al., 2003). Both the BCoV and the SARS-CoV S glycoprotein
contain multiple analogous motifs as well as unique motifs to their corresponding
antigenic group. Comparison of the S1 portions reveals no apparent homologies with
respect to potential domains that function in receptor binding indicating that there is
substantial divergence with respect to this function. However, a comparison between the
amino acid sequences between the SARS-CoV and BCoV S proteins reveal a variety of
regions that are conserved throughout the majority of the coronavirus families whose
exact functions have yet to be determined. One such highly conserved amino acid
sequence (KWPWYVWL) can be found proximal to the transmembrane region (Fig 4.3).
The exact function of this eight-residue sequence has yet to be elucidated, but it may play
a structural role in the stabilization of the protein in the membrane. The S2 ectodomain
portion of the coronavirus S glycoprotein was found to have a higher degree of homology
(38% homology) when compared to that of the S1 subunit (16% homology), the
transmembrane region (22% homology), or the endodomain of the S2 subunit (16%
homology). Heptad repeats (HR), a sequence motif characteristic of coiled-coils, are
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Figure 4.3. Alignment of the membrane spanning domain and endodomain of the spike glycoprotein from ten different coronaviruses (Abraham et al., 1990; Binns et al., 1985; Delmas et al., 1992; Kunkel and Herrler, 1993; Luytjes et al., 1987; Marra et al., 2003; Mounir and Talbot, 1993; Parker, Gallagher, and Buchmeier, 1989; Raabe, Schelle-Prinz, and Siddell, 1990; Rasschaert and Laude, 1987). A schematic diagram of the SARS-CoV S protein is shown on top from amino acid 1 to amino acid 1255. A vertical line demarcates the approximate location of the division between the S1 and S2 subunits of the protein. The carboxyl terminus (amino acids 1193 to 1255) of the SARS-CoV S glycoprotein is shown enlarged below and is aligned with the same region of the S glycoprotein from nine other coronaviruses. Viruses from antigenic group I (feline infectious peritonitis virus [FIPV], transmissible gastroenteritis virus [TGEV’, human coronavirus 229E [HCoV-229E]), antigenic group II (three different mouse hepatitis virus strains [A59, JHM, and MHV2], bovine coronavirus [BCoV], and human coronavirus OC43 [HCoV-OC43]), and antigenic group III (infectious bronchitis virus [IBV]) are represented in the alignment. The membrane spanning domain and the cytoplasmic tail are denoted with arrows above the alignment. Residues conserved in at least eight of the ten coronaviruses represented are indicated by the shaded residues. Cysteines that are highly conserved throughout all of the S proteins are noted by asterisks.
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found in the S2 portion of the S molecule and are common motifs in many viral and
cellular fusion proteins (Skehel and Wiley, 1998) (Fig 4.4).These repeat regions allow the
protein to fold back upon itself. This event is critical to the function of the protein in that
it is the first step in initiating the membrane fusion event. The BCoV and SARS-CoV
HR regions are highly homologous to one another with the amino most HR region (HR1)
having 56% homology and the HR region proximal to the transmembrane sequence
(HR2) having 39% homology between the two viruses.
Comparison of the Endodomain of the SARS-CoV and BCoV Spike Glycoproteins
A common motif that is found in the coronavirus spike protein is the presence of a
charge-rich region in the endodomain. Analysis of the endodomains indicate that both
the BCoV and SARS-CoV S glycoprotein contain a homologous charged region (Fig
4.2). The charged rich region of the SARS-CoV S protein contains six out of seven
charged residues and has been shown to be a potent endocytosis signal that may be
stabilized by the rest of the endodomain (Petit et al., 2005). The charged rich region of
the BCoV S protein contains ten out of twenty-four charged residues and is located on the
carboxyl terminus of the protein’s endodomain. Although the entire amino acid sequence
of S contains little homology between different strains of coronaviruses, all S
glycoproteins contain a cysteine-rich region in their endodomain. Contained within the
BCoV and SARS-CoV S glycoprotein endodomains are regions of eight out of eighteen
and nine out of twenty amino acids that are cysteine residues, respectively. Many of
these cysteine residues located in the S protein are conserved throughout all of the
coronavirus families (Fig 4.3).
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Figure 4.4. Alignment heptad repeat regions of the S1 subunit of the spike glycoprotein from SARS-CoV and BCoV (Abraham et al., 1990; Marra et al., 2003). A schematic diagram of the SARS-CoV S protein is shown on top from amino acid 1 to amino acid 1255. The transmembrane region of the protein is noted with a black box labeled “Tm”. The two heptad repeat regions are indicated with striped boxes and labels HR1 and HR2 respectively. A vertical line demarcates the approximate location of the division between the S1 and S2 subunits of the protein. The two regions containing the heptad repeats (amino acids 879 to 980) of the SARS-CoV S glycoprotein is shown enlarged below and is aligned with the same region of the S glycoprotein from BCoV. Residues conserved between the two viruses represented are overlayed by shaded boxes.
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It has been shown for other viral class I fusion proteins that the carboxyl terminus
plays a regulatory role in membrane fusion (Bagai and Lamb, 1996; Sergel and Morrison,
1995; Seth, Vincent, and Compans, 2003; Tong et al., 2002; Yao and Compans, 1995).
Specifically for coronaviruses, the MHV S glycoprotein endodomain contains charged-
rich and cysteine-rich regions, which are critical for fusion of infected cells (Bos et al.,
1995; Chang, Sheng, and Gombold, 2000; Ye, Montalto-Morrison, and Masters, 2004).
To elucidate the role of the specific motifs found in both the BCoV and the SARS-CoV
spike proteins, a series of truncations targeting specific motifs in both proteins were made
and tested for their cell-to-cell fusion activity (Fig 4.5).
Effect of Mutations of S-mediated Cell-to-Cell Fusion
Wild-type SARS-CoV S is able to cause extensive cell-to-cell fusion (syncytial
formation) in a transient system especially when overlayed with cells expressing the
SARS-CoV ACE2 receptor (Li et al., 2003). To determine the ability of each truncation
mutant to cause cell-to-cell fusion, fused cells were first labeled by
immunohistochemistry using the anti-FLAG antibody (Fig 4.6). This
immunohistochemical analysis serves to visualize both protein expression as well as
approximate cell-cell fusion activity. The extent of cell-to-cell fusion caused by each
mutant glycoprotein was quantified by calculating the average size of approximately 300
syncytia using digital analysis software. The average syncytium size for each mutant was
then normalized to that found in wild type S transfected cells (see Materials and
Methods) (Fig 4.7). For the truncations made in the SARS-CoV S glycoprotein, a range
of different phenotypes were observed (see Chapter 2). For the T1214 (86%) and T1247
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Figure 4.5. Schematic diagram of the SARS-CoV and BCoV S glycoprotein endodomains. Amino acid sequences of the carboxyl termini are shown for the wild-type S glycoproteins as well as truncated proteins. Each SARS-CoV S truncation is coupled with its corresponding BCoV S truncation. The cysteine cluster and the charged rich regions of the S wild-type proteins are encompassed in brackets and appropriately labeled. The shaded residues represent the transmembrane portion of the endodomain. Amino acids that serve as predicted phosphorylation sites are denoted by asterisks.
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Figure 4.6. Immunohistochemical detection of total expression of the BCoV S wild-type and truncated proteins. Vero cells were transfected with T1359 (Panel A), T1355 (Panel B), T1346 (Panel C), and T1333 (Panel D), and wild-type respiratory (BCoV-Lun So 3xF) (Panel E) and enteric (BCoV-End So 3xF) (Panel F) strains of the BCoV optimized S) labeled with an amino terminus 3xFLAG tag. At 48 hours post-transfection, cells were immunohistochemically processed wither under permeabilized conditions to show total expression with anti-FLAG antibody.
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Figure 4.7. Quantitation of the extent of S-mediated cell fusion. The average size of syncytia for each mutant was determined by digitally analyzing the area of approximately 300 syncytia stained by immunohistochemistry for S glycoprotein expression using the Image Pro Plus 5.0 software package (see Materials and Methods). The fusion levels quantitated for the VERO-E6 cell line are represented by black bars while the levels for VERO cells are represented by grey bars. Error bars shown represent the standard deviation calculated through comparison of the data from each of three experiments.
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(66%) mutants, the formation of syncytia was inhibited by the percentages indicated. The
T1229 truncation had less impact on cell-to-cell fusion reducing the average size of
syncytia by 22%. In contrast, the T1238 mutant produced on the average 43% larger
syncytia than that of the wild-type SARS-CoV S.
For the BCoV Spike, transiently expression of codon optimized S is only able to
produce moderate amounts of fusion. Unlike the SARS-CoV, the receptor for BCoV has
yet to be elucidated. There is no significant difference in cell-to-cell fusion between S
proteins of the respiratory and enteric strains of the virus (Fig 4.7). Two different cell
lines, VERO and VERO-E6 were used since the BCoV receptor is not available to
augment fusion in order to gain a consensus of fusion activity. Levels of fusion were
reduced greater for VERO-E6 cells when compared to VERO cells. The T1359
truncation, which is closest to the carboxyl end of the protein and had the smallest impact
on cell-to-cell fusion, reduced the size of the syncytium by 10% and 3% for VERO-E6
and VERO cells respectively. The T1346 and T1333 truncations had similar effects on
the level fusion observed with reductions in fusion by 15% and 13% for VERO-E6 cells
and 9% and 9% for VERO cells respectively. The truncation that had the most impact on
cell-cell fusion was that T1355, which reduced syncytium formation by 17% and 9% for
VERO-E6 and VERO cells respectively.
DISCUSSION
Comparison Between the SARS-CoV and BCoV Spike Proteins
As stated above, there is significantly less homology in the S1 portion between
different strains of coronaviruses when compared to that of the S2 proteins. A possible
explanation for this lack of similarity between the two viruses in this region is that the S1
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subunit of the S protein contains a domain which is the determinant of viral tropism. The
SARS-CoV S protein that ACE2 is able to bind to a 193 amino acid fragment (amino
acids 318-510 of the SARS-CoV protein) in the S1 portion of the S protein which
indicates that this region is potentially the receptor binding domain primarily responsible
for virus binding to the target cell (Babcock et al., 2004; Chakraborti et al., 2005; Wong
et al., 2004; Xiao et al., 2003). The receptor binding domain for BCoV is currently
unknown. However for the MHV S protein, a protein highly homologous to the BCoV S,
the receptor binding domain has been localized to the amino terminal 330 amino acids of
S1 (Kubo, Yamada, and Taguchi, 1994; Suzuki and Taguchi, 1996; Taguchi et al.,
1995). It is probably because of virus receptor specificity that these regions are not very
well conserved.
The SARS-CoV S2 ectodomain was found to have a high degree of homology
(38% homologous) when compared that of the BCoV S2 ectodomain. This region is
probably more highly conserved because of the HR domains found in this portion of the
S glycoprotein. Virus infectivity depends on the folding back mechanism, which is
dependent on the two HR regions, in order to facilitate virus entry. Also, this portion of
the protein maybe limited structurally in how many amino acid changes, additions, and
deletions it can accommodate and still function efficiently to initiate the virus-to-cell
fusion event. The differences seen in the amount of fusion generated by these viruses
may be linked to structural differences found between the two proteins. Activation
leading to membrane fusion may occur more efficiently with the SARS-CoV as
compared with BCoV. Conversely, the fusion caused by the BCoV S may in fact be
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more regulated and controlled as opposed to the fusion event involving the SARS-CoV S
protein which may be somewhat deregulated.
For the endodomains of the two S proteins, there were two important domains that
were found in both proteins. These two domains were the charged rich region and the
cysteine rich domain. For the charged rich region, there was not a high degree of
homology in that same specific amino acids were present but rather the homology was
achieved through the presence of charged residues found in high concentrations in the
same areas. The charged-rich region of the murine hepatitis virus (MHV) S endodomain,
which has high sequence homology to the BCoV S glycoprotein, has been found to play a
major role in determining the ability of the protein to be assembled into virions (Ye,
Montalto-Morrison, and Masters, 2004). There are high concentrations of cysteine
residues found in both proteins’ endodomains. These cysteine residues are often
conserved throughout a majority of the coronavirus S glycoproteins. For MHV and the
SARS-CoV, studies have shown the cysteine rich domain if required for coronavirus-
induced membrane fusion (Bos et al., 1995; Chang, Sheng, and Gombold, 2000; Godeke
et al., 2000; Petit et al., 2005; Ye, Montalto-Morrison, and Masters, 2004) (Fig 4.4).
Furthermore, substituting the wild-type cytoplasmic portion of the cysteine rich region
and cytoplasmic tail with the cytoplasmic tail of the VSV-G abolished MHV S
glycoprotein mediated cell-cell fusion (Bos et al., 1995).
S-mediated Cell-to-Cell Fusion
Several studies have shown that the intracytoplasmic endodomain of the class I
fusion proteins may play an more important role in intracellular transport as well as virus-
induced cell-to-cell fusion (Bagai and Lamb, 1996; Bos et al., 1995; Chang, Sheng, and
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Gombold, 2000; Lontok, Corse, and Machamer, 2004; Petit et al., 2005; Schwegmann-
Wessels et al., 2004; Sergel and Morrison, 1995; Tong et al., 2002; Waning et al., 2004;
Yao and Compans, 1995). To compare predicted functional domains in the BCoV and
SARS-CoV S glycoproteins, serial truncations were made at sites that were predicted to
contain domains that may contribute to the function and transport of the entire protein.
The ability of the BCoV truncated proteins to produce S-mediated cell-to-cell fusion was
then quantitated and compared to its SARS-CoV S counterpart.
The T1359 truncation targeted the sequence in the BCoV S protein that had been
previously found to play a role intracellular S trafficking as well as a site that is predicted
by the NetPhos 2.0 software to be phosphorylated (Blom, Gammeltoft, and Brunak, 1999;
Lontok, Corse, and Machamer, 2004). The truncation, T1359, had a slight effect on
protein mediated fusion with a reduction from the wild-type fusion activity of 10% on
VERO-E6 cells. Another truncation introduced into the BCoV S protein was designed to
bring the charged region to a more proximal position to the carboxyl end of the protein.
This truncation, T1355, showed the biggest reduction of fusion out of all the truncation
tested for BCoV S (83% of the wild-type fusion for VERO-E6 cells).The corresponding
SARS-CoV S truncation, T1247, had a very significant impact on the proteins ability to
cause fusion. It was speculated that the T1247 truncation exposed an acidic cluster that
in turn greatly enhanced endocytosis. This enhancement of endocytosis led to a reduction
of fusion by 66%. The charged cluster in the BCoV S is less concentrated than that of the
SARS-CoV which seems to support the idea that it is exactly this concentration of
charged residues in the SARS-CoV S protein that is responsible for the substantial
aberration in fusion levels. The T1346 truncation for BCoV was designed to truncate as
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much of the charged region as possible without disturbing the cysteine rich region. The
SARS-CoV counterpart truncation targeted the charged region of SARS-CoV S
glycoprotein. The T1238 truncation of the SARS-CoV actually increase the size of
syncytia formation by 40% while the T1346 truncation made on the BCoV S protein
caused a reduction in fusion by 15% for VERO-E6 cells. This increase in fusion may be
attributed to destabilization of the endodomain by the truncation made. Stabilization of
the carboxyl terminus has been shown to decrease the fusion activity of the vesicular
stomatitis virus G glycoprotein (Waning et al., 2004), therefore, conversely, it is possible
that destabilization of the carboxyl terminus may cause an increase in fusion.
The final truncation made in the BCoV and SARS-CoV cleaved off most of the
protein’s endodomain. For the SARS-CoV, the 1214T truncation was found to not be
efficiently expressed on the surface of the cell. This inability of the protein to be
expressed on the cell surface may account for its severe reduction (15% when compared
to the wild-type) in S mediated fusion. The corresponding BCoV truncation, T1333,
reduced the size of syncytia formation by 13% in VERO-E6. This discrepancy in fusion
levels possibly may only be due to the disruption in glycoprotein transport as it is the case
with the corresponding mutations of the SARS-CoV S protein.
No differences in fusion levels were observed between the RBCoV and EBCoV S
proteins. It should be noted that between the two strains of BCoV there are seven amino
acid differences when comparing RBCoV and EBCoV (Fig. 4.1). Four of these amino
acid changes occur in the S1 portion of the protein, two of which are in the domain that
could potentially bind to the receptor on the cell surface. This change in the potential
receptor binding domain may account for the differences in virus tropism seen between
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the two viruses (Chouljenko et al., 2001). The other three amino acid changes occur in
the S2 portion which is known to function in fusion since it contains the fusion peptide as
well as the two heptad repeat regions. These three amino acids, however, are not located
in regions known to play a role in membrane fusion. All amino acid changes in S2 occur
at locations between the two heptad repeat regions. This could potentially explain why
there are no detectable differences in level of fusion caused by the two different proteins.
A possible explanation for the differences in fusion levels may be due to the lack
of other viral proteins. In the case of SARS-CoV, the S protein seems to be the main
determinance of virus fusion activity; for the BCoV however, the hemagglutinnin-
esterase protein may play a role in virus fusion activity as well as virulence. Expressing
the S in the context of the entire virus or at least in the context of other structural proteins
may augment fusion in order to make quantification more accurate. Another potential
cause of the differences noted between the BCoV and SARS-CoV S truncations may be
due to the fundamental differences in each of the wild-type protein’s ability to cause cell-
to-cell fusion. The SARS-CoV S, when overlayed with cells expressing the ACE2
receptor, is able to induce very large syncytia formation; however, the BCoV S is not
very fusogenic. Since the BCoV receptor remains unknown, receptor mediated
enhancement of syncytia formation in is not possible. Different cell lines were used in
this study in order to establish a consensus about the levels of fusion caused by each
truncation. VERO-E6 cells were more prone to fusion than VERO cells which made
comparison of fusion levels more accurate than their VERO counterparts; therefore
VERO-E6 fusion levels were used when comparing the BCoV S and its counterpart
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truncation. This low level of fusion activity may hamper further studies on the
functional domains of the BCoV S glycoprotein.
MATERIALS AND METHODS
Cells
African green monkey kidney (Vero) cells were obtained from the American Type
Culture Collection (Rockville, Md.). Cells were propagated and maintained in Dulbecco
modified Eagle medium (Sigma Chemical Co., St. Louis, Mo.) containing sodium
bicarbonate and 15 mM HEPES supplemented with 10% heat-inactivated fetal bovine
serum.
Plasmids
The DNAworks program (Hoover and Lubkowski, 2002) was used to design
synthetic oligonucleotides used in a PCR-based system in order to generate a codon
optimized BCoV S glycoprotein for the respiratory strain of the virus. Since the S
proteins only differ between the respiratory and enteric strain by seven amino acids, PCR
overlap extension (Aiyar, Xiang, and Leis, 1996) was used to construct the codon
optimized enteric strain S glycoprotein. The BCoV-Lun So 3xF and BCoV-Ent So 3xF
plasmids were generated by cloning their respective codon-optimized BCoV S gene,
without the DNA sequence coding for the signal peptide, into the p3XFLAG-CMV-9
plasmid vector (Sigma). In order to construct the truncation mutants, primers were
designed that incorporated a stop codon and an Xba I restriction site at the appropriate
gene site (Fig 4.2). Restriction endonuclease sites EcoRV and XbaI were then used to
clone the gene construct into the BCoV-Lun So 3xF plasmid.
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Total Protein Immunohistochemistry
Vero cell monolayers in six-well plates were transfected with the indicated plasmids
utilizing the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s
directions. At 48 h post transfection, the cells were washed with TBS-Ca/Mg and either
fixed with iced cold methanol. Immunohistochemistry was performed by utilizing the
Vector Laboratories Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA)
essentially as described in the manufacturer’s directions. Briefly, cells were washed with
TBS-Ca/Mg and incubated in TBS supplemented with 5% normal horse serum and 5%
bovine serum albumin at room temperature for 1 h. After blocking, cells were reacted
with anti-FLAG antibody (1:500) in TBS blocking buffer for 3 h, washed four times with
TBS-Ca/Mg, and incubated with biotinylated horse anti-mouse antibody. Excess antibody
was removed by four washes with TBS-Ca/Mg and subsequently incubated with
Vectastain Elite ABC reagent for 30 min. Finally, cells were washed three times with
TBS-Ca/Mg, and reactions were developed with NovaRed substrate (Vector
Laboratories) according to the manufacturer’s directions.
Quantitation of the Extent of S-mediated Cell Fusion
Vero cell monolayers in six-well plates were transfected in triplicate with the
indicated plasmids utilizing the Lipofectamine 2000 reagent (Invitrogen) according to the
manufacturer’s directions. Concurrently, Vero cell monolayers in six-well plates were
transfected with the plasmid encoding the ACE2 receptor protein utilizing the
Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s directions. At
24 h post transfection, cells containing the mutant plasmids, the ACE2 receptor, and
normal untransfected cells were washed with TBS-Ca/Mg, trypsinized, and overlayed in
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a single well of a six-well plate at a ratio of 2 ml (cells transfected with the ACE2
receptor) : 0.5 ml (cells transfected with the mutant) : 1.5 ml (untransfected cells). All of
the cells transfected with ACE2 were pooled to ensure that every well had an equal
amount of cells with receptor expressed on their surface. After incubation for 24 h, the
cells were washed with TBS-Ca/Mg and fixed with ice cold methanol.
Immunohistochemistry was performed by utilizing the Vector Laboratories Vectastain
Elite ABC kit essentially as described in the manufacturer’s directions. Briefly, cells were
washed with TBS-Ca/Mg and incubated in TBS blocking buffer supplemented with
normal horse serum at room temperature for 1 h. After blocking, cells were reacted with
anti-FLAG antibody (1:500) in TBS blocking buffer for 3 h, washed four times with
TBS-Ca/Mg, and incubated with biotinylated horse anti-mouse antibody. Excess antibody
was removed by four washes with TBS-Ca/Mg and subsequently incubated with
Vectastain Elite ABC reagent for 30 min. Finally, cells were washed three times with
TBS-Ca/Mg, and reactions were developed with NovaRed substrate (Vector
Laboratories) according to the manufacturer’s directions. The average size of syncytia for
each mutant was determined by analyzing the area of approximately 300 syncytia, from
digital images, using the Image Pro Plus 5.0 software package. The averages were then
converted to percentages of the average syncytia size of the wild type SARS-CoV S.
Error bars shown represent the standard deviations calculated through comparison of the
data from each of three experiments.
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CHAPTER V
CONCLUDING REMARKS
SUMMARY
The SARS-CoV is a recently discovered coronavirus that is able to produce very
high mortality rates in subsets of the population (Anand et al., 2003). A key target for
analysis of how the SARS-CoV was able to achieve such a high mortality rate is the viral
spike (S) glycoprotein. Unfortunately, the ability to transiently express the S protein had
been hindered by the proteins massive size of 1255 amino acids (Marra et al., 2003)
along with the predicted high degree of secondary structure of the RNA encoding the
protein.
Recently, however, the protein has been codon optimized in order to allow the
expression of the S protein using a transient system (Li et al., 2003). This process of
codon optimization, in which not only are all codons replaced with the specific codon
most commonly used to encode the amino acid but the secondary structure of the RNA is
significantly reduced, has allowed the efficient in vitro expression of the protein. The
work included in this dissertation has focused on the genetic alteration and manipulation
of the optimized SARS-CoV S protein in order to elucidate the role of several putative
functional and conserved domains in SARS-CoV S mediated cell-cell fusion as well as
intracellular transport. In addition, we generated an optimized clone of the bovine
coronavirus (BCoV) S glycoprotein in order to compare similar mutations made in both
proteins.
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These investigations have shown that there are several regions that are
indispensable for the proper functioning and transport of this protein. The amino
terminal heptad repeat as well as regions adjacent to the heptad repeat are important
structural domains for the correct functioning of the protein in S-mediated cell-cell
fusion. In order to investigate these regions, mutations designed to collapse the alpha
helical structure of these domains were introduced into the protein. These mutations
produced a severe impact on S-mediated cell-cell fusion but allowed for the appropriate
synthesis and transport found in the wild-type protein.
The role of the SARS-CoV endodomain was investigated by the construction of a
series of carboxyl terminal truncations as well as cluster to alanine mutations targeting
specific conserved motifs in the endodomain of the protein. The endodomain of the
protein is able to affect the function of the proteins commonly thought to be associated
with the ectodomain (Bagai and Lamb, 1996; Bos et al., 1995; Chang, Sheng, and
Gombold, 2000; Lontok, Corse, and Machamer, 2004; Schwegmann-Wessels et al., 2004;
Seth, Vincent, and Compans, 2003; Tong et al., 2002; Waning et al., 2004; Yao and
Compans, 1995). Endo-domain truncations of the SARS-CoV S protein specifically
targeted the acidic cluster and the cysteine rich clusters found within the SARS-CoV S
endodomain. The SARS-CoV S protein is divergent from other coronavirus S proteins in
that the acidic cluster is a separate domain from the cysteine rich domain as opposed to
the overlapping of these domains as seen in other coronaviruses such as mouse hepatitis
virus (MHV). Cluster to alanine mutations were introduced targeting the acidic cluster,
the cysteine rich domains, and a predicted phosphorylation site in the endodomain.
Mutations affecting regions of the endodomain led to a wide array of phenotypes. These
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phenotypes include a decrease in S-mediated cell-cell fusion, an increase in S-mediated
cell-cell fusion, altered endocytosis patterns, altered protein recycling, and altered
intracellular protein transport. This wide variety of results demonstrate how incredibly
important the endodomain of type I membrane fusion proteins are in the regulation of
protein function and transport.
The bovine coronavirus (BCoV) is able to produce a disease state similar to that
of the SARS-CoV in cattle (Storz et al., 2000a; Storz et al., 2000b). Given this
resemblance to SARS, a comparison study between the two S glycoproteins was
performed to better understand any homologies shared between the two proteins. This
was accomplished by constructing a panel of BCoV S truncations and comparing their
fusion activity to the activity of a panel of SARS-CoV S truncations. The absence of
correlation in impact of protein function between the truncations may point to domains
that are unique to the SARS-CoV S. However, there were correlations between some
SARS-CoV S truncations with similar truncations of the MHV S glycoprotein (Chang,
Sheng, and Gombold, 2000). Further studies of the BCoV S glycoprotein are needed in
order to make any supported claims about the functional domains of the BCoV S
endodomain.
In conclusion, this dissertation work has capitalized on the recent development of
the codon optimization of the SARS-CoV and BCoV S glycoproteins in order to
introduce targeted mutations and truncations to both of these proteins. Currently, the
work produced herein has contributed to the understanding of potent motifs in the S
endodomain in their functioning in S-mediated cell-cell fusion as well as intracellular
transport.
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CURRENT AND FUTURE RESEARCH CHALLENGES
The high mortality rate for patients infected with the SARS-CoV and the probable
link between this high mortality with the S glycoprotein make the study of the protein
tremendously important to the field of viral pathogenesis. The SARS-CoV is the most
recently identified human coronavirus whose possible impact on worldwide public health
has led to an intense research effort in the field. The elucidation of functional domains
found in the protein may ultimately lead to new drug development as well as new
potential vaccine development for the disease.
These investigations clearly show that the mutations introduced into the predicted
functional domains of the protein had a significant impact on protein transport and
function. One aspect of particular interest would be these mutants impact on viral
assembly. Virus like particles have been produced for other coronaviruses using transient
systems (Corse and Machamer, 2003; de Haan et al., 1998; Huang et al., 2004; Vennema
et al., 1996). If a virion like particle assembly system could be established, each of the
mutants and truncations could be assayed for their ability to be incorporated into the
virion particles using immunoelectron microscopy. Through the elucidation of the
SARS-CoV packaging signal, which is already known for several coronaviruses
(Cologna and Hogue, 2000; Escors et al., 2003; Fosmire, Hwang, and Makino, 1992;
Izeta et al., 1999; Woo et al., 1997), a potential virus packaging system could also be
established. Once incorporated into the virus like particles, the mutants and truncations
could also be assayed for their ability of mediating virus entry by using a reporter dye
199
enclosed by the particle or potentially by packaging a reporter gene within the virus like
particle. Another use of the particle packaging system could be in vaccine development
and production.
The construction of an infectious cDNA clone (Almazan et al., 2000; Gonzalez et
al., 2002; Yount, Curtis, and Baric, 2000; Yount et al., 2003) may provide a tool to
observe the constructed mutants and truncations constructed in the research presented in
this dissertation ability to function in the context of the entire virus. Specific
measurements concerning viral entry kinetics, viral replication, virus pathology, and viral
egress could be ascertained for the entire panel of mutants constructed. However it
should be noted that the incorporation of mutations in the cDNA clone could potentially
be problematic because of the enormous size of the infectious clone. A potential solution
for this problem would be to create an infectious cDNA clone without the S glycoprotein
where the S glycoprotein could be provided in trans. This would allow the S mutants to
be made on a separate more manageable plasmid. This S-null infectious clone would
also be substantially more safe and easier to handle than a fully infectious clone. An
alternate possibility would be to construct an infectious clone using a bacterial artificial
chromosome (BAC) system in which mutations could be introduced into the BAC using a
Sac B/Rec A mutagenesis system . The introduction of the mutations in the context of
the virus could potentially lead to very significant finding which would significantly
progress the area of coronavirus research.
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Loan, R. W. (2000b). Isolation of respiratory bovine coronavirus, other cytocidal viruses, and Pasteurella spp from cattle involved in two natural outbreaks of shipping fever. J Am Vet Med Assoc 216(10), 1599-604.
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Vennema, H., Godeke, G. J., Rossen, J. W., Voorhout, W. F., Horzinek, M. C., Opstelten, D. J., and Rottier, P. J. (1996). Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes. Embo J 15(8), 2020-8.
Waning, D. L., Russell, C. J., Jardetzky, T. S., and Lamb, R. A. (2004). Activation of a
paramyxovirus fusion protein is modulated by inside-out signaling from the cytoplasmic tail. Proc Natl Acad Sci U S A 101(25), 9217-22.
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APPENDIX A
ADDITIONAL WORK
VIRUS LIKE PARTICLE AND PACKAGING SYSTEM
The etiological agent for the recently recognized severe acute respiratory
syndrome (SARS) was discovered to be the SARS coronavirus (SARS-CoV) (Drosten et
al., 2003; Ksiazek et al., 2003). An enveloped positive stranded virus, this coronavirus
displays a host range divergent from other members of the coronavirus family. This
divergence from typical coronavirus may play a role in its dramatically increased
pathogenicity. Most coronaviruses can replicate in either respiratory and/or
gastrointestinal tract (Holmes, 2001; Li et al., 2003), however the SARS-CoV has
evolved the ability to replicate in both places during the viral infection.
Another coronavirus of particular interest is the bovine coronavirus (BCoV).
BCoV has a significant impact on the cattle industry resulting in serious economic losses.
Infection by the virus cause epidemics of acute diarrhea in calves and adult cattle (Mebus
et al., 1973; Saif et al., 1988; Taniguti et al., 1986; Tsunemitsu et al., 1991; Weisberg,
1975) which results in reduced milk production in dairy cows and/or death (Saif et al.,
1988; Takahashi, Inaba, and Sato, 1980; Taniguti et al., 1986).
The typical coronavirus encodes for four structural proteins. The protein
responsible for binding and attachment is the spike (S) glycoprotein. It is the largest
encoded structural protein found in the coronavirus genome. It functions as a typical type
I membrane glycoprotein. The most abundant protein contained in the virion envelope is
the matrix (M) glycoprotein. It is a triple spanning membrane protein that has a long
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carboxyl terminal cytoplasmic domain inside the virion envelope and a short amino
terminal domain located outside of the virion (Holmes, 2001; Locker et al., 1992;
Narayanan et al., 2000). The other protein that comprises the virion membrane is the
envelope (E) protein. This protein is expressed at significantly lower levels than the that
of the M protein (Godet et al., 1992; Liu and Inglis, 1991; Siddell, 1995b; Yu et al.,
1994). The final structural protein found in the typical coronavirus is the nucleocapsid
(N) protein. The N protein is an internal phosphoprotein that interacts with the genomic
RNA to form the virion core. BCoV, as with the SARS-CoV, encoded for these four
structural proteins that are typically found in coronaviruses. Unlike the SARS-CoV
however, the BCoV also has a hemmagglutinin-esterase protein encoded into its genome
that is only found in a certain subset of coronaviruses.
It has been shown that the M and E proteins are instrumental in virion assembly.
In fact, studies have revealed that expression of the M and E proteins are enough to
produce noninfectious membrane bound particles of similar size and shape to the normal
wild-type virion (Corse and Machamer, 2003; de Haan et al., 1998; Huang et al., 2004;
Vennema et al., 1996). Expressing S, along with M and E, produced VLPs that were not
only similar in size and shape but were also infectious. This indicates that the S protein is
not required for virion assembly but is required for infectivity. Similar roles of structural
proteins have also been elucidated for other coronaviruses including the infectious
bronchitis virus (IBV) (Corse and Machamer, 2000). For the SARS-CoV, the expression
of M and E does not lead to particle formation; however the expression of M and N is
able to produce VLPs (Huang et al., 2004).
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In this study, attempts were made to create virus like particles (VLPs) for BCoV
and to repeat the study in which VLPs for the SARS-CoV were synthesized. These
particles could then be used to create potential vaccines for the viruses that the structural
proteins were derived from. The definitive goal of this series of experiments was to
produce BCoV or SARS-CoV particles in which a foreign gene could be packaged.
Particularly for BCoV, particles created in this packaging system could be loaded with a
gene that encodes for a cytolytic protein or a protein that is able to elicit a targeted
immune response. Since BCoV has a predilection for rectal cells, a BCoV virus like
particle packaged with a gene encoding a cytolytic protein could be used as treatment
against rectal (colon) cancer.
For the BCoV, the M, E and S proteins were cloned into an expression vector
under the cytomegalovirus promoter. Once cloned into the vector, the genes were then
transfected into cells in order to check for the appropriate protein synthesis. The M and E
proteins were able to be efficiently expressed; however, the S glycoprotein was not able
to be expressed by transient expression. Several different methods were attempted in
order to correct the problem of the S protein not able to be expressed. Among the
methods employed were cloning the S protein into T7 vaccinia driven expression system,
cloning the vector into a variety of different vectors under different promoters, and
separately cloning the S1 and S2 subunits of the S protein into two different vectors and
transfecting the two subunits into the same cell. None of these methods were able to
produce transiently expressed S protein without killing the cells. It is probable that the S
protein was so large that the RNA encoding the protein produced from the transfected
plasmid was unable to get out of the nucleus. Since coronaviruses are purely
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cytoplasmic, no mechanism evolved that would allow such a large RNA to be transported
out of the nucleus. Another problem is that the RNA encoding S protein has an
extremely high degree of 2° structure which would further inhibit the RNA from exiting
the nucleus.
Recently, the SARS-CoV S protein was able to be transiently expressed through
the technique of codon optimization (ACE2). Codon optimization takes advantage of the
degenerate code used to encode amino acids. Certain codons are more prevalent for
encoding the same amino acid than other degenerate codons. Codon optimization is the
process of reconstructing the nucleotide sequence in such a way that all of the codons
used in the optimized gene are the most prevalent used codons. The optimization
process, usually done with the assistance of computing software, can also produce a
sequence that not only uses all of the optimal codons but abolishes most of the 2°
structure associated with the RNA. In order to be able to express the BCoV S protein, a
condon optimized version of the gene was synthesized which allowed transient
expression of the protein.
Once all of the proteins were able to be efficiently expressed, different
combinations of the structural proteins were transfected into a variety of cell lines. The
essential combinations transfected for the SARS-CoV were M and N and M, N, and S,
while for BCoV the most important combinations were M and E and M, E, and S. Once
the plasmids were cotransfected, the cells were allowed to incubate for 48 hours before
processing them for visualization using electron microscopy. VLPs were not able to be
visualized using this method.
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A possible reason that no VLPs were able to be visualized could be that the only
codon optimized proteins used in the experiment was the S glycoprotein. In the previous
study in which SARS-CoV proteins cotransfected were able to produce VLPS, all of the
structural proteins used were codon optimized. This codon optimization would lead to
much higher expression of protein from all of the structural proteins which would also
lead to higher levels of VLP production. Our combination of plasmids may not have
been able to produce enough levels of protein in order to be able to visualize VLP
formation. Codon optimization of the BCoV M and E and the SARS-CoV N and M may
be able to increase expression levels so that enough protein is produced to be able to form
coronavirus like particles. Another possible explanation could be the difficulty in
visualizing the particles themselves. Coronavirus particles are approximately the same
size and shape as a wide variety of cellular structures. This in combination with low
protein expression or inefficient transfection levels may make it very difficult to visualize
coronavirus like particles using electron microscopy. Different transfection reagents in
combination with the use of cells more susceptible to transfection may be able to alleviate
this particular problem.
BOVINE CORONAVIRUS INFECTIOUS CLONE
The development of full-length cDNA clones has remarkably advanced the ability
to perform molecular genetic analysis on the structure and function of RNA viruses. This
cDNA clone, once transfected into a permissive cell line, becomes the source of
infectious RNA transcripts (Ahlquist et al., 1984; Boyer and Haenni, 1994). These
infectious clones have been constructed for several viruses including caliciviruses,
arteriviruses, flaviviruses, picronavirures, and alphaviruses whose positive sensed RNA
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genomes that range in size from approximately 7-15 kb in length (Agapov et al., 1998;
Davis et al., 1989; Racaniello and Baltimore, 1981; Rice et al., 1989; Rice et al., 1987;
Sosnovtsev and Green, 1995; Sumiyoshi, Hoke, and Trent, 1992; van Dinten et al., 1997).
These clones have made new methods and approaches in studying RNA viruses available
that have not been previously available before.
The family Coronaviridae has the largest RNA viral genomes found in nature
(Lai and Cavanagh, 1997; Siddell, 1995a). It is this enormous length of the coronavirus
genome and stabilization issues with plasmids encoding coronavirus replicase sequences
have made the construction of a full-length cDNA clone very cumbersome (Ahlquist et
al., 1984; Eleouet et al., 1995; Enjuanes and Van der Zaijst, 1995; Lai and Cavanagh,
1997; Siddell, 1995a). These problems were overcome when a full-length cDNA clone
of the transmissible gastroenteritis virus (TGEV) was constructed and stably cloned into a
bacterial artificial chromosome (BAC) vector (Almazan et al., 2000).
In order to be able to take advantage of molecular genetic analysis, a full-length
cDNA clone of the bovine coronavirus (BCoV) was attempted to be constructed using
recombinant technology. Specifically, a cDNA clone could have a dramatic impact on
studies involving the comparison of two strains of BCoV that were found to have
different tropisms. This comparison study was between a respiratory strain of BCoV and
an enteric strain of BCoV that were isolated from the same animal with fatal shipping
pneumonia. It has been suggested that the difference in viral tropism is due to slight
variation in the S1 subunit of the spike (S) glycoprotein (Chouljenko et al., 1998; Gelinas
et al., 2001; Hasoksuz et al., 2002; Rekik and Dea, 1994). There are only 4 amino acids
in the S1 subunit of the S protein that are different between the respiratory and enteric
209
strains of BCoV. It is known that the main determinance of tissue tropism is attributed to
the S1 portion of the S protein. It is possible that a single amino acid change in the S1
subunit may shift the BCoV tropism from the respiratory tract to the enteric tract. Once
constructed, mutagenesis of the infectious clone could isolate with amino acid(s) that
plays a role in this difference in tropism.
A full-length infectious clone was constructed for both the respiratory and enteric
strains of BCoV. This infectious clone was then transfected into a variety of cells
including HRT-18G, VERO, BHK, and COS-7. These cells were tested for their ability
to produce infectious virus after being transfected with the cDNA clone using
immunohistochemistry, indirect immunofluorensence, and observing the cells for the
cytopathic effect of the virus. Once transfected, several rounds of passaging the potential
virus were made in order to increase virus production to detectable levels. Also in order
to prevent false positive results, two silent mutations were introduced into the viral
genomes so that they could be distinguished from the wild-type virus. All attempts to
transfect the cDNA clone and produce infectious virus turned up negative by
immunohistochemistry, indirect immunofluorensence, and signs of cytopathic effect
created by the virus. One method employed to try and augment the efficiency of virus
production was the use of a T7 in vitro transcription system. First, several sequences in
the cDNA clone were mutated because they were know to interfere with T7 transcription.
The mutations placed in these areas were silent mutations so that the same amino acids
would be used in translation of the cDNA clone. Once the infectious clone was free of
possible interfering sequences, the T7 system was used to transcribe that cDNA into
RNA that could then be directly transfected into the cell lines. This was thought to make
210
virus production more efficient since the RNA has already been transcribed. All attempts
at isolating infectious virus were unsuccessful.
One potential problem in producing infectious virus from both the cDNA and
RNA transfection system could have been the cell lines used. The most commonly used
cell line for BCoV propagation is HRT-18G (Storz et al., 1996). This cell line, while
being the best at virus production has very poor transfection efficiency. HRT-18G cells
are difficult to transfect under optimal conditions much less when dealing with such a
large construct. The enormous size of the genetic material in conjunction with the given
inability of the cells to be efficiently transfected, make infectious virus recovery virtually
impossible. Other cells lines that are able to be efficiently transfected are not very
hospitable for virus production. Another problem may be the lack of actual transcription
or translation of the genetic construct. Such an enormous RNA would have a very high
degree of 2° structure which may prevent translation or transcription. Also, the cDNA
copy of the virus is unlikely to be efficiently transcribed or translated. During viral
replication, viral proteins may play a role in enhancing transcription and/or translation.
Without the aid of these proteins, expression of the viral genome may not be efficient
enough to produce an amount of virus that could be isolated.
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APPENDIX B
PERMISSION TO INCLUDE PUBLISHED WORK
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218
VITA
Chad Michael Petit was born on March 26, 1978, to Kevin and Gwyneth Petit in
Metairie, Louisiana. Growing up, Chad had interests in art and music. He fulfilled these
interests by learning how to play the guitar, trumpet, and baritone and also by being in the
gifted and talented art program in high school. In 1996, Chad graduated 11th in his class
of 300 from Hahnville High School. Chad was then awarded a chancellor’s aid
scholarship to attend Louisiana State University and pursued a major in biochemistry.
While attending LSU, Chad served as the treasurer for the Student Affiliation of the
American Cancer Society, a member of the student election board, and was a member of
the Louisiana State University Tiger Marching Band. Upon graduation, Chad was
awarded three Bachelor of Science degrees in the fields of biochemistry, chemistry, and
microbiology by earning 190 total credit hours. Always having an interest in viruses,
Chad joined the laboratory of Dr. K. G. Kousoulas to pursue a doctoral degree in the area
of research of coronaviruses.