influenza pathway

Report for Pathway "Influenza Infection"

Luo, F, Squires, B, Scheuermann, RH, Gillespie, ME, Bortz, E, Garcia-Sastre, A, Steel, J, Marsh, G.

1 Influenza Infection

Authors

Luo, F, Squires, B, Scheuermann, RH, 2006-01-05 15:13:12.

Editors

D'Eustachio, P, Gillespie, ME, 2006-01-07 21:50:17.

Reviewers

Garcia-Sastre, A, Squires, B, 2006-10-30 21:55:36.

Description

For centuries influenza epidemics have plagued man, and influenza was probably the disease described by Hippocrates in 412 BC. Today itremains a major cause of morbidity and mortality worldwide with large segments of the human population affected every year. Many animalspecies can be infected by influenza viruses, often with catastrophic consequences. A continuing threat is the possibility of a pandemic similar tothat experienced in 1918, estimated to have been responsible for 50 million deaths worldwide.

Influenza viruses belong to the family of Orthomyxoviridae; viruses with segmented RNA genomes that are negative sense and single-stranded(Baltimore 1971).

Influenza virus strains are named according to their type (A, B, or C), the species from which the virus was isolated (omitted if human), locationof isolate, the number of the isolate, the year of isolation, and in the case of influenza A viruses, the hemagglutinin (H) and neuraminidase (N)subtype. For example, the virus of H5N1 subtype isolated from chickens in Hong Kong in 1997 is: influenza A/chicken/Hong Kong/220/97(H5N1)virus. Currently 16 different hemagglutinin (H1 to H16) subtypes and 9 different neuraminidase (N1 to N9) subtypes are known for influenza Aviruses. Most human disease is due to Influenza viruses of the A type, so the events of Influenza infection have been annotated in Reactomewith reference to this type.

References

P Palese, ML Shaw, "Orthomyxoviridae: The Viruses and Their Replication", Fields Virology, 5th edition D.M. Knipe and P.M. Howley, Editors.2006, Lippencott Williams and Wilkins: Philadelphia ISBN-10: 0-7817-6060-7, 2001, 1647-1689.

RM Krug, RA Lamb, "Orthomyxoviridae: The Viruses and Their Replication", Fields Virology. 4th edition, editors: Knipe DM, Howley PM,.Philadelphia: Lippincott Williams & Wilkins. ISBN: 0-7817-1832-5, 2001.

1.1 Influenza Life Cycle

Reviewers

Garcia-Sastre, A, 2004-05-12 19:00:00, Garcia-Sastre, A, Squires, B, 2006-10-30 21:55:36, Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

The virus particle initially associates with a human host cell by binding to sialic acid-containing receptors on the host cell surface. The boundvirus is endocytosed by one of four distinct mechanisms. The low endosomal pH sets in motion a number of steps that lead to viral membranefusion mediated by the viral hemagglutinin (HA) protein, and the eventual release of the uncoated viral ribonucleoprotein complex into thecytosol of the host cell. The ribonucleoprotein complex is transported through the nuclear pore into the nucleus. Once in the nucleus, theincoming negative-sense viral RNA (vRNA) is transcribed into messenger RNA (mRNA) by a primer-dependent mechanism. Replication occursvia a two step process. A full-length complementary RNA (cRNA), a positive-sense copy of the vRNA, is first made and this in turn is used as atemplate to produce more vRNA. The viral proteins are expressed and processed and eventually assemble with vRNAs at budding sites withinthe host cell membrane. The viral protein complexes and ribonucleoproteins are assembled into viral particles and bud from the host cell,enveloped in the host cell's membrane.

This release contains a framework for the further annotation of the viral life-cycle.

References



1.1.1 Binding of the influenza virion to the host cell

Description

Influenza viruses bind via their surface HA (hemagglutinin) to sialic acid in alpha 2,3 or alpha 2,6 linkage with galactose on the host cell surface.Sialic acid in 2,6 linkages is characteristic of human cells while 2,3 linkages are characteristic of avian cells. The specificity of influenza HA forsialic acid in alpha 2,6 or alpha 2,3 linkages is a feature restricting the transfer of influenza viruses between avian species and humans. Thisspecies barrier can be overcome, however. Notably, passaged viruses adapt to their host through mutation in the receptor binding site of theviral HA gene.

References

L Mochalova, A Gambaryan, J Romanova, A Tuzikov, A Chinarev, D Katinger, H Katinger, A Egorov, N Bovin, "Receptor-binding properties ofmodern human influenza viruses primarily isolated in Vero and MDCK cells and chicken embryonated eggs", Virology, 313, 2003, 473-80.


RJ Connor, Y Kawaoka, RG Webster, JC Paulson, "Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates",Virology, 205, 1994, 17-23.

AS Gambaryan, JS Robertson, MN Matrosovich, "Effects of egg-adaptation on the receptor-binding properties of human influenza A and Bviruses", Virology, 258, 1999, 232-9.

Reaction

1.1.2 Entry of Influenza Virion into Host Cell via Endocytosis

Description

Virus particles bound to the cell surface can be internalized by four mechanisms. Most internalization appears to be mediated by clathrin-coatedpits, but internalization via caveolae, macropinocytosis, and by non-clathrin, non-caveolae pathways has also been described for influenzaviruses.

References


1.1.2.1 Clathrin-Mediated Pit Formation And Endocytosis Of The Influenza Virion

Description

Virus particles bound to the cell surface can be internalized by four mechanisms. Most internalization appears to be mediated by clathrin-coatedpits.

References


KS Matlin, H Reggio, A Helenius, K Simons, "Infectious entry pathway of influenza virus in a canine kidney cell line", J Cell Biol, 91, 1981,601-13.

Reaction

1.1.3 Fusion and Uncoating of the Influenza Virion

Description

Uncoating of viral particles takes place in the host cell endosome. Acidification of the endosome promotes fusion of the viral and endosomalmembranes, causing a structural change in the viral hemagglutinin (HA) and freeing the fusion peptide of its HA2 subunit to interact with theendosome membrane. The concerted structural change of several HA molecules opens up a pore through which the viral RNP passes into thecytosol of the cell. The precise timing and the location of uncoating (early vs. late endosomes) depends on the pH-mediated transition of thespecific HA molecule involved. The virus-associated M2 ion channel protein allows the influx of H+ ions into the virion, which disruptsprotein-protein interactions, resulting in the release of RNP free of the viral M1 matrix protein. Thus the HA mediated fusion of the viralmembrane with the endosomal membrane and the M2-mediated release of the RNP results in the release of the RNP complex into the cytosol.Amantadine and rimantadine have been shown to block the ion channel activity of the M2 protein and thus interfere with uncoating.

References

JJ Skehel, AJ Hay, JA Armstrong, "On the mechanism of inhibition of influenza virus replication by amantadine hydrochloride", J Gen Virol, 38,1978, 97-110.


T Stegmann, HW Morselt, J Scholma, J Wilschut, "Fusion of influenza virus in an intracellular acidic compartment measured by fluorescencedequenching", Biochim Biophys Acta, 904, 1987, 165-70.

M Marsh, A Helenius, "Virus entry into animal cells", Adv Virus Res, 36, 1989, 107-51.

IV Chizhmakov, FM Geraghty, DC Ogden, A Hayhurst, M Antoniou, AJ Hay, "Selective proton permeability and pH regulation of the influenzavirus M2 channel expressed in mouse erythroleukaemia cells", J Physiol, 494, 1996, 329-36.

1.1.3.1 Fusion of the Influenza Virion to the Host Cell Endosome

Description

After the virus binds to the target cell surface and is endocytosed, the low pH of the endosome causes the viral HA (hemagglutinin) to undergo astructural change which frees the fusion peptide of its HA2 subunit allowing it to interact with the endosome membrane. The transmembranedomain of the HA2 (inserted into the viral membrane) and the fusion peptide (inserted into the endosomal membrane) are in juxtaposition in theacidic pH structure of HA. The concerted structural change of several hemagglutinin molecules then opens a pore through which the viral RNPwill be able to pass into the host cell cytosol.

References



SB Sieczkarski, GR Whittaker, "Viral entry", Curr Top Microbiol Immunol, 285, 2005, 1-23.

1.1.3.1.1 Conformation change in hemagglutinin freeing the fusion peptide of HA2

Description

The low pH of the endosome causes the viral HA (hemagglutinin) to undergo a structural change which frees the fusion peptide of its HA2subunit.

References


T Stegmann, "Membrane fusion mechanisms: the influenza hemagglutinin paradigm and its implications for intracellular fusion", Traffic, 1, 2000,598-604.

Reaction

1.1.3.1.2 Fusion of the influenza virion HA2 protein transmembrane domain to the host cell endosome membrane

Description

The fusion peptide of its HA2 subunit interacts with the endosome membrane. The transmembrane domain of the HA2 is inserted into the viralmembrane and the fusion peptide is inserted into the endosomal membrane. In the acidic pH structure of HA the two ends of the HA complexare in juxtaposition.

References


Reaction

1.1.3.1.3 Concerted hemagglutinin pore formation

Description

The concerted structural change of several hemagglutinin molecules opens a pore through which the viral RNP will be able to pass into the hostcell cytosol.

References



Reaction

1.1.3.2 Uncoating of the Influenza Virion

Description

The precise timing and location of uncoating (early vs. late endosomes) depends on the pH-mediated transition of the specific viralhemagglutinin (HA) molecule involved. The uncoating of influenza viruses in endosomes is blocked by changes in pH caused by weak bases(e.g. ammonium chloride and chloroquine) or ionophores (e.g. monensin). Effective uncoating is also dependent on the presence of the viral M2ion channel protein. Early on it was recognized that amantadine and rimantadine inhibit replication immediately following virus infection. Later itwas found that the virus-associated M2 protein allows the influx of H+ ions into the virion, which disrupts protein-protein interactions, resulting inthe release of viral RNP free of the viral matrix (M1) protein. Amantadine and rimantadine have been shown to block the ion channel activity ofthe M2 protein and thus uncoating. The HA mediated fusion of the viral membrane with the endosomal membrane and the M2-mediated releaseof the RNP results in the appearance of free RNP complexes in the cytosol. This completes the uncoating process. The time frame for theuncoating process has been examined by inhibiting virus penetration with ammonium chloride. Typically, virus particles show a penetration halftime of about 25 minutes after viral adsorption. Ten minutes later (half time of 34 minutes after adsorption) RNP complexes are found in thenucleus. Uptake of RNP molecules through nuclear pores is an active process, involving the nucleo-cytoplasmic trafficking machinery of the hostcell.

References

K Martin, A Helenius, "Transport of incoming influenza virus nucleocapsids into the nucleus", J Virol, 65, 1991, 232-44.

C Wang, K Takeuchi, LH Pinto, RA Lamb, "Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block", J Virol,67, 1993, 5585-94.


1.1.3.2.1 Virion-associated M2 protein mediated ion infusion

Description

The uncoating of influenza viruses in endosomes is blocked by changes in pH caused by weak bases (e.g. ammonium chloride and chloroquine)or ionophores (e.g. monensin). Effective uncoating is also dependent on the presence of the viral M2 ion channel protein. Early on it wasrecognized that amantadine and rimantadine inhibit replication immediately following virus infection. Later it was found that the virus-associatedM2 protein allows the influx of H+ ions from the endosome into the virion. This disrupts protein-protein interactions, resulting in the release ofviral RNP free of the viral matrix (M1) protein. Amantadine and rimantadine have been shown to block the ion channel activity of the M2 proteinand thus uncoating.

References




Reaction

1.1.3.2.2 Ribonucleoprotein release from M1 proteins

Description

The influx of H+ ions into the virion disrupts protein-protein interactions, resulting in the release of the viral RNP from the viral matrix (M1)protein. The uncoating process is complete with the appearance of free RNP complexes in the cytosol.

References


Reaction

1.1.4 Transport of Ribonucleoproteins into the Host Nucleus

Authors

Gillespie, ME, 2005-11-14 17:18:07.

Reviewers

Squires, B, 2006-10-29 16:42:09.

Description

An unusual characteristic of the influenza virus life cycle is its dependence on the nucleus. Trafficking of the viral genome into and out of thenucleus is a tightly regulated process with all viral RNA synthesis occurring in the nucleus. The eight influenza virus genome segments neverexist as naked RNA but are associated with four viral proteins to form viral ribonucleoprotein complexes (vRNPs). The major viral protein in theRNP complex is the nucleocapsid protein (NP), which coats the RNA. The remaining proteins PB1, PB2 and PA bind to the partiallycomplementary ends of the viral RNA, creating the distinctive panhandle structure. These RNPs (10-20nm wide) are too large to passivelydiffuse into the nucleus and therefore, once released from an incoming particle must rely on the active import mechanism of the host cell nuclearpore complex. All proteins in the RNP complex can independently localize to the nucleus due to the presence of nuclear localization signals(NLSs) which mediate their interaction with the nuclear import machinery, including the RanGTPase (Fodor, 2004; Deng et al., 2006). Howeverthe signals on NP have been shown to be both sufficient and necessary for the import of viral RNA.

References

E Fodor, M Smith, "The PA subunit is required for efficient nuclear accumulation of the PB1 subunit of the influenza A virus RNA polymerasecomplex", J Virol, 78, 2004, 9144-53.

ST Nath, DP Nayak, "Function of two discrete regions is required for nuclear localization of polymerase basic protein 1 of A/WSN/33 influenzavirus (H1 N1)", Mol Cell Biol, 10, 1990, 4139-45.


T Deng, OG Engelhardt, B Thomas, AV Akoulitchev, GG Brownlee, E Fodor, "Role of ran binding protein 5 in nuclear import and assembly of theinfluenza virus RNA polymerase complex", J Virol, 80, 2006, 11911-9.

A Nieto, S de la Luna, J Barcena, A Portela, J Ortin, "Complex structure of the nuclear translocation signal of influenza virus polymerase PAsubunit", J Gen Virol, 75, 1994, 29-36.

JF Cros, A Garcia-Sastre, P Palese, "An unconventional NLS is critical for the nuclear import of the influenza A virus nucleoprotein andribonucleoprotein", Traffic, 6, 2005, 205-13.

1.1.4.1 Recognition of the Nuclear Localization Signal (NLS) by a Karyopherin Alpha Family Protein

Authors

Gillespie, ME, 2005-11-14 17:18:07.

Reviewers

Squires, B, 2006-10-29 16:42:09.

Description

The eight influenza virus genome segments never exist as naked RNA but are associated with four viral proteins to form viral ribonucleoproteincomplexes (vRNPs). The major viral protein in the RNP complex is the nucleocapsid protein (NP), which coats the RNA. The remaining proteinsPB1, PB2 and PA bind to the partially complementary ends of the viral RNA, creating the distinctive panhandle structure. The influenza viral NPbehaves like a nuclear localization sequence (NLS) containing protein. The RNP docks at the nuclear envelope only in the presence of theheterodimeric karyopherin alpha and beta complex. Here karyopherin alpha recognizes the RNP.

References

RE O'Neill, R Jaskunas, G Blobel, P Palese, J Moroianu, "Nuclear import of influenza virus RNA can be mediated by viral nucleoprotein andtransport factors required for protein import", J Biol Chem, 270, 1995, 22701-4.


Reaction

1.1.4.2 Recruitment of Karyopherin Beta to form a Trimeric Complex

Authors

Gillespie, ME, 2005-11-14 17:18:07.

Reviewers

Squires, B, 2006-10-29 16:42:09.

Description

The eight influenza virus genome segments are associated with four viral proteins to form viral ribonucleoprotein complexes (vRNPs). The majorviral protein in the RNP complex is the nucleocapsid protein (NP), which coats the RNA. The remaining proteins PB1, PB2 and PA bind to thepartially complementary ends of the viral RNA. The influenza viral NP behaves like a nuclear localization sequence (NLS) containing protein.The RNP docks at the nuclear envelope only in the presence of the heterodimeric karyopherin alpha and beta complex. Once the NLS isrecognized by karyopherin alpha the karyopherin beta subunit joins the complex.

References


Reaction

1.1.4.3 Docking and transport of the RNP:Karyopherin complex through the nuclear pore

Authors

Gillespie, ME, 2005-11-14 17:18:07.

Reviewers

Squires, B, 2006-10-29 16:42:09.

Description

These RNPs (10-20nm wide) are too large to passively diffuse into the nucleus and therefore, once released from an incoming particle they mustrely on the active import mechanism of the host cell nuclear pore complex (NPC). Once the RNP heterodimeric karyopherin complex docks atthe NPC, it is transported into the nucleus.

References

J Mukaigawa, DP Nayak, "Two signals mediate nuclear localization of influenza virus (A/WSN/33) polymerase basic protein 2", J Virol, 65, 1991,245-53.

Reaction

1.1.4.4 Release of the RNP into the host cell nucleus

Authors

Gillespie, ME, 2005-11-14 17:18:07.

Reviewers

Squires, B, 2006-10-29 16:42:09.

Description

Once the viral RNP and heterodimeric karyopherin complex has been transported into the nucleus the RNP dissasociates from the heterodimerickaryopherins.

References


Reaction

1.1.5 Influenza Viral RNA Transcription and Replication

Authors

Bortz, E, Garcia-Sastre, A, 2007-02-12 19:39:05.

Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

In the host cell nucleus, the viral negative-strand RNA (vRNA) serves as a template for the synthesis both of capped, polyadenylated viralmessenger RNA and of full-length positive-strand RNA or complementary RNA (cRNA). The cRNA is associated with the same viral proteins asthe vRNA. It serves as a template for the synthesis of new vRNA molecules, which in turn serve as a template for mRNA particularly early ininfection, and cRNA. Viral RNA polymerase subunits (PB1, PB2, and PA) and nucleoprotein (NP) enter the host cell nucleus and catalyze allthree of these reactions. During initial infection, these proteins enter the nucleus as part of the viral RNP complex. After the first round of viralmRNA synthesis (primary transcription) and translation, newly synthesized viral polymerase proteins and NP localize to the nucleus to catalyzefurther mRNA transcription and vRNA/cRNA replication. Late in the infection process, the synthesis of vRNA and nuclear export of newlysynthesized vRNP (vRNA complexed with NP and viral polymerase) is increased relative to transcription (Krug, 1981; Braam, 1983; Kawakami,1983; Huang, 1990; Cros, 2003; Fodor, 2004; Deng, 2005; Amorim, 2006; reviewed in Neumann, 2004; Engelhardt, 2006; Buolo, 2006).

References


K Kawakami, A Ishihama, "RNA polymerase of influenza virus. III. Isolation of RNA polymerase-RNA complexes from influenza virus PR8.", JBiochem (Tokyo), 93, 1983, 989-96.

T Deng, J Sharps, E Fodor, GG Brownlee, "In vitro assembly of PB2 with a PB1-PA dimer supports a new model of assembly of influenza Avirus polymerase subunits into a functional trimeric complex", J Virol, 79, 2005, 8669-74.

JF Cros, P Palese, "Trafficking of viral genomic RNA into and out of the nucleus: influenza, Thogoto and Borna disease viruses", Virus Res, 95,2003, 3-12.

S Boulo, H Akarsu, RW Ruigrok, F Baudin, "Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes", Virus Res, 124, 2006,12-21.

RM Krug, "Priming of influenza viral RNA transcription by capped heterologous RNAs", Curr Top Microbiol Immunol, 93, 1981, 125-49.

TS Huang, P Palese, M Krystal, "Determination of influenza virus proteins required for genome replication", J Virol, 64, 1990, 5669-73.

J Braam, I Ulmanen, RM Krug, "Molecular model of a eucaryotic transcription complex: functions and movements of influenza P proteins duringcapped RNA-primed transcription", Cell, 34, 1983, 609-18.

G Neumann, GG Brownlee, E Fodor, Y Kawaoka, "Orthomyxovirus replication, transcription, and polyadenylation", Curr Top Microbiol Immunol,283, 2004, 121-43.

MJ Amorim, P Digard, "Influenza A virus and the cell nucleus", Vaccine, 24, 2006, 6651-5.

OG Engelhardt, E Fodor, "Functional association between viral and cellular transcription during influenza virus infection", Rev Med Virol, 16,2006, 329-45.

1.1.5.1 vRNP Assembly

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

For each of eight gene segments, a viral ribonucleoprotein (vRNP), containing a viral negative-sense RNA (vRNA) segment complexed withnucleoprotein (NP) and the trimeric influenza polymerase (PB1, PB2, and PA), is assembled in the nucleus (Braam, 1983; Jones, 1986; Cros,2003; reviewed in Buolo, 2006). The vRNP functions in three modes (reviewed in Mikulasova, 2000; Neumann, 2004): (1) transcription, whichsynthesizes viral messenger RNA from the vRNA template using as primers 5' ends of cellular mRNAs containing the cap; (2) replication, whichproduces positive-sense complementary RNA (cRNA) and subsequently vRNA, both complexed with NP and the trimeric polymerase; or (3), thevRNP is exported from the nucleus into the cytoplasm and is incorporated into assembling virions at the plasma membrane.

References



A Mikulasova, E Vareckova, E Fodor, "Transcription and replication of the influenza a virus genome", Acta Virol, 44, 2000, 273-82.



IM Jones, PA Reay, KL Philpott, "Nuclear location of all three influenza polymerase proteins and a nuclear signal in polymerase PB2", EMBO J,5, 1986, 2371-6.

1.1.5.1.1 Viral Polymerase Assembly

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

The mature ternary influenza viral polymerase complex consists of PB1, PB2, and PA. The N-terminus of PB1 (residues 1-48) interacts withPB2, and amino acids 506-659 in PB1 interact with the PA subunit (Gonzalez, 1996; Perez, 2001). Although monomeric PB1, PB2 and PA, aswell as PB1-PB2 and PB1-PA dimers are likely to exist in infected cells, it is believed that most of the polymerase proteins are assembled intothe trimeric PB1-PB2-PA complex (Detjen, 1987). Newly synthesized subunits of the polymerase are imported into the nucleus through nuclearlocalization signals (NLS), which interact with cellular importin family proteins (Jones, 1986; Buolo, 2006). Importin beta-3 (Ran binding protein 5)facilitates nuclear import of PB1 and a PB1-PA dimer (Deng, 2006); coexpression of PA with PB1 was shown to enhance the import of PB1(Fodor, 2004). A PB1-PB2 dimer has been found to interact with the molecular chaperone heat shock protein 90 (HSP90) to facilitate import(Naito, 2007). The three subunits assembled in the nucleus form a mature ternary polymerase complex that binds viral vRNA or cRNA (Jones,1986; Buolo, 2006).

References


S Gonzalez, T Zurcher, J Ortin, "Identification of two separate domains in the influenza virus PB1 protein involved in the interaction with the PB2and PA subunits: a model for the viral RNA polymerase structure", Nucleic Acids Res, 24, 1996, 4456-63.

BM Detjen, Angelo C St, MG Katze, RM Krug, "The three influenza virus polymerase (P) proteins not associated with viral nucleocapsids in theinfected cell are in the form of a complex", J Virol, 61, 1987, 16-22.

T Naito, F Momose, A Kawaguchi, K Nagata, "Involvement of hsp90 in assembly and nuclear import of influenza virus RNA polymerasesubunits", J Virol, 81, 2007, 1339-49.



DR Perez, RO Donis, "Functional analysis of PA binding by influenza a virus PB1: effects on polymerase activity and viral infectivity", J Virol, 75,2001, 8127-36.


Reaction

1.1.5.1.2 NP binds vRNA

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

Viral genomic RNA (vRNA) and complementary RNA (cRNA) are likely bound by the influenza nucleoprotein (NP) immediately upon synthesis.Although two nuclear localization signals have been mapped in the NP, an unconventional N-terminal NLS and a bipartite NLS within aminoacids 198-216 (Wang, 1997; Neumann, 1997; Ozawa, 2007), the crystal structure of the NP suggests that only the unconventional NLS isexposed and can be used as a functional NLS (Ye, 2006). This unconvenetional NLS interacts with importins alpha-1 and -2 (Cros et al., 2005;Wang et al., 1997; Buolo et al., 2006). The three-dimensional structure of NP has revealed that NP molecules associate as a trimer, interactingthrough beta-sheets b5, b6, and b7 in the C-terminal domain of the protein; the viral RNA likely wraps around the outside of the complex (Ye,2006).

References

P Wang, P Palese, RE O'Neill, "The NPI-1/NPI-3 (karyopherin alpha) binding site on the influenza a virus nucleoprotein NP is a nonconventionalnuclear localization signal", J Virol, 71, 1997, 1850-6.

Q Ye, RM Krug, YJ Tao, "The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA", Nature, 444, 2006,1078-82.


G Neumann, MR Castrucci, Y Kawaoka, "Nuclear import and export of influenza virus nucleoprotein", J Virol, 71, 1997, 9690-700.

M Ozawa, K Fujii, Y Muramoto, S Yamada, S Yamayoshi, A Takada, H Goto, T Horimoto, Y Kawaoka, "Contributions of two nuclear localizationsignals of influenza A virus nucleoprotein to viral replication", J Virol, 81, 2007, 30-41.


Reaction

1.1.5.2 Viral Messenger RNA Synthesis

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

Like the mRNAs of the host cell, influenza virus mRNAs are capped and polyadenylated (reviewed in Neumann, 2004). The methylated caps,however, are scavenged from host cell mRNAs and serve as primers for viral RNA synthesis, a process termed 'cap-snatching' (Krug, 1981;Hagen, 1994). The PB2 polymerase protein binds the cap, activating endonucleolytic cleavage of the host mRNA by PB1. The 3' poly-A tracts onviral messages are generated by polymerase stuttering on poly-U tracts near the 5' end of the template vRNA (Robertson, 1981; Zheng, 1999).The second process allows polyadenylation of viral mRNAs when the host cell polyadenylation process has been inhibited (Engelhardt, 2006;Amorim, 2006). Notably, early transcripts (including NP and NS1) accumulate in the cytoplasm before late transcripts (M1, HA, and NS2), and invarying abundances, suggesting additional control mechanisms regulating viral gene expression (Shapiro, 1987; Hatada, 1989; Amorim, 2006).

References

GI Shapiro, T Jr Gurney, RM Krug, "Influenza virus gene expression: control mechanisms at early and late times of infection andnuclear-cytoplasmic transport of virus-specific RNAs", J Virol, 61, 1987, 764-73.

H Zheng, HA Lee, P Palese, A Garcia-Sastre, "Influenza A virus RNA polymerase has the ability to stutter at the polyadenylation site of a viralRNA template during RNA replication", J Virol, 73, 1999, 5240-3.

E Hatada, M Hasegawa, J Mukaigawa, K Shimizu, R Fukuda, "Control of influenza virus gene expression: quantitative analysis of each viralRNA species in infected cells", J Biochem (Tokyo), 105, 1989, 537-46.

JS Robertson, M Schubert, RA Lazzarini, "Polyadenylation sites for influenza virus mRNA", J Virol, 38, 1981, 157-63.



M Hagen, TD Chung, JA Butcher, M Krystal, "Recombinant influenza virus polymerase: requirement of both 5' and 3' viral ends for endonucleaseactivity", J Virol, 68, 1994, 1509-15.



1.1.5.2.1 Assembly of an Active Transcription Complex

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

The 5' end of the vRNA associates with a binding site on the PB1 subunit of the viral RNA polymerase, distinct from the 3' vRNA binding site,which is subsequenty bound forming a loop. These binding events set off allosteric conformational changes in the trimeric polymerase complexthat induce PB2 binding of the methylated cap on a host pre-mRNA (Plotch, 1981; Cianci, 1995; Li, 1998; Brownlee, 2002; Kolpashchikov,2004). PB2 amino acids 242-282 and 538-577 are involved in cap binding (Honda, 1999). Direct or indirect interaction with active, transcribinghost RNA polymerase II is thought to supply host mRNA for the caps (Bouloy, 1978; Engelhardt, 2005).

References

GG Brownlee, JL Sharps, "The RNA polymerase of influenza a virus is stabilized by interaction with its viral RNA promoter", J Virol, 76, 2002,7103-13.

M Bouloy, SJ Plotch, RM Krug, "Globin mRNAs are primers for the transcription of influenza viral RNA in vitro", Proc Natl Acad Sci U S A, 75,1978, 4886-90.

OG Engelhardt, M Smith, E Fodor, "Association of the influenza A virus RNA-dependent RNA polymerase with cellular RNA polymerase II", JVirol, 79, 2005, 5812-8.

ML Li, BC Ramirez, RM Krug, "RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the sameprotein subunit constitute the two required RNA-binding sites", EMBO J, 17, 1998, 5844-52.

DM Kolpashchikov, A Honda, A Ishihama, "Structure-function relationship of the influenza virus RNA polymerase: primer-binding site on the PB1subunit", Biochemistry, 43, 2004, 5882-7.

C Cianci, L Tiley, M Krystal, "Differential activation of the influenza virus polymerase via template RNA binding", J Virol, 69, 1995, 3995-9.

SJ Plotch, M Bouloy, I Ulmanen, RM Krug, "A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs togenerate the primers that initiate viral RNA transcription", Cell, 23, 1981, 847-58.

A Honda, K Mizumoto, A Ishihama, "Two separate sequences of PB2 subunit constitute the RNA cap-binding site of influenza virus RNApolymerase", Genes Cells, 4, 1999, 475-85.

Reaction

1.1.5.2.2 Priming and Initiation of Transcription

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

The host cell mRNA bound to viral RNA polymerase PB2 subunit is cleaved by the viral RNA polymerase PB1 subunit's endonuclease activity,and the capped 5' end plus 10-13 nucleotides of the host mRNA remains bound to the polymerase complex (Plotch, 1981; Krug, 1981; Hagen,1994; Cianci, 1995, Li, 1998; Li, 2001). Viral mRNA may be protected against cap-snatching by the polymerase complex itself, which tightlybinds capped viral mRNA (Shih, 1996). A guanine residue, complementary to a cytosine in the vRNA, is added to the host-derived cap,catalyzed by the RNA polymerase activity of the PB1 viral RNA polymerase subunit (Beaton, 1981; Toyoda, 1986).

References

ML Li, P Rao, RM Krug, "The active sites of the influenza cap-dependent endonuclease are on different polymerase subunits", EMBO J, 20,2001, 2078-86.

AR Beaton, RM Krug, "Selected host cell capped RNA fragments prime influenza viral RNA transcription in vivo", Nucleic Acids Res, 9, 1981,4423-36.


T Toyoda, M Kobayashi, S Nakada, A Ishihama, "Molecular dissection of influenza virus RNA polymerase: PB1 subunit alone is able to catalyzeRNA synthesis", Virus Genes, 12, 1996, 155-63.



SR Shih, RM Krug, "Surprising function of the three influenza viral polymerase proteins: selective protection of viral mRNAs against thecap-snatching reaction catalyzed by the same polymerase proteins", Virology, 226, 1996, 430-5.


Reaction

1.1.5.2.3 Elongation of viral mRNA

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

Catalyzed by the RNA polymerase activity of the viral PB1 subunit, an mRNA complementary to the bound vRNA is synthesized (Plotch, 1977).PA and PB2 move down the growing mRNA in complex with PB1, with PB2 possibly dissociating from the cap (Braam, 1983). However, the5â€™ end of the vRNA may remain bound during elongation as the template is threaded through in a 3â€™ to 5â€™ direction until apolyadenylation signal is encountered (Poon, 1998; Zheng, 1999).

References

LL Poon, DC Pritlove, J Sharps, GG Brownlee, "The RNA polymerase of influenza virus, bound to the 5' end of virion RNA, acts in cis topolyadenylate mRNA", J Virol, 72, 1998, 8214-9.


SJ Plotch, RM Krug, "Influenza virion transcriptase: synthesis in vitro of large, polyadenylic acid-containing complementary RNA", J Virol, 21,1977, 24-34.


Reaction

1.1.5.2.4 Polyadenylation and Termination

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

A poly-uridine sequence motif, consisting in most cases of 5-7 U residues, abuts the "panhandle" duplex structure in the vRNA; this sequence isapproximately 16 nucleotides from the 5' end of this RNA duplex structure within the vRNA promoter. Encountering this signal, the viral RNApolymerase stutters, leading to the synthesis of a poly-A tail on the viral mRNA (Robertson, 1981; Luo, 1991; Li,1994; Poon, 1998; Zheng et al.1999).

References

GX Luo, W Luytjes, M Enami, P Palese, "The polyadenylation signal of influenza virus RNA involves a stretch of uridines followed by the RNAduplex of the panhandle structure", J Virol, 65, 1991, 2861-7.

X Li, P Palese, "Characterization of the polyadenylation signal of influenza virus RNA", J Virol, 68, 1994, 1245-9.




Reaction

1.1.5.2.5 Viral mRNA Splicing (M, NS segments)

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

The viral polymerase complex produces positive-sense viral mRNA with host-cell derived 5' methyl caps. Alternately spliced mRNA transcribedfrom M and NS vRNA segments 7 and 8, producing the spliced mRNA for M2 and NEP/NS2, respectively, are thought to be coupled to thecellular splicing and export mechanisms (Lamb, 1980; Lamb, 1981; Chen, 2000; Li, 2001). As segments 7 and 8 each encode two proteins,splicing must be regulated allowing for alternative mRNAs, with the spliced products in the minority (approximately 10%). M1 splicing may beregulated by the viral polymerase and the cellular SR splicing protein SF2/ASF (Shih, 1995; Shih, 1996); while NS1 splicing appears to beregulated by the viral mRNA intrinsically (Alonso-Caplen, 1991; Valcarel, 1991).

References

SR Shih, RM Krug, "Novel exploitation of a nuclear function by influenza virus: the cellular SF2/ASF splicing factor controls the amount of theessential viral M2 ion channel protein in infected cells", EMBO J, 15, 1996, 5415-27.

J Valcarcel, A Portela, J Ortin, "Regulated M1 mRNA splicing in influenza virus-infected cells", J Gen Virol, 1991, 1301-8.

RA Lamb, CJ Lai, PW Choppin, "Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: colinear and interruptedmRNAs code for overlapping proteins", Proc Natl Acad Sci U S A, 78, 1981, 4170-4.

RA Lamb, PW Choppin, RM Chanock, CJ Lai, "Mapping of the two overlapping genes for polypeptides NS1 and NS2 on RNA segment 8 ofinfluenza virus genome", Proc Natl Acad Sci U S A, 77, 1980, 1857-61.

SR Shih, ME Nemeroff, RM Krug, "The choice of alternative 5' splice sites in influenza virus M1 mRNA is regulated by the viral polymerasecomplex", Proc Natl Acad Sci U S A, 92, 1995, 6324-8.

FV Alonso-Caplen, RM Krug, "Regulation of the extent of splicing of influenza virus NS1 mRNA: role of the rates of splicing and of thenucleocytoplasmic transport of NS1 mRNA", Mol Cell Biol, 11, 1991, 1092-8.

Z Chen, RM Krug, "Selective nuclear export of viral mRNAs in influenza-virus-infected cells", Trends Microbiol, 8, 2000, 376-83.

Y Li, ZY Chen, W Wang, CC Baker, RM Krug, "The 3'-end-processing factor CPSF is required for the splicing of single-intron pre-mRNAs invivo", RNA, 7, 2001, 920-31.

Reaction

1.1.5.2.6 Export of Spliced Viral mRNA

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

In the cases of spliced, polyadenylated mRNA transcribed from M (segment 7) and NS (segment 8) vRNA templates (producing the splicedmRNA for M2 and NS2/NEP, respectively), export may be coupled to aspects of the cellular splicing and export mechanisms (Chen, 2000;Alonso-Caplan et al, 1992; Amorim, 2006). Simultaneously, the export of cellular mRNA appear to be inhibited by the viral NS1 protein, whichbinds to the cellular cleavage and polyadenylation specificity factor (CPSF), preventing polyadenylation and completion of pre-mRNA processing(Nemerof et al., 1998; Fortes, 1994; Lu, 1994; Li, 2001).

References

Y Lu, XY Qian, RM Krug, "The influenza virus NS1 protein: a novel inhibitor of pre-mRNA splicing", Genes Dev, 8, 1994, 1817-28.

P Fortes, A Beloso, J Ortin, "Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport", EMBO J,13, 1994, 704-12.

FV Alonso-Caplen, ME Nemeroff, Y Qiu, RM Krug, "Nucleocytoplasmic transport: the influenza virus NS1 protein regulates the transport ofspliced NS2 mRNA and its precursor NS1 mRNA", Genes Dev, 6, 1992, 255-67.

ME Nemeroff, SM Barabino, Y Li, W Keller, RM Krug, "Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits3'end formation of cellular pre-mRNAs", Mol Cell, 1, 1998, 991-1000.



Reaction

1.1.5.2.7 Viral mRNA Export

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

The viral polymerase complex produces positive-sense viral mRNA with host-cell derived 5' methyl caps. Capped viral mRNAs are selectivelyexported from the host cell nucleus through a currently unclear mechanism that may rely on components of the host cell mRNA exportmachinery (Chen, 2000; Engelhardt, 2006). Polyadenylation of viral mRNA appears be required for influenza mRNA export (Poon, 2000). Acoupling of viral mRNA export with cellular pre-mRNA processing complexes, recruited by phosphorylation of host RNA polymerase II C-terminaldomain which interacts with the viral polymerase (Engelhardt, 2005), has been proposed as controlling the export of a subset (M1, HA, and NS1,but not NP) of viral mRNA from the nucleus (Amorim, 2007).

References


MJ Amorim, EK Read, RM Dalton, L Medcalf, P Digard, "Nuclear Export of Influenza A Virus mRNAs Requires Ongoing RNA Polymerase IIActivity", Traffic, 8, 2007, 1-11.

LL Poon, E Fodor, GG Brownlee, "Polyuridylated mRNA synthesized by a recombinant influenza virus is defective in nuclear export", J Virol, 74,2000, 418-27.



Reaction

1.1.5.3 cRNA Synthesis

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

Synthesis of full length complementary viral RNA (cRNA) requires that vRNA transcription initiates without the help of a host cell methyl RNA capas a primer (Crow, 2004; Vreede, 2004; Deng, 2006), and that it proceeds to the 5' end of the vRNA template without stuttering on thesub-terminal poly-U sequence. Free viral NP protein appears to play a central role in enabling both of these features of cRNA synthesis,although the molecular details of its role remain unclear (Shapiro, 1988; Medcalf, 1999; Mullin, 2004).

References

GI Shapiro, RM Krug, "Influenza virus RNA replication in vitro: synthesis of viral template RNAs and virion RNAs in the absence of an addedprimer", J Virol, 62, 1988, 2285-90.

AE Mullin, RM Dalton, MJ Amorim, D Elton, P Digard, "Increased amounts of the influenza virus nucleoprotein do not promote higher levels ofviral genome replication", J Gen Virol, 85, 2004, 3689-98.

T Deng, FT Vreede, GG Brownlee, "Different de novo initiation strategies are used by influenza virus RNA polymerase on its cRNA and viralRNA promoters during viral RNA replication", J Virol, 80, 2006, 2337-48.

M Crow, T Deng, M Addley, GG Brownlee, "Mutational analysis of the influenza virus cRNA promoter and identification of nucleotides critical forreplication", J Virol, 78, 2004, 6263-70.

L Medcalf, E Poole, D Elton, P Digard, "Temperature-sensitive lesions in two influenza A viruses defective for replicative transcription disruptRNA binding by the nucleoprotein", J Virol, 73, 1999, 7349-56.

FT Vreede, TE Jung, GG Brownlee, "Model suggesting that replication of influenza virus is regulated by stabilization of replicative intermediates",J Virol, 78, 2004, 9568-72.

1.1.5.3.1 Initiation of cRNA Synthesis

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

Viral vRNA, complexed with NP protein, is bound by the trimeric viral polymerase complex in a stable secondary structure-dependent manner,referred to as a panhandle, fork or cork-screw (Fodor, 1994; Brownlee, 2002; Park, 2003; Crow, 2004). This RNA structure is made of both the5Ã¢â‚¬â„¢ and 3' ends of the vRNA. The polymerase is thought to first bind the 5' end of the vRNA and then the 3' end. Synthesis of cRNAinitiates without a host cell methylated RNA cap as a primer (Beaton, 1986; Galarza, 1996; Deng, 2006; Engehardt, 2006).

References




E Fodor, DC Pritlove, GG Brownlee, "The influenza virus panhandle is involved in the initiation of transcription", J Virol, 68, 1994, 4092-6.

JM Galarza, Q Peng, L Shi, DF Summers, "Influenza A virus RNA-dependent RNA polymerase: analysis of RNA synthesis in vitro", J Virol, 70,1996, 2360-8.

CJ Park, SH Bae, MK Lee, G Varani, BS Choi, "Solution structure of the influenza A virus cRNA promoter: implications for differential recognitionof viral promoter structures by RNA-dependent RNA polymerase", Nucleic Acids Res, 31, 2003, 2824-32.

AR Beaton, RM Krug, "Transcription antitermination during influenza viral template RNA synthesis requires the nucleocapsid protein and theabsence of a 5' capped end", Proc Natl Acad Sci U S A, 83, 1986, 6282-6.


Reaction

1.1.5.3.2 cRNA Extension

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

Virion vRNP is capable of synthesizing cRNA immediately following entry into the cell nucleus (Vreede, 2006). The PB1 subunit principallycatalyzes extension (Nakagawa, 1996). However, cRNA does not accumulate until later in the infection process, possiby requiring NP and thetrimeric polymerase for stabilization (Vreede, 2004). The vRNA template is released.

References

Y Nakagawa, K Oda, S Nakada, "The PB1 subunit alone can catalyze cRNA synthesis, and the PA subunit in addition to the PB1 subunit isrequired for viral RNA synthesis in replication of the influenza virus genome", J Virol, 70, 1996, 6390-4.

FT Vreede, GG Brownlee, "Influenza virion-derived viral ribonucleoproteins synthesize both mRNA and cRNA in vitro", J Virol, 2006.


Reaction

1.1.5.4 vRNA Synthesis

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

The synthesis of full-length negative strand viral RNA from a cRNA template is believed to follow the same principles as the synthesis of cRNAfrom a vRNA template. The cRNA, complexed with viral nucleocapsid (NP) protein, is used as template by the trimeric viral polymerase (Pritlove,1995; Vreede, 2004; Crow, 2004), and newly synthesized vRNA molecules are immediately packaged with NP molecules to formribonucleoprotein complexes (Vreede, 2004). There is some evidence that the production of vRNA-containing vRNP occurs in the nuclear matrixas well as the nucleoplasm (Takizawa, 2006).

References

N Takizawa, K Watanabe, K Nouno, N Kobayashi, K Nagata, "Association of functional influenza viral proteins and RNAs with nuclear chromatinand sub-chromatin structure", Microbes Infect, 8, 2006, 823-33.



DC Pritlove, E Fodor, BL Seong, GG Brownlee, "In vitro transcription and polymerase binding studies of the termini of influenza A virus cRNA:evidence for a cRNA panhandle", J Gen Virol, 1995, 2205-13.

1.1.5.4.1 Initiation of vRNA Synthesis

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

Initiation of synthesis of the viral genomic RNA (vRNA) is thought to require hairpin (or panhandle/corkscrew) RNA loop structures formed byboth the 5' and 3' ends of the cRNA (Pritlove, 1995; Crow, 2004; Park, 2003; Deng, 2006). The cRNA promoter has a similar structure to thevRNA promoter, but slight sequence differences are believed to result in a stronger cRNA promoter. As with the vRNA promoter, the polymeraseis thought to first bind to the 5' end of the cRNA, then to the 3' end, and subsequently initiate RNA synthesis.

References





Reaction

1.1.5.4.2 vRNA Extension

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

vRNA is synthesized from the complementary cRNA strand by the trimeric polymerase complex, and bound by free NP protein (Honda, 1988;Mikulasova, 2000; Neumann, 2004). The PB1 subunit, with PA, catalyzes extension (Nakagawa, 1996). The cRNA is released.

References


A Honda, K Ueda, K Nagata, A Ishihama, "RNA polymerase of influenza virus: role of NP in RNA chain elongation", J Biochem (Tokyo), 104,1988, 1021-6.



Reaction

1.1.5.5 Viral mRNA Translation

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

Spliced and unspliced viral mRNA in the cytoplasm are translated by host cell ribosomal translation machinery (reviewed in Kash, 2006). At leastten viral proteins are synthesized: HA, NA, PB1, PB2, PA, NP, NS1, NEP/NS2, M1, and M2. Viral mRNA translation is believed to be enhancedby conserved 5'UTR sequences that interact with the ribosomal machinery and at least one cellular RNA-binding protein, G-rich sequence factor1 (GRSF-1), has been found to specifically interact with the viral 5' UTRs. (Park, 1995; Park, 1999). The viral NS1 protein and the cellular proteinP58(IPK) enhance viral translation indirectly by preventing the activation of the translational inhibitor PKR (Salvatore, 2002; Goodman, 2006).The viral NS1 protein has also been proposed to specifically enhance translation through interaction with host poly(A)-binding protein 1 (PABP1)(Burgui, 2003). Simultaneously, host cell protein synthesis is downregulated in influenza virus infection through still uncharacterized mechanisms(Katze, 1986; Garfinkel, 1992; Kash, 2006). In most human influenza A strains (such as PR8), the PB1 mRNA segment is capable of producinga second protein, PB1-F2, from a short +1 open reading frame initiating downstream of the PB1 ORF initiation codon (Chen, 2001).

References

M Salvatore, CF Basler, JP Parisien, CM Horvath, S Bourmakina, H Zheng, T Muster, P Palese, A Garcia-Sastre, "Effects of influenza A virusNS1 protein on protein expression: the NS1 protein enhances translation and is not required for shutoff of host protein synthesis", J Virol, 76,2002, 1206-12.

I Burgui, T Aragon, J Ortin, A Nieto, "PABP1 and eIF4GI associate with influenza virus NS1 protein in viral mRNA translation initiationcomplexes", J Gen Virol, 84, 2003, 3263-74.

MS Garfinkel, MG Katze, "Translational control by influenza virus. Selective and cap-dependent translation of viral mRNAs in infected cells.", JBiol Chem, 267, 1992, 9383-90.

JC Kash, DM Cunningham, MW Smit, Y Park, D Fritz, J Wilusz, MG Katze, "Selective translation of eukaryotic mRNAs: functional molecularanalysis of GRSF-1, a positive regulator of influenza virus protein synthesis", J Virol, 76, 2002, 10417-26.

W Chen, PA Calvo, D Malide, J Gibbs, U Schubert, I Bacik, S Basta, R O'Neill, J Schickli, P Palese, P Henklein, JR Bennink, JW Yewdell, "Anovel influenza A virus mitochondrial protein that induces cell death", Nat Med, 7, 2001, 1306-12.

YW Park, J Wilusz, MG Katze, "Regulation of eukaryotic protein synthesis: selective influenza viral mRNA translation is mediated by the cellular

RNA-binding protein GRSF-1", Proc Natl Acad Sci U S A, 96, 1999, 6694-9.

AG Goodman, JA Smith, S Balachandran, O Perwitasari, SC Proll, MJ Thomas, MJ Korth, GN Barber, LA Schiff, MG Katze, "The cellular proteinP58IPK regulates influenza virus mRNA translation and replication through a PKR-mediated mechanism", J Virol, 81, 2007, 2221-30.

MG Katze, D DeCorato, RM Krug, "Cellular mRNA translation is blocked at both initiation and elongation after infection by influenza virus oradenovirus", J Virol, 60, 1986, 1027-39.

YW Park, MG Katze, "Translational control by influenza virus. Identification of cis-acting sequences and trans-acting factors which may regulateselective viral mRNA translation.", J Biol Chem, 270, 1995, 28433-9.

JC Kash, AG Goodman, MJ Korth, MG Katze, "Hijacking of the host-cell response and translational control during influenza virus infection", VirusRes, 119, 2006, 111-20.

1.1.5.5.1 Synthesis of PB1-F2

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

For most influenza A strains (such as PR8), the PB1 mRNA segment produces a second protein, PB1-F2, from the +1 open reading frame(Chen, 2001). PB1-F2 is a pro-apoptotic, mitochondria-localized protein (Chen, 2001; Gibbs, 2003) that oligomerizes (Bruns, 2007) andsensitizes cells to death in concert with the mitochondrial ANT3 and VDAC proteins (Zamarin, 2005).

References

D Zamarin, A Garcia-Sastre, X Xiao, R Wang, P Palese, "Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 andVDAC1", PLoS Pathog, 1, 2005, e4.


K Bruns, N Studtrucker, A Sharma, T Fossen, D Mitzner, A Eissmann, U Tessmer, R Roder, P Henklein, V Wray, U Schubert, "Structuralcharacterization and oligomerization of PB1-F2, a proapoptotic influenza A virus protein", J Biol Chem, 282, 2007, 353-63.

JS Gibbs, D Malide, F Hornung, JR Bennink, JW Yewdell, "The influenza A virus PB1-F2 protein targets the inner mitochondrial membrane via apredicted basic amphipathic helix that disrupts mitochondrial function", J Virol, 77, 2003, 7214-24.

Reaction

1.1.5.5.2 Viral Protein Synthesis

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

Spliced and unspliced viral mRNA exported into the cytoplasm are translated by the host cell ribosomal translation machinery (reviewed in Kash,2006). At least ten viral proteins are synthesized: HA, NA, PB1, PB2, PA, NP, NS1, NEP/NS2 (from spliced NS mRNA), M1, and M2 (fromspliced M mRNA). The abundance of each of these proteins is thought to be controlled by differential mRNA abundances and stability (Tekamp,1980; Hatada, 1989). As the localization of the nascent polypeptides is different between viral proteins with transmembrane domains (HA, NAand M2, which translocate to the ER and are transported through the Golgi to the plasma membrane) and soluble viral proteins (such as NP, thepolymerase subunits, and NS1), mechanisms linking the translation of particular viral mRNA with subsequent protein localization rely on signalsequences recognized by the cell.

References



PA Tekamp, EE Penhoet, "Quantification of influenza virus messenger RNAs", J Gen Virol, 47, 1980, 449-59.


Reaction

1.1.6 Export of Viral Ribonucleoproteins from Nucleus

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

Influenza genomic RNA (vRNA), synthesized in the nucleus of the infected host cell, is packaged into ribonucleoprotein (RNP) complexescontaining viral polymerase proteins and NP (nucleocapsid). NP trimers bind the sugar phosphate backbone of the vRNA. As influenza viral RNPcomplexes are too large for passive diffusion out of the nucleus, utilization of the cellular nuclear export machinery is achieved by viral adaptorproteins. Matrix protein (M1) is critical for export of the complex from the nucleus, mediating the interaction of the RNP complex with the viralNEP/NS2 protein, which in turn interacts with host cell CRM1/exportin-1 nuclear export protein (Martin, 1991; O'Neill, 1998; Neumann et al.,2000; Elton, 2001; Cros, 2003; Ye, 2006; reviewed in Boulo, 2006).

References

G Neumann, MT Hughes, Y Kawaoka, "Influenza A virus NS2 protein mediates vRNP nuclear export through NES-independent interaction withhCRM1", EMBO J, 19, 2000, 6751-8.

D Elton, M Simpson-Holley, K Archer, L Medcalf, R Hallam, J McCauley, P Digard, "Interaction of the influenza virus nucleoprotein with thecellular CRM1-mediated nuclear export pathway", J Virol, 75, 2001, 408-19.



1.1.6.1 Viral RNP Complexes in the Host Cell Nucleus

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

Viral RNP is assembled in the host cell nucleus through the interaction of full-length negative strand viral RNA (vRNA) and the viral nucleocapsid(NP) and matrix (M1) proteins. Studies of interactions of the purified components in vitro and of tissue culture model systems expressing variouscombinations of the components have established roles for both NP and M1 proteins in the assembly of a complex that has the physicalproperties of vRNP purified from virions and that can be exported from the host cell nucleus (Whittaker, 1996; Huang, 2001; Baudin, 2001). ViralRNP complexes have been found in the nucleoplasm, and also in the nuclear periphery, associated with the nuclear matrix or chromatin,particularly for vRNA-containing complexes and M1 protein (Elton, 2005; Garcia-Robles, 2005; Takizawa et al., 2006).

References


D Elton, MJ Amorim, L Medcalf, P Digard, "'Genome gating'; polarized intranuclear trafficking of influenza virus RNPs", Biol Lett, 1, 2005, 113-7.

I Garcia-Robles, H Akarsu, CW Muller, RW Ruigrok, F Baudin, "Interaction of influenza virus proteins with nucleosomes", Virology, 332, 2005,329-36.

X Huang, T Liu, J Muller, RA Levandowski, Z Ye, "Effect of influenza virus matrix protein and viral RNA on ribonucleoprotein formation andnuclear export", Virology, 287, 2001, 405-16.

1.1.6.1.1 Newly synthesized vRNP for export

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

The nascent vRNP complexes, one for each gene segment, contain the negative-sense viral RNA and polymerase proteins (PB1, PB2, PA, andNP). In a model using negative-sense viral RNP reconstituted from transfected cells, there are multiple NP complexes and one polymerasecomplex arranged along a closed vRNA loop (Area et al., 2004). The three-dimensional structure of NP has revealed that three NP moleculesform a stable trimer, interacting through beta-sheets b5, b6, and b7 in the C-terminal domain of the protein (Ye, 2006), with the viral RNAwrapping around the outside of the complex. Viral RNA from purified virions is present in an RNase-sensitive complex with NP and PB1, PB2,and PA, consistent with this structural model (Baudin et al, 1994; Ruigrok et al., 1995; Klumpp et al., 1997). It is not clear what controls the fateof vRNP, whether it is destined to become a template for transcription, for replication, or for export into the cytoplasm for packaging into virionsat the plasma membrane, nor how distinct sub-nuclear localization and NP distribution at the nuclear matrix might mark, or polarize, a vRNP forexport (Elton, 2005; Takizawa et al., 2006).

References


RW Ruigrok, F Baudin, "Structure of influenza virus ribonucleoprotein particles. II. Purified RNA-free influenza virus ribonucleoprotein formsstructures that are indistinguishable from the intact influenza virus ribonucleoprotein particles.", J Gen Virol, 1995, 1009-14.



K Klumpp, RW Ruigrok, F Baudin, "Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure", EMBO J,16, 1997, 1248-57.

F Baudin, C Bach, S Cusack, RW Ruigrok, "Structure of influenza virus RNP. I. Influenza virus nucleoprotein melts secondary structure inpanhandle RNA and exposes the bases to the solvent.", EMBO J, 13, 1994, 3158-65.

E Area, J Martin-Benito, P Gastaminza, E Torreira, JM Valpuesta, JL Carrascosa, J Ortin, "3D structure of the influenza virus polymerasecomplex: localization of subunit domains", Proc Natl Acad Sci U S A, 101, 2004, 308-13.

Reaction

1.1.6.1.2 Binding of M1 to vRNP

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

M1 protein binds to viral RNP through its C-terminal domain (Baudin, 2001). The influenza M1 protein accumulates in the infected cell nucleusthrough a nuclear localization signal (NLS) RKLKR (residues 101-105) in its N-terminus (Ye, 1999). A host cell protein, HSP70, is thought toinhibit M1 binding at nonpermissive temperatures (Hirayama et al., 2004).

References

Z Ye, T Liu, DP Offringa, J McInnis, RA Levandowski, "Association of influenza virus matrix protein with ribonucleoproteins", J Virol, 73, 1999,7467-73.

T Liu, J Muller, Z Ye, "Association of influenza virus matrix protein with ribonucleoproteins may control viral growth and morphology", Virology,304, 2002, 89-96.

F Baudin, I Petit, W Weissenhorn, RW Ruigrok, "In vitro dissection of the membrane and RNP binding activities of influenza virus M1 protein",Virology, 281, 2001, 102-8.

M Bui, EG Wills, A Helenius, GR Whittaker, "Role of the influenza virus M1 protein in nuclear export of viral ribonucleoproteins", J Virol, 74,2000, 1781-6.

E Hirayama, H Atagi, A Hiraki, J Kim, "Heat shock protein 70 is related to thermal inhibition of nuclear export of the influenza virusribonucleoprotein complex", J Virol, 78, 2004, 1263-70.


Reaction

1.1.6.1.3 Binding of NEP/NS2 to vRNP:M1

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

Structural characterization of NEP/NS2 suggests that acidic residues in the C-terminus of NEP/NS2 bind to M1, with Trp78 critical for interaction(Ward, 1995; Yasuda, 1993; Akarsu, 2003).

References

J Yasuda, S Nakada, A Kato, T Toyoda, A Ishihama, "Molecular assembly of influenza virus: association of the NS2 protein with virion matrix",Virology, 196, 1993, 249-55.

AC Ward, LA Castelli, AC Lucantoni, JF White, AA Azad, IG Macreadie, "Expression and analysis of the NS2 protein of influenza A virus", ArchVirol, 140, 1995, 2067-73.

Reaction

1.1.6.2 NEP/NS2 Interacts with the Cellular Export Machinery

Authors


Reviewers

Squires, B, 2007-02-12 19:39:22.

Description

The viral RNP complex is exported from the nucleus via the host cell CRM1 export pathway (Fukuda, 1997; Neumann, 2000; reviewed in Buolo,2006). The vRNP complex does not interact directly with CRM1 to form an export complex. Rather, an additional viral protein, nuclear exportprotein (NEP/NS2), acts as an adaptor, binding the viral matrix M1 protein and CRM1, thus linking the viral RNP with CRM1 (Martin, 1991;O'Neill, 1998; Neumann, 2000; Akarsu, 2003). The CRM1/exportin-1 complex recruits additional host cell proteins, and traverses the nuclearpore into the cytosol.

References


K Martin, A Helenius, "Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import",Cell, 67, 1991, 117-30.

H Akarsu, WP Burmeister, C Petosa, I Petit, CW Muller, RW Ruigrok, F Baudin, "Crystal structure of the M1 protein-binding domain of theinfluenza A virus nuclear export protein (NEP/NS2)", EMBO J, 22, 2003, 4646-55.


RE O'Neill, J Talon, P Palese, "The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins", EMBO J, 17,1998, 288-96.

1.1.6.2.1 Binding of vRNP:M1:NEP complex to CRM1 export receptor

Description

Virus NEP/NS2 interacts with human CRM1 (hCRM1), possibly dependent on a nuclear export signal (NES) motif in the NEP/NS2 N-terminalregion (O'Neill, 1998; Neumann, 2000). The CRM1/exportin-1 pathway is a cellular mechanism for nuclear export, with CRM1 interacting with theRan small GTPase and a cargo molecule's leucine-rich NES (Fukuda, 1997; Petosa, 2004). Leptomycin B, which specifically inhibits hCRM1,blocks export of viral RNP (Elton, 2001; Ma, 2001; Watanabe, 2001). Thus, NEP/NS2 interaction with cellular nuclear export machinery isessential for nuclear export of vRNP complexes and influenza virus release. A role for NP protein interaction with export machinery has alsobeen proposed (Elton, 2001).

References

K Ma, AM Roy, GR Whittaker, "Nuclear export of influenza virus ribonucleoproteins: identification of an export intermediate at the nuclearperiphery", Virology, 282, 2001, 215-20.



K Watanabe, N Takizawa, M Katoh, K Hoshida, N Kobayashi, K Nagata, "Inhibition of nuclear export of ribonucleoprotein complexes of influenzavirus by leptomycin B", Virus Res, 77, 2001, 31-42.

Reaction

1.1.6.2.2 vRNP Export through the nuclear pore

Description

Viral RNP, bound by M1 and NEP/NS2 interacting with CRM1, are shuttled through the nuclear pore into the cytoplasm (Martin, 1991; O'Neill,1998; Buolo, 2006). This mechanism may resemble export of HIV-1 ribonucleoprotein, where the HIV-1 Rev export protein interacts with CRM1(Askjaer, 1998). A number of cofactors are implicated in CRM1-mediated export, including the small GTPase Ran, Ran-binding proteins 1 and 3,and a guanine nucleotide exchange factor (Nilsson, 2001; Nemergut, 2002; Petosa, 2004). Ternary CRM1-cofactor-cargo complexes likelyinteract transiently with nuclear pore proteins (nucleoporins) as they traverse the pore (reviewed in Suntharalingam, 2003). RanGTP ishydrolyzed to RanGDP in the cytoplasm, an activity that can be stimulated by NEP/NS2 (Akarsu, 2003). Influenza infection activatesRaf/MEK/ERK signaling, which is necessary for NEP/NS2-mediated export of viral RNP (Pleschka, 2001; Marjuki, 2006). Influenza vRNPcomplexes released into the cytoplasm do not re-enter the nucleus, as they are thought to remain bound by M1, preventing re-import (Martin,1991). It has been suggested that M1 binding of zinc cations could distinguish M1 bound to the vRNP from polymerized, matrix M1 present innascent virions (Elster, 1994).

References


J Nilsson, P Askjaer, J Kjems, "A role for the basic patch and the C terminus of RanGTP in regulating the dynamic interactions with importinbeta, CRM1 and RanBP1", J Mol Biol, 305, 2001, 231-43.

H Marjuki, MI Alam, C Ehrhardt, R Wagner, O Planz, HD Klenk, S Ludwig, S Pleschka, "Membrane accumulation of influenza A virushemagglutinin triggers nuclear export of the viral genome via protein kinase Calpha-mediated activation of ERK signaling", J Biol Chem, 281,2006, 16707-15.

C Petosa, G Schoehn, P Askjaer, U Bauer, M Moulin, U Steuerwald, M Soler-Lopez, F Baudin, IW Mattaj, CW Muller, "Architecture ofCRM1/Exportin1 suggests how cooperativity is achieved during formation of a nuclear export complex", Mol Cell, 16, 2004, 761-75.


S Pleschka, T Wolff, C Ehrhardt, G Hobom, O Planz, UR Rapp, S Ludwig, "Influenza virus propagation is impaired by inhibition of theRaf/MEK/ERK signalling cascade", Nat Cell Biol, 3, 2001, 301-5.

ME Nemergut, ME Lindsay, AM Brownawell, IG Macara, "Ran-binding protein 3 links Crm1 to the Ran guanine nucleotide exchange factor", JBiol Chem, 277, 2002, 17385-8.

P Askjaer, TH Jensen, J Nilsson, L Englmeier, J Kjems, "The specificity of the CRM1-Rev nuclear export signal interaction is mediated byRanGTP", J Biol Chem, 273, 1998, 33414-22.

C Elster, E Fourest, F Baudin, K Larsen, S Cusack, RW Ruigrok, "A small percentage of influenza virus M1 protein contains zinc but zinc doesnot influence in vitro M1-RNA interaction", J Gen Virol, 1994, 37-42.


Reaction

1.1.7 Virus Assembly and Release

Description

Influenza viruses assemble and bud from the apical plasma membrane of polarized cells e.g. lung epithelial cells of the infected host. Thisasymmetrical process (i.e. apical [Influenza virus] or basolateral [Marburg virus]) is thought to have an important role in viral pathogenesis andtissue tropism. In most cases the individual viral envelope proteins are seen to accumulate at the same polar surface from which virus buddingoccurs, suggesting that they determine the maturation site

References


AP Schmitt, RA Lamb, "Influenza virus assembly and budding at the viral budozone", Adv Virus Res, 64, 2005, 383-416.

ER Boulan, DD Sabatini, "Asymmetric budding of viruses in epithelial monlayers: a model system for study of epithelial polarity", Proc Natl AcadSci U S A, 75, 1978, 5071-5.

1.1.7.1 Assembly of Viral Components at the Budding Site

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

Following synthesis on membrane-bound ribosomes, the three viral integral membrane proteins, HA (hemagglutinin), NA (neuraminidase) andM2 (ion channel) enter the host endoplasmic reticulum (ER) where all three are folded and HA and NA are glycosylated. Subsequently HA isassembled into a trimer. HA, NA and M2 are transported to the Golgi apparatus where cysteine residues on HA and M2 are palmitoylated. Furincleaves HA into HA1 and HA2 subunits and all three proteins are directed to the virus assembly site on the apical plasma membrane via apicalsorting signals. The signals for HA and NA reside on the transmembrane domains (TMD) while the sorting signal for M2 is not yet characterized.The TMDs of HA and NA also contain the signals for lipid raft association. Lipid rafts are non-ionic detergent-resistant lipid microdomains withinthe plasma membrane that are rich in sphingolipids and cholesterol. Examination of purified virus particles indicates that influenza virus budspreferentially from these microdomains.

References


1.1.7.1.1 Entry of M2 into the endoplasmic reticulum

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

The integral membrane protein M2 is synthesized on membrane-bound ribosomes and subsequently transported across the ER, where it isfolded and assembled into a tetramer.

References

RW Doms, RA Lamb, JK Rose, A Helenius, "Folding and assembly of viral membrane proteins", Virology, 193, 1993, 545-62.

Reaction

1.1.7.1.2 Assembly of M2 tetramers

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

The M2 from influenza A virus is a 97-residue protein with a single transmembrane helix that associates to form a tetramer in the endoplasmicreticulum (Salom et al, 2000). A 15-20-residue segment C-terminal to the membrane-spanning region has been postulated to aid in thestabilization of the tetrameric assembly (Kochendoerfer et al 1999).

References

GG Kochendoerfer, D Salom, JD Lear, R Wilk-Orescan, SB Kent, WF DeGrado, "Total chemical synthesis of the integral membrane proteininfluenza A virus M2: role of its C-terminal domain in tetramer assembly", Biochemistry, 38, 1999, 11905-13.

D Salom, BR Hill, JD Lear, WF DeGrado, "pH-dependent tetramerization and amantadine binding of the transmembrane helix of M2 from theinfluenza A virus", Biochemistry, 39, 2000, 14160-70.

Reaction

1.1.7.1.3 Entry of NA into the endoplasmic reticulum

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

The integral membrane protein NA is synthesized on membrane-bound ribosomes and subsequently transported across the ER where it isfolded and glycosylated. Subsequently NA is assembled into a tetramer.

References


Reaction

1.1.7.1.4 Glycosylation of NA

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

Glycosylation of NA occurs within the endoplasmic reticulum and is believed to be neccessary for proper tetramerization of the NA dimers. Sugarresidues become attached to four of the five potential glycosylation sites in the head of N1 neuraminidase (Hausman et al., 1997).

References

L Markoff, BC Lin, MM Sveda, CJ Lai, "Glycosylation and surface expression of the influenza virus neuraminidase requires the N-terminalhydrophobic region", Mol Cell Biol, 4, 1984, 8-16.

DJ Hulse, RG Webster, RJ Russell, DR Perez, "Molecular determinants within the surface proteins involved in the pathogenicity of H5N1influenza viruses in chickens", J Virol, 78, 2004, 9954-64.

T Saito, G Taylor, RG Webster, "Steps in maturation of influenza A virus neuraminidase", J Virol, 69, 1995, 5011-7.

J Hausmann, E Kretzschmar, W Garten, HD Klenk, "Biosynthesis, intracellular transport and enzymatic activity of an avian influenza A virusneuraminidase: role of unpaired cysteines and individual oligosaccharides", J Gen Virol, 1997, 3233-45.

Reaction

1.1.7.1.5 Assembly of NA tetramers

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

Tetramerisation of the NA occurs in the ER following an initial dimerisation step. Tetramerisation is believed to be dependant on glycosylation ofthe NA molecules

References



Reaction

1.1.7.1.6 Entry of HA into the endoplasmic reticulum

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

The integral membrane protein HA is synthesized on membrane-bound ribosomes and subsequently transported across the endoplasmicreticulum, where it is folded, glycosylated, and assembled into a trimer.

References


Reaction

1.1.7.1.7 Glycosylation and Folding of HA

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

The ectodomain of HA is translocated into the ER lumen, where it undergoes a series of folding events mediated by the formation of disulfidebonds and glycosylation reactions. The formation of a discrete intermediate species of highly folded monomeric protein preceeds trimerisation.The folding process is efficient and rapid, with greater than 90% of the protein trafficked to the golgi apparatus; and mature HA0 subunitsappearing in a matter of a few minutes. Calnexin and calreticulin have been identified as cellular lectins which interact transiently with newlysynthesized HA by attaching to partially trimmed N-linked oligosaccharides (Herbert et al., 1997), facilitating correct folding of the HA molecule.

References

C Hammond, I Braakman, A Helenius, "Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding andquality control", Proc Natl Acad Sci U S A, 91, 1994, 913-7.

M Molinari, A Helenius, "Chaperone selection during glycoprotein translocation into the endoplasmic reticulum", Science, 288, 2000, 331-3.

CS Copeland, KP Zimmer, KR Wagner, GA Healey, I Mellman, A Helenius, "Folding, trimerization, and transport are sequential events in thebiogenesis of influenza virus hemagglutinin", Cell, 53, 1988, 197-209.

PJ Gallagher, JM Henneberry, JF Sambrook, MJ Gething, "Glycosylation requirements for intracellular transport and function of thehemagglutinin of influenza virus", J Virol, 66, 1992, 7136-45.

PC Roberts, W Garten, HD Klenk, "Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin", J Virol,67, 1993, 3048-60.

W Chen, A Helenius, "Role of ribosome and translocon complex during folding of influenza hemagglutinin in the endoplasmic reticulum of livingcells", Mol Biol Cell, 11, 2000, 765-72.

I Singh, RW Doms, KR Wagner, A Helenius, "Intracellular transport of soluble and membrane-bound glycoproteins: folding, assembly andsecretion of anchor-free influenza hemagglutinin", EMBO J, 9, 1990, 631-9.

MS Segal, JM Bye, JF Sambrook, MJ Gething, "Disulfide bond formation during the folding of influenza virus hemagglutinin", J Cell Biol, 118,1992, 227-44.

W Chen, J Helenius, I Braakman, A Helenius, "Cotranslational folding and calnexin binding during glycoprotein synthesis", Proc Natl Acad Sci US A, 92, 1995, 6229-33.

JR Peterson, A Ora, PN Van, A Helenius, "Transient, lectin-like association of calreticulin with folding intermediates of cellular and viralglycoproteins", Mol Biol Cell, 6, 1995, 1173-84.

DN Hebert, JX Zhang, W Chen, B Foellmer, A Helenius, "The number and location of glycans on influenza hemagglutinin determine folding andassociation with calnexin and calreticulin", J Cell Biol, 139, 1997, 613-23.

R Daniels, B Kurowski, AE Johnson, DN Hebert, "N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin", MolCell, 11, 2003, 79-90.

Reaction

1.1.7.1.8 Trimerization of HA

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

Trimerisation of the fully folded and fully oxidised HA monomer is thought to occur in the endoplasmic reticulum and ERGIC compartment,following dissociation of HA from calnexin. Trimerisation is generally thought to be the final step in HA maturation occurring in the endoplasmicreticulum before transport to the Golgi apparatus, although Yewdell et al (1988) provide data suggesing that trimerisation may occur within theGolgi.

References

CS Copeland, RW Doms, EM Bolzau, RG Webster, A Helenius, "Assembly of influenza hemagglutinin trimers and its role in intracellulartransport", J Cell Biol, 103, 1986, 1179-91.

MJ Gething, K McCammon, J Sambrook, "Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellulartransport", Cell, 46, 1986, 939-50.

U Tatu, C Hammond, A Helenius, "Folding and oligomerization of influenza hemagglutinin in the ER and the intermediate compartment", EMBOJ, 14, 1995, 1340-8.


A Ceriotti, A Colman, "Trimer formation determines the rate of influenza virus haemagglutinin transport in the early stages of secretion inXenopus oocytes", J Cell Biol, 111, 1990, 409-20.

J Hearing, MJ Gething, J Sambrook, "Addition of truncated oligosaccharides to influenza virus hemagglutinin results in itstemperature-conditional cell-surface expression", J Cell Biol, 108, 1989, 355-65.

JW Yewdell, A Yellen, T Bachi, "Monoclonal antibodies localize events in the folding, assembly, and intracellular transport of the influenza virushemagglutinin glycoprotein", Cell, 52, 1988, 843-52.


Reaction

1.1.7.1.9 Transport of HA trimer, NA tetramer and M2 tetramer from the endoplasmic reticulum to the Golgi Apparatus

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

Processed viral proteins are transported from the endoplasmic reticulum to the Golgi apparatus.

References


1.1.7.1.9.1 Budding of vesicle with the HA trimer, NA tetramer and M2 tetramer from the endoplasmic reticulum into the cytosol

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

Viral proteins are packaged into a golgi apparatus bound transport vesicle.

References


Reaction

1.1.7.1.9.2 Fusion of vesicle containing the HA trimer, NA tetramer and M2 tetramer to the Golgi apparatus

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

Once the tranport vesicle arrives at the golgi apparatus, it docks and dumps its contents into the golgi lumen.

References


Reaction

1.1.7.1.10 Palmitoylation of cysteine residues on HA in the cis-Golgi network

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

The hemagglutinin of influenza virus is palmitoylated with long-chain fatty acids.

Palmitoylation of HA is believed to occur in the cis golgi network (Veit 1993), shortly after trimerisation of the molecule, and before cleavage ofthe HA into HA1 and HA2. HA is palmitoylated through thioester linkages at three cysteine residues located in the cytoplasmic domain and at thecarboxy-terminal end of the transmembrane region. Lack of acylation has no obvious influence on the biological activities of HA.

References

E Ponimaskin, MF Schmidt, "Domain-structure of cytoplasmic border region is main determinant for palmitoylation of influenza virushemagglutinin (H7)", Virology, 249, 1998, 325-35.

G Russ, JR Bennink, T Bachi, JW Yewdell, "Influenza virus hemagglutinin trimers and monomers maintain distinct biochemical modifications andintracellular distribution in brefeldin A-treated cells", Cell Regul, 2, 1991, 549-63.

H Jin, K Subbarao, S Bagai, GP Leser, BR Murphy, RA Lamb, "Palmitylation of the influenza virus hemagglutinin (H3) is not essential for virusassembly or infectivity", J Virol, 70, 1996, 1406-14.

BJ Chen, M Takeda, RA Lamb, "Influenza virus hemagglutinin (H3 subtype) requires palmitoylation of its cytoplasmic tail for assembly: M1proteins of two subtypes differ in their ability to support assembly", J Virol, 79, 2005, 13673-84.

M Veit, H Reverey, MF Schmidt, "Cytoplasmic tail length influences fatty acid selection for acylation of viral glycoproteins", Biochem J, 1996,163-72.

M Veit, MF Schmidt, "Timing of palmitoylation of influenza virus hemagglutinin", FEBS Lett, 336, 1993, 243-7.

HY Naim, B Amarneh, NT Ktistakis, MG Roth, "Effects of altering palmitylation sites on biosynthesis and function of the influenza virushemagglutinin", J Virol, 66, 1992, 7585-8.

P Portincasa, G Conti, C Chezzi, "Role of acylation of viral haemagglutinin during the influenza virus infectious cycle", Res Virol, 143, 1992,401-6.

M Veit, E Kretzschmar, K Kuroda, W Garten, MF Schmidt, HD Klenk, R Rott, "Site-specific mutagenesis identifies three cysteine residues in thecytoplasmic tail as acylation sites of influenza virus hemagglutinin", J Virol, 65, 1991, 2491-500.

DA Steinhauer, SA Wharton, DC Wiley, JJ Skehel, "Deacylation of the hemagglutinin of influenza A/Aichi/2/68 has no effect on membrane fusionproperties", Virology, 184, 1991, 445-8.

M Veit, G Herrler, MF Schmidt, R Rott, HD Klenk, "The hemagglutinating glycoproteins of influenza B and C viruses are acylated with differentfatty acids", Virology, 177, 1990, 807-11.

Reaction

1.1.7.1.11 Palmitoylation of cysteine residues on M2 in the cis-golgi network

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

Palmitoylation of influenza A M2 occurs in the ER, or cis golgi network, following tetramerisation. The palmitoylation reaction proceeds via alabile thioester type bond at a specific residue of M2 (Sugrue et al., 1990).

References

RJ Sugrue, RB Belshe, AJ Hay, "Palmitoylation of the influenza A virus M2 protein", Virology, 179, 1990, 51-6.

M Veit, HD Klenk, A Kendal, R Rott, "The M2 protein of influenza A virus is acylated", J Gen Virol, 72, 1991, 1461-5.

Reaction

1.1.7.1.12 Association of HA into rafts

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

Influenza virus buds preferentially from lipid rafts (Scheiffele et al, 1999). NA protein individually accumulates at, and is selectively incorporatedinto rafts (Kundu et al., 1996). The signals for raft association lie within the transmembranse domain (TMD), (Barman et al., 2001, Barman et al.,2004), and raft association of NA has been shown to be essential for efficient virus replication. This is believed to be due to a requirement for aconcentration of NA at specific areas of the plasma membrane to support a level of NA incorporation into budding particles sufficient to allow forefficient virus release (Barman et al., 2004).

References

S Barman, A Ali, EK Hui, L Adhikary, DP Nayak, "Transport of viral proteins to the apical membranes and interaction of matrix protein withglycoproteins in the assembly of influenza viruses", Virus Res, 77, 2001, 61-9.

DP Nayak, EK Hui, S Barman, "Assembly and budding of influenza virus", Virus Res, 106, 2004, 147-65.

A Kundu, RT Avalos, CM Sanderson, DP Nayak, "Transmembrane domain of influenza virus neuraminidase, a type II protein, possesses anapical sorting signal in polarized MDCK cells", J Virol, 70, 1996, 6508-15.

M Takeda, GP Leser, CJ Russell, RA Lamb, "Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion", ProcNatl Acad Sci U S A, 100, 2003, 14610-7.

S Barman, L Adhikary, AK Chakrabarti, C Bernas, Y Kawaoka, DP Nayak, "Role of transmembrane domain and cytoplasmic tail amino acidsequences of influenza a virus neuraminidase in raft association and virus budding", J Virol, 78, 2004, 5258-69.

P Scheiffele, MG Roth, K Simons, "Interaction of influenza virus haemagglutinin with sphingolipid-cholesterol membrane domains via itstransmembrane domain", EMBO J, 16, 1997, 5501-8.

P Scheiffele, A Rietveld, T Wilk, K Simons, "Influenza viruses select ordered lipid domains during budding from the plasma membrane", J BiolChem, 274, 1999, 2038-44.

Reaction

1.1.7.1.13 Association of NP into rafts

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

There is evidence that NP alone is intrinsically targeted to the apical plasma membrane and associates with lipid rafts in a cholesterol-dependentmanner, which suggests that RNPs could reach the assembly site independently of the other viral components.

References

M Carrasco, MJ Amorim, P Digard, "Lipid raft-dependent targeting of the influenza A virus nucleoprotein to the apical plasma membrane",Traffic, 5, 2004, 979-92.

RT Avalos, Z Yu, DP Nayak, "Association of influenza virus NP and M1 proteins with cellular cytoskeletal elements in influenza virus-infectedcells", J Virol, 71, 1997, 2947-58.

M Simpson-Holley, D Ellis, D Fisher, D Elton, J McCauley, P Digard, "A functional link between the actin cytoskeleton and lipid rafts duringbudding of filamentous influenza virions", Virology, 301, 2002, 212-25.

Reaction

1.1.7.1.14 Association of NA into rafts

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

Influenza virus buds preferentially from lipid rafts (Scheiffele et al, 1999). NA protein individually accumulates at, and is selectively incorporatedinto rafts (Kundu et al., 1996). The signals for raft association lie within the transmembranse domain (TMD), (Barman et al., 2001, Barman et al.,2004), and raft association of NA has been shown to be essential for efficient virus replication. This is believed to be due to a requirement for aconcentration of NA at specific areas of the plasma membrane to support a level of NA incorporation into budding particles sufficient to allow forefficient virus release (Barman et al., 2004).

References






Reaction

1.1.7.1.15 Transport of processed viral proteins to the cell membrane

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

Once processed, the viral proteins are transported from the golgi apparatus to the plasma membrane.

References

S Heino, S Lusa, P Somerharju, C Ehnholm, VM Olkkonen, E Ikonen, "Dissecting the role of the golgi complex and lipid rafts in biosynthetictransport of cholesterol to the cell surface", Proc Natl Acad Sci U S A, 97, 2000, 8375-80.

Reaction

1.1.7.1.16 Accumulation of M1 at the inner leaflet of the lipid bilayer

Authors

Steel, J, 2007-04-30 20:49:24.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

There is evidence for the association of M1 with lipid rafts in influenza infected cells, whereas M1 expressed alone remains soluble (Ali et al.,2000; Zhang and Lamb, 1996), suggesting association of M1 with other viral proteins in targetting to the cell membrane. Coexpression of HA andNA together with M1 has been shown to promote raft association of M1. This association requires the TMD and cytoplasmic tails of HA and NA(Ali et al, 2000; Zhang et al, 2000). This is consistent with M1 becoming associated with HA and NA during their passage through the exocyticpathway to raft domains in the apical membrane. alternatively M1 may use the cytoskeleton to reach the virus assembly site, as M1 interactswith cytoskeletal components (Alvalos et al., 1997). The M1 interaction depends on the presence of RNP and is most likely mediated by directbinding of F-actin by NP (Digard et al., 1999).

References

J Zhang, RA Lamb, "Characterization of the membrane association of the influenza virus matrix protein in living cells", Virology, 225, 1996,255-66.


J Zhang, A Pekosz, RA Lamb, "Influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins", JVirol, 74, 2000, 4634-44.

P Digard, D Elton, K Bishop, E Medcalf, A Weeds, B Pope, "Modulation of nuclear localization of the influenza virus nucleoprotein throughinteraction with actin filaments", J Virol, 73, 1999, 2222-31.

A Ali, RT Avalos, E Ponimaskin, DP Nayak, "Influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of M1protein", J Virol, 74, 2000, 8709-19.

Reaction

1.1.7.2 Packaging of Eight RNA Segments

Authors

Marsh, G, 2007-04-30 20:49:40.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

For a budding influenza virus to be fully infectious is it essential that it contains a full complement of the eight vRNA segments. Two differentmodels have been proposed for packaging of the vRNPs into newly assembling virus particles; the random incorporation model and the selectiveincorporation model.

The random incorporation model as its name suggests proposes that there is no selection at all on which vRNPs are packaged. It is assumedthat each vRNP has equal probability of being packaged, and that if enough vRNPS are packaged a particular percentage of budding virions willreceive at least one copy of each genome segment. This model is supported by evidence that infectious virions may possess more than eightvRNPs assuring the presence of a full complement of eight vRNPs in a significant percentage of virus particles. Mathematical analysis ofpackaging suggested that twelve RNA segments would need to be packaged in order to obtain approximately 10% of virus particles that are fullyinfectious (Enami, 1991), a number that is compatible with experimental data (Donald, 1954). Due to the low amount of RNA per virion(estimated at 1-2% w/w), enumeration of the precise number of RNAs packaged in a virion is difficult.

The selective incorporation model, suggests that each vRNA segment contains a unique "packaging signal" allowing it to act independently, with

each vRNA segment being packaged selectively. There is increasing evidence to support the theory of a packaging signal within the codingregions at both the 5' and 3' end of the genomic RNA, with signals being reported for all segments except segment 7 (Ozawa 2007, Muramoto2006, Fujii 2005, Fujii 2003, Watanabe 2003, Liang 2005). The exact method by which individual vRNP segments are packaged is not known butit has been hypothesized to occur via specific RNA-RNA or protein-RNA interactions. This model is also supported by thin section electronmicroscopy images of influenza particles that show eight distinct "dots", presumably vRNPs within virus particles (Noda 2006).

References

Y Muramoto, A Takada, K Fujii, T Noda, K Iwatsuki-Horimoto, S Watanabe, T Horimoto, H Kida, Y Kawaoka, "Hierarchy among viral RNA(vRNA) segments in their role in vRNA incorporation into influenza A virions", J Virol, 80, 2006, 2318-25.


Y Liang, Y Hong, TG Parslow, "cis-Acting packaging signals in the influenza virus PB1, PB2, and PA genomic RNA segments", J Virol, 79, 2005,10348-55.

Y Fujii, H Goto, T Watanabe, T Yoshida, Y Kawaoka, "Selective incorporation of influenza virus RNA segments into virions", Proc Natl Acad SciU S A, 100, 2003, 2002-7.

HB DONALD, A ISAACS, "Counts of influenza virus particles", J Gen Microbiol, 10, 1954, 457-64.

K Fujii, Y Fujii, T Noda, Y Muramoto, T Watanabe, A Takada, H Goto, T Horimoto, Y Kawaoka, "Importance of both the coding and thesegment-specific noncoding regions of the influenza A virus NS segment for its efficient incorporation into virions", J Virol, 79, 2005, 3766-74.

T Noda, H Sagara, A Yen, A Takada, H Kida, RH Cheng, Y Kawaoka, "Architecture of ribonucleoprotein complexes in influenza A virusparticles", Nature, 439, 2006, 490-2.

T Watanabe, S Watanabe, T Noda, Y Fujii, Y Kawaoka, "Exploitation of nucleic acid packaging signals to generate a novel influenza virus-basedvector stably expressing two foreign genes", J Virol, 77, 2003, 10575-83.


M Enami, G Sharma, C Benham, P Palese, "An influenza virus containing nine different RNA segments", Virology, 185, 1991, 291-8.

1.1.7.2.1 RNP association

Authors

Marsh, G, 2007-04-30 20:49:40.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

The random incorporation model as its name suggests proposes that there is no selection at all on which vRNPs are packaged. It is assumedthat each vRNP has equal probability of being packaged, and that if enough vRNPS are packaged a particular percentage of budding virions willreceive at least one copy of each genome segment. This model is supported by evidence that infectious virions may possess more than eightvRNPs assuring the presence of a full complement of eight vRNPs in a significant percentage of virus particles. Mathematical analysis ofpackaging suggested that twelve RNA segments would need to be packaged in order to obtain approximately 10% of virus particles that are fullyinfectious (Enami, 1991), a number that is compatible with experimental data (Donald, 1954). Due to the low amount of RNA per virion(estimated at 1-2% w/w), enumeration of the precise number of RNAs packaged in a virion is difficult.

References



Reaction

1.1.7.2.2 Association with M1 at cell membrane

Authors

Marsh, G, 2007-04-30 20:49:40.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

As influenza viruses bud from the plasma membrane of infected cells, complete virions are not seen inside cells. In polarized epithelial cells,assembly and budding of influenza occurs from the apical plasma membrane (Schmitt, 2004). For efficient assembly, all virion components mustaccumulate at the budding site, and it is believed that the viral glycoprotein accumulation determines the site of virus assembly and budding(Nayak, 2004). M1 is thought to be the bridge between the envelope glycoproteins and the RNPs for assembly (Schmitt, 2004). M2 is alsorequired, because if it is not present RNPs are not packaged into budding virions (McCown, 2005), however it role is not known.

References


MF McCown, A Pekosz, "The influenza A virus M2 cytoplasmic tail is required for infectious virus production and efficient genome packaging", JVirol, 79, 2005, 3595-605.

AP Schmitt, RA Lamb, "Escaping from the cell: assembly and budding of negative-strand RNA viruses", Curr Top Microbiol Immunol, 283, 2004,145-96.

Reaction

1.1.7.3 Budding

Authors

Marsh, G, 2007-04-30 20:49:40.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

The process by which influenza virus particles bud from an infected cell is not very well understood. Accumulation of M1 at the inner leaflet of theplasma membrane is thought to be the trigger for the initiation of bud formation. This bud formation continues until the inner core of the virus iscompletely enveloped. Completion of the budding process requires the membrane at the base of the bud to fuse. Although M1 is thought to bethe driving force for bud formation, other viral and cellular proteins have been demonstrated to affect size and shape of the virus particle.Generally, influenza virus particles are either spherical or filamentous and this characteristic morphology is genetically linked to the M segment(Bourmakina, 2003; Roberts, 1998). Host factors such as polarization and the actin cytoskeleton play a critical role in determining the shape offilamentous particles (Roberts, 1998; Simpson-Holley, 2002).

References

SV Bourmakina, A Garcia-Sastre, "Reverse genetics studies on the filamentous morphology of influenza A virus", J Gen Virol, 84, 2003, 517-27.



PC Roberts, RW Compans, "Host cell dependence of viral morphology", Proc Natl Acad Sci U S A, 95, 1998, 5746-51.

PC Roberts, RA Lamb, RW Compans, "The M1 and M2 proteins of influenza A virus are important determinants in filamentous particleformation", Virology, 240, 1998, 127-37.


1.1.7.3.1 Membrane fusion

Authors

Marsh, G, 2007-04-30 20:49:40.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

The final step in the budding process is the fusion of the lipid membrane surrounding the virion core, producing an extracellular enveloped virusparticle.

Reaction

1.1.7.4 Release

Authors

Marsh, G, 2007-04-30 20:49:40.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

Once the viral envelope has separated from the cell membrane Influenza virus particles are actively released to complete the budding process.HA (hemagglutinin) anchors the virus to the cell by binding to sialic acid-containing receptors on the cell surface. The enzymatic activity of theneuraminidase (NA) protein removes the sialic acid and releases the virus from the host cell. NA activity is also required to remove sialic acidfrom the carbohydrates present on the viral glycoproteins to prevent the viral particles from aggregating.

References



1.1.7.4.1 Neuraminidase enzymatic release from sialic acid

Authors

Marsh, G, 2007-04-30 20:49:40.

Reviewers

Squires, B, Rush, MG, 2007-04-30 20:48:45.

Description

The release of influenza virus particles after seperation of the virus and infected cell membrane is an active process. During the buddingprocess, HA on the surface of the newly budding virion binds to cell surface molecules containing sialic acid residues as seen during attachment.The NA glycoproteins neuraminidase activity is essential to cleave the link between the HA and sialic acid on the surface of the host cell fromwhich the budding virus is emeging from. Thus the NA mediated cleavage of sialic acid residues terminally linked to glycoproteins and glycolipidsis the final step in releasing the virus particle from the host cell. This essential role of NA in release of virus particle has been demonstrated withthe use of NA inhibitors (Palese, 1976; Luo, 1999; Garman, 2004), ts NA mutant viruses (Palese, 1974) and with viruses lacking NA activity (Liu,1995). In all cases, viruses remain bound to the cell surface in clumps in the absence of NA enzymatic activity, resulting in loss of infectivity.Addition of exogenous sialidase results in virus release and recovery of infectivity. The sialidase activity of the NA is also important for removingsialic acid from the HA on virus particles, if this is not removed, virus particles aggregate.

References

C Liu, MC Eichelberger, RW Compans, GM Air, "Influenza type A virus neuraminidase does not play a role in viral entry, replication, assembly,or budding", J Virol, 69, 1995, 1099-106.

C Luo, E Nobusawa, K Nakajima, "An analysis of the role of neuraminidase in the receptor-binding activity of influenza B virus: the inhibitoryeffect of Zanamivir on haemadsorption", J Gen Virol, 1999, 2969-76.

P Palese, RW Compans, "Inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid(FANA): mechanism of action", J Gen Virol, 33, 1976, 159-63.

P Palese, K Tobita, M Ueda, RW Compans, "Characterization of temperature sensitive influenza virus mutants defective in neuraminidase",Virology, 61, 1974, 397-410.

E Garman, G Laver, "Controlling influenza by inhibiting the virus's neuraminidase", Curr Drug Targets, 5, 2004, 119-36.

Reaction

1.2 Host Interactions with Influenza Factors

Reviewers

Gale M, Jr, 2004-05-12 19:00:00.

Description

Infection of a human host cell with influenza virus triggers an array of host processes that interfere with viral replication, notably the production oftype I interferon. The viral NS1 protein plays a central role in these virus-host interactions.

References



1.2.1 NS1 Mediated Effects on Host Pathways

Description

Viral NS1 protein is a nuclear, dimeric protein that is highly expressed in infected cells and has dsRNA-binding activity. The RNA-binding domainlies within the N-terminal portion of the protein. The NS1 RNA-binding domain forms a symmetric homodimer with a six-helical fold. Mutationalanalysis has demonstrated that dimer formation is crucial for RNA-binding. The basic residues are believed to make contact with the phosphatebackbone of the RNA which is consistent with an observed lack of sequence specificity. Neither NS1 nor its bound RNA undergo any significantstructural changes upon binding. The NS1 dimer spans the minor groove of canonical A-form dsRNA. The non-RNA binding portion of NS1 hasbeen termed the effector domain and includes binding sites for host cell poly (A)-binding protein II (PABII) and the 30kDa subunit of cleavageand polyadenylation specificity factor (CPSF).

References


ME Nemeroff, XY Qian, RM Krug, "The influenza virus NS1 protein forms multimers in vitro and in vivo", Virology, 212, 1995, 422-8.

XY Qian, CY Chien, Y Lu, GT Montelione, RM Krug, "An amino-terminal polypeptide fragment of the influenza virus NS1 protein possessesspecific RNA-binding activity and largely helical backbone structure", RNA, 1, 1995, 948-56.

1.2.1.1 Inhibition of Host mRNA Processing and RNA Silencing

Description

The Influenza Virus NS1 protein inhibits the cleavage and polyadenylation specificity factor CPSF and the PABII components of the host cell 3'end processing machinery, preventing efficient 3' end processing of host pre-mRNAs. NS1 also inhibits the splicing of pre-mRNAs, resulting intheir retention within the host cell nucleus.

References



Z Chen, Y Li, RM Krug, "Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 3'-end processing machinery", EMBO J,18, 1999, 2273-83.

1.2.1.1.1 Binding of NS1 to cleavage and host polyadenylation specificity factor (CPSF)

Description

Influenza virus's non-structural protein (NS1) binds to the host cell's cleavage and host polyadenylation specificity factor (CPSF), inhibiting theability of CPSF to bind to pre-mRNAs and thus preventing efficient 3' end processing and export of host cell mRNAs out of the nucleus.

References

K Shimizu, A Iguchi, R Gomyou, Y Ono, "Influenza virus inhibits cleavage of the HSP70 pre-mRNAs at the polyadenylation site", Virology, 254,1999, 213-9.

DL Noah, KY Twu, RM Krug, "Cellular antiviral responses against influenza A virus are countered at the posttranscriptional level by the viralNS1A protein via its binding to a cellular protein required for the 3' end processing of cellular pre-mRNAS", Virology, 307, 2003, 386-95.



Reaction

1.2.1.1.2 Binding of NS1 to poly(A)-binding protein II (PABII)

Description

The influenza virus non-structural protein 1 (NS1) binds to the host cell's poly(A)-binding protein II (PABII) thus preventing PABII from properlyextending the poly-A tail of pre-mRNA within the host cell nucleus. These pre-mRNAs are then prevented from exiting the nucleus.

References


Reaction

1.2.1.2 Inhibition of Interferon Synthesis

Description

Interferon Synthesis is inhibited.

1.2.1.2.1 Inhibition of IFN-beta

Description

Since the presence of intracellular dsRNA serves as the signal for virus infection and triggers host interferon (IFN) synthesis the simplest modelfor viral NS1 protein function is that it sequesters dsRNA and thus prevents the downstream signaling required to activate IRF-3, NF-kB andAP-1. These findings are strongly supported by mutational analyses of NS1 that indicate that the IFN antagonist properties of NS1 depend on itsability to bind dsRNA. However, a compensatory mutation (S42G), which was acquired during the passaging of the mutant RNA-binding virus,results in partial restoration of wild-type phenotype but does not restore RNA binding. This indicates that the ability of NS1 to inhibit IFNsynthesis is not solely dependent on dsRNA binding and that additional mechanisms may be involved.

References

NR Donelan, CF Basler, A Garcia-Sastre, "A recombinant influenza A virus expressing an RNA-binding-defective NS1 protein induces highlevels of beta interferon and is attenuated in mice", J Virol, 77, 2003, 13257-66.

1.2.1.2.1.1 Binding of NS1 to dsRNA

Description

The ability of viral non-structural protein 1 (NS1) to sequester dsRNA is believed to be one of the primary mechanisms by which NS1 preventsactivation of downstream anti-viral signaling pathways.

References


Reaction

1.2.1.3 Inhibition of PKR

Description

The key role played by PKR in the innate response to virus infection is emphasized by the large number of viruses that encode PKR inhibitors.

1.2.1.3.1 Binding of NS1 to dsRNA

Please refer to section 1.2.1.2.1.1 for full details of "Binding of NS1 to dsRNA"

Reaction

1.2.1.3.2 Binding of NS1 to PKR

Description

At the beginning of this reaction, 1 molecule of 'NS1 Homodimer', and 1 molecule of 'PKR human' are present. At the end of this reaction, 1molecule of 'NS1 Homodimer:PKR Complex' is present.

Reaction

1.2.2 Influenza Virus Induced Apoptosis

Description

Influenza A virus induces apoptosis in a variety of ways including activation of host TGF-beta by expression of viral NA, M1 and M2 proteins, andby the binding of viral PB1-F2 to host mitochondrial adenine nucleotide translocator 3 (ANT3).

References


S Schultz-Cherry, N Dybdahl-Sissoko, G Neumann, Y Kawaoka, VS Hinshaw, "Influenza virus ns1 protein induces apoptosis in cultured cells", JVirol, 75, 2001, 7875-81.

OP Zhirnov, TE Konakova, T Wolff, HD Klenk, "NS1 protein of influenza A virus down-regulates apoptosis", J Virol, 76, 2002, 1617-25.

T Takizawa, S Matsukawa, Y Higuchi, S Nakamura, Y Nakanishi, R Fukuda, "Induction of programmed cell death (apoptosis) by influenza virusinfection in tissue culture cells", J Gen Virol, 74, 1993, 2347-55.

SJ Morris, GE Price, JM Barnett, SA Hiscox, H Smith, C Sweet, "Role of neuraminidase in influenza virus-induced apoptosis", J Gen Virol, 80,1999, 137-46.

WJ Wurzer, C Ehrhardt, S Pleschka, F Berberich-Siebelt, T Wolff, H Walczak, O Planz, S Ludwig, "NF-kappaB-dependent induction of tumornecrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas/FasL is crucial for efficient influenza virus propagation", J Biol Chem, 279,2004, 30931-7.

SJ Morris, H Smith, C Sweet, "Exploitation of the Herpes simplex virus translocating protein VP22 to carry influenza virus proteins into cells forstudies of apoptosis: direct confirmation that neuraminidase induces apoptosis and indications that other proteins may have a role", Arch Virol,

147, 2002, 961-79.

1.2.2.1 NA activation of TGF-beta

Description

Influenza A virus induces apoptosis in a variety of ways including by activation of host TGF-beta by viral neuraminidase (NA).

References

S Schultz-Cherry, VS Hinshaw, "Influenza virus neuraminidase activates latent transforming growth factor beta", J Virol, 70, 1996, 8624-9.

S Oh, JM McCaffery, MC Eichelberger, "Dose-dependent changes in influenza virus-infected dendritic cells result in increased allogeneic T-cellproliferation at low, but not high, doses of virus", J Virol, 74, 2000, 5460-9.


Reaction

1.2.2.2 PB1-F2 binds to the mitochondrial adenine nucleotide translocator 3 ANT3, inducing apoptosis

Description

Influenza A virus induces apoptosis in a variety of ways including binding of viral PB1-F2 to host mitochondrial adenine nucleotide translocator 3(ANT3).

References

AN Chanturiya, G Basanez, U Schubert, P Henklein, JW Yewdell, J Zimmerberg, "PB1-F2, an influenza A virus-encoded proapoptoticmitochondrial protein, creates variably sized pores in planar lipid membranes", J Virol, 78, 2004, 6304-12.


Reaction

Full List of Literature References for Pathway "Influenza Infection"

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XY Qian, CY Chien, Y Lu, GT Montelione, RM Krug, "An amino-terminal polypeptide fragment of the influenza virus NS1 protein possessesspecific RNA-binding activity and largely helical backbone structure", RNA, 1, 1995, 948-56.

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