JOURNAL OF VIROLOGY, May 1993, p. 2546-2551 Vol. 67, No. 50022-538X/93/052546-06$02.00/0Copyright © 1993, American Society for Microbiology

Site-Directed Mutations in the Sindbis Virus E2 GlycoproteinIdentify Palmitoylation Sites and Affect Virus Budding

LIDIA IVANOVA AND MILTON J. SCHLESINGER*Department ofMolecular Microbiology, Washington University School ofMedicine,

St. Louis, Missouri 63110-1093

Received 16 December 1992/Accepted 29 January 1993

The assembly and budding of Sindbis virus, a prototypic member of the alphavirus subgroup in the familyTogaviridae, requires a specific interaction between the nucleocapsid core and the membrane-embeddedglycoproteins El and E2. These glycoproteins are modified posttranslationally by the addition of palmitic acid,and inhibitors of acylation interfere with this budding process (M. J. Schlesinger and C. Malfer, J. Biol. Chem.257:9887-9890, 1982). This report describes the use of site-directed mutagenesis to identify two of the acylationsites in the E2 glycoprotein as the cysteines near the carboxyl terminus of the protein which is oriented to thecytoplasmic domain of this type 1 transmembrane protein. Additional mutations were made at two prolineswithin a hydrophobic sequence of E2 that is highly conserved among several alphaviruses, and the mutantviruses were aberrant in assembly and particle formation. These data support earlier studies indicating that thenative structure of the cytoplasmic domain of E2 is essential for proper assembly of this enveloped virus.

The three major Sindbis virus structural proteins, capsid,El, and E2, are expressed in the infected cell by co- andposttranslational proteolytic processing of a polyproteinwhose synthesis initiates at a single site on a subgenomicspecies of viral RNA (2). The first protein formed is thecapsid, which is released from the nascent polypeptide by anautoprotease activity residing in the capsid sequences (1, 8).Continued translation produces a transmembrane signal se-quence that directs the insertion of the growing polypeptideinto the lumen of the endoplasmic reticulum (20, 24). Trans-location of nascent chains ceases at a stop-transfer sequencewhich forms the transmembrane domain for the E2 spikeglycoprotein located near the carboxyl terminus of thisprotein (16). After the stop-transfer sequence KARRE, thereis a sequence of about 28 predominantly hydrophobic aminoacids (18), which appears to be reinserted into the membranesince this portion of the protein can serve as a signalsequence (13). The signalase, localized to the lumen of theendoplasmic reticulum, proteolytically releases E2 from thepolyprotein (16), and then posttranslationally the carboxylterminus becomes reoriented to the cytoplasmic face of thebilayer. As the glycoprotein moves from the endoplasmicreticulum to the Golgi, palmitate groups are added to cys-teines in the E2 protein, but the specific sites have not beendetermined (14, 22). Fatty acylation of the two cysteinesclose to the carboxyl terminus of E2 might be coupled to thereorientation of the 28-amino-acid portion of E2. To deter-mine whether these amino acids were, in fact, sites ofacylation, we used site-directed mutagenesis.The ability to use reverse genetics and site-directed mu-

tagenesis became available for Sindbis virus as a result of thesuccessful construction of a cDNA encoding the entiregenomic sequence of the virus and the demonstration thatRNA transcribed in vitro from the cDNA could be trans-fected into cells and produce infectious progeny virions (17).In our initial studies, mutations were made to replace singleamino acids in a part of the cytoplasmic domain of E2 thatwas highly conserved among several related alphaviruses.

* Corresponding author.

One double mutant was constructed to replace the twoconserved adjacent cysteines positioned close to the car-boxyl terminus of the protein (5) because we had shown thatthe Sindbis virus E2 protein was posttranslationally modifiedin its cytoplasmic domain with three fatty acids that ap-peared to be linked covalently via thioester bonds (14, 22).Furthermore, blocking the fatty acylation reaction inhibitedvirus assembly and budding (21). However, the doublecysteine mutant was not viable: it produced no progeny eventhough the transfected RNA made virus structural proteins(5). A new set of mutants were constructed to replace thecysteines individually, and these mutated RNAs proved tobe viable. Here, we describe the phenotypes of these twomutations and show that they are deficient in fatty acylgroups. In addition, we prepared four other mutants withchanges in conserved prolines in the E2 sequence and at onesite where a small hydrophobic residue, alanine, was substi-tuted with either isoleucine or lysine.

MATERIALS AND METHODS

Materials. Secondary cultures of chicken embryo fibro-blasts (CEFs) were grown in Earl's minimal essential me-dium supplemented with 3% fetal bovine serum. The babyhamster kidney (BHK21) cell line was grown in the samemedium supplemented with 10% fetal bovine serum, and theC7-10 mosquito cell line was grown in Dulbecco's modifiedEagle's medium supplemented with nonessential amino ac-ids and 10% fetal bovine serum. The plasmids containing theentire Sindbis virus genome were kindly supplied by C. Riceand have been described previously (4). Restriction enzymesand reagents for recombinant DNA experiments were fromNew England Biolabs and Bethesda Research LaboratoriesLife Technologies, Inc., and were used as specified by themanufacturer. All isotopes were from Amersham, Inc. Oli-gonucleotides for mutant preparations were obtained fromthe Howard Hughes Medical Institute, Washington Univer-sity School of Medicine.

Site-directed mutagenesis and isolation of mutants. Theprocedures for preparing single site-directed mutations in theE2 cytoplasmic domain were identical to those previously

2546

ACYLATION SITES IN SINDBIS VIRUS E2 GLYCOPROTEIN 2547

MUTANT SEQUENCE

395 423ECLTPYALAPNAVIPTSLALLCCVRSANA

P399 G .....GA401I ......

A401K K...P404G .G...C416A AC417A ......A

FIG. 1. Amino acid sequence of the cytoplasmic domain of theSindbis virus E2 glycoprotein and replacements by site-directedmutagenesis. The numbering is based on the sequence published byStrauss and Strauss (23).

viable. The specific infectivity of transfected mutant RNAsranged from 2 x 106 to 6 x 106 PFU/ng of RNA for five of thesix mutants; it was 2 x 105 PFU/ng of RNA for mutantC417A, and the plaques for this transfected RNA weresmall. Plaque-purified virus was then obtained for eachmutant and tested for growth potential on secondary CEFs,BHK21 cells, and C7-10 mosquito cells. From these results,

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described in detail (4, 10, 15). The portion of the DNA (577nucleotides) containing the mutation and subsequently in-serted into the virus cDNA was sequenced to confirm thatonly the desired mutation was formed (19). Linearized viruscDNA was transcribed into genome-length RNA from theSP6 promoter, isolated, and used for transfection experi-ments as previously described (4, 5). Mutant strains wereplaque purified, and then their properties were examined.Virus growth and labeling of cells. In most experiments,

monolayers of ca. 106 cells, grown almost to confluence,were infected with virus at a multiplicity of infection of 20.Infected cells were usually labeled with 10 to 25 ,uCi of the[35S]methionine or [35S]cysteine isotope in a medium lackingeither methionine or cysteine. For [3H]palmitate labeling,the fatty acid was dried under a stream of nitrogen to removethe solvent and resuspended in the complete culture me-dium. After a labeling period, the medium was collected andcell monolayers were washed thoroughly with cold physio-logical saline, scraped, and collected by pelleting in a micro-centrifuge tube. The pellets were boiled in 1% sodiumdodecyl sulfate (SDS), and extracts were applied to gelsdirectly or diluted 10-fold for precipitation with antibodiesby previously described methods (4, 5). Extracellular viruswas collected by adsorption to Cellufine (Amicon, Inc.) and,after being thoroughly washed, released by boiling the resinwith small volumes of gel-loading buffer, as described pre-viously (4, 5).

Gel electrophoresis and quantitation of radioactive proteinbands. Samples of SDS-denatured, labeled proteins wereseparated by SDS-polyacrylamide electrophoresis (PAGE)(11), and fluorographs were prepared. The locations ofprotein bands in gels were identified from the fluorograms,and the bands were excised from the gels for scintillationcounting. This method of quantitation was determined tohave an accuracy of ±5%. In the experiments to measurelevels of [35S]cysteine and [3H]palmitate, quantitation ofradioactive proteins was determined by densitometry of thefluorograms.

RESULTS

Sites of mutations and growth characteristics. The sixmutations studied here are noted in Fig. 1; they include thesingle-site substitution of cysteines 416 and 417 to alanine,prolines 399 and 404 to glycine, and alanine 401 to eitherisoleucine or lysine. Transfection of RNAs with these mu-tations produced progeny viruses that were phenotypicallydistinct from the wild-type virus. Two in vitro transcriptswere prepared from each mutated cDNA, and three separatetransfections were carried out to ensure that mutants were

2 4 6 8Time in hours

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from infected primary CEFs (A), BHK21 cells (B), and C7-10mosquito cells (C). Monolayers were infected at a multiplicity ofinfection of 20, and titers were determined at the different timespostinfection by plaque formation on CEFs. Symbols: 0, C417A;------, C416A; , wild type; o, P399G; * , P404G; *, A401I;O, A401K.

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2548 IVANOVA AND SCHLESINGER

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FIG. 3. Palmitoylation of Sindbis virus glycoproteins in wild-type and mutant virus-infected cells. CEFs were labeled for 15 minat 4 h postinfection. Extracts were analyzed by SDS-PAGE, andradioactivity in bands was quantitated by densitometry. The valuesare expressed as relative amounts of radioactivity incorporated intothe specific protein band. Symbols: *, wild type; E:, C416A; E0,C417A; X, P399G; X, P404G.

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FIG. 4. Intracellular formation of virus-specific proteins in CEFslabeled with [35S]methionine. Cells (ca. 106) were infected at amultiplicity of infection of 20 and labeled for 15 min with 20 ,uCi of[35S]methionine (A). Extracts were prepared and analyzed by SDS-PAGE (10% polyacrylamide) followed by fluorography. In separatemonolayers, the label was removed and extracts were prepared aftera 60-min chase (B). Numbers above the lanes indicate the mutants.The Sindbis virus p62, El, E2, and capsid are noted, and molecularmass markers are shown. C, capsid.

the mutants could be divided into three classes. Class Iincluded mutants P399G, P404G, A401I, and A401K, whichreleased almost normal amounts of infectious virus frominfected CEFs or BHK21 cells but slightly smaller amountsfrom infected C7-10 cells grown at 30°C. Class II includedthe single mutant C416A, which released virus somewhatmore slowly than the other mutants and wild-type virus frominfected CEFs and BHK21 cells and was much slower thanthese when grown on C7-10 cells. Class III included themutant C417A, which was highly defective in virus releasefrom infected CEFs, BHK21 cells, and C7-10 cells (Fig. 2).This last mutant also produced small plaques on CEFs,consistent with its slower growth in these cells. A moredetailed analysis of the growth of these six mutants on CEFsas determined by the rate of infectious virus release, re-corded as PFU per hour, showed that all mutants formedvirus somewhat more slowly than the wild type prior to 6 to7 h postinfection but that only the class III mutant, C417A,persisted in slow virus release beyond this time (data notshown).

Identification of cysteines as palmitoylation sites. Fromearlier studies our laboratory postulated that cysteines in thecytoplasm might be sites for posttranslational attachment of

TABLE 1. Analysis of palmitoylation in wild-type and mutantSindbis virus glycoproteinsa

Ratio of radioactivity in":

Virus p62/El p62/El normalized toVirus ~~~~~~~~~~~wildtypeCysteine Palmitic Cytie Palmitic

Cysteine acid Cysteine acid

Wild type 0.9 1.6 1.0 1.0C416A 0.7 0.7 0.8 0.4C417A 0.6 0.6 0.7 0.4P399G 0.9 1.7 1.0 1.1P404G 0.9 1.8 1.0 1.1

a Extracts from infected cells labeled for 15 min with either [35S]cysteine or[3H]palmitic acid were subjected to SDS-PAGE.

b The radioactivity in protein bands corresponding to p62, El, and E2 wasquantitatively evaluated by densitometric analysis of fluorograms. The rela-tive values for cysteine and palmitate are noted in Fig. 3.

palmitic acid (14, 22). In our initial attempts to replace thesecysteines, we prepared a double mutant lacking both cys-teine 416 and 417, but this mutant was unable to produceprogeny virus (5). The single mutations, however, wereviable, and cells infected with the mutants could be analyzedfor the amounts of palmitate and cysteine in the glycopro-teins. The data are shown in Fig. 3, and the analysis ispresented in Table 1. Values were expressed on the basis ofthe amounts of labeled cysteine and palmitic acid in El; thisglycoprotein was not altered in these mutants (see below).We normalized these ratios to that of the wild type and foundthat both cysteine mutants had about half as much label inpalmitate as the wild type did (Table 1). On the basis ofprevious data indicating three acylation sites in the p62cytoplasmic domain (14), we would have expected a valuecloser to 0.67. The lower, measured value may be due to anallosteric effect of the mutation; i.e., the substituted aminoacid could affect this part of the polypeptide such that theadjacent cysteine was unavailable for palmitoylation. Thenumber of cysteines should be about 6% smaller than in the

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FIG. 5. Formation of intracellular and extracellular virus pro-teins in CEFs infected with wild-type and mutant viruses. Infectedcells were labeled with [35S]methionine for 15 min at 3 h postinfec-tion and then chased for 60 min. Details are given in Materials andMethods. The total amounts of virus-labeled proteins include thep62, El, E2, and capsid polypeptides, determined by quantitation ofprotein bands separated by SDS-PAGE. Symbols: *, wild type;E3, P399G; El, A4011; 03, A401K; El, P404G; *, C416A; E,C417A.

J. VIROL.

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ACYLATION SITES IN SINDBIS VIRUS E2 GLYCOPROTEIN 2549

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A4011. The electron-microscopic analysis was performed by Marilyn Aach Levy, Department of Cell Biology and Physiology, WashingtonUniversity School of Medicine. Arrows indicate multicored virus particles. Bar, 0.2 p.m.

wild type (1 fewer cysteine out of a total of 17 in the entireprotein), and our data gave a somewhat greater value. Twoother mutants with substitutions for proline were similar towild-type virus in ratios of cysteine and palmitate label.

Other phenotypes of these mutants. The effect of these twocysteine substitutions and the four other mutations withinthe cytoplasmic domain of E2 were tested by labelinginfected cells with [35S]methionine or [35S]cysteine for eithera 15-min pulse or a 15-min pulse plus a 60-min chase (Fig. 4Aand B, respectively). When compared with the wild-type

virus pattern of glycoproteins, p62 and El, the mutantsshowed an additional slower-migrating band of about 65kDa. This band was immunoprecipitated by an anti-E2antibody but not an anti-El antibody (data not shown), andwe postulated that it was an uncleaved p62-6K protein. Aftera 60-min chase, the 65-kDa band in C417A accounted for 20to 25% of the total label in the glycoproteins. For wild-typevirus this value was only 2 to 3% of the total glycoproteinlabel. When measured by continuous incorporation of[35S]methionine over a 6-h period, the 65-kDa protein accu-

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2550 IVANOVA AND SCHLESINGER

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ER Membrane Palmitoylation

Cytoplasm NH3 ++ NH3COO-

FIG. 7. Postulated model of reorientation of the Sindbis virus E2cytoplasmic domain. Symbols: 0, *, normal and acylated cys-teines, respectively; 4, palmitoyl groups. Abbreviation: ER, en-

doplasmic reticulum.

mulated in all mutants, although the levels were much higherin the C417A mutant (data not shown). Very little of thisprotein accumulated in wild-type virus-infected cells underthese conditions. Also after the chase, the rate of E2formation in cells infected with C417A was 45 to 50% thatmeasured for wild-type virus. The other five mutants showeda rate of E2 formation not significantly different from that ofwild-type virus. Mutant-infected cells also showed accumu-lation of small amounts (3 to 7%) of a larger unprocessedSindbis virus polyprotein consisting of p62-6K-El, whereaswild-type virus showed less than 2% of this protein (theexposure of the fluorograms depicted in Fig. 4 was too shortto reveal this band).The amounts of virus proteins formed intracellularly and

the amounts released into the culture medium during a60-min chase following a 15-min pulse of [35S]methioninelabel are displayed in Fig. 5. It is clear that all mutantssecreted fewer particles than the wild-type virus, and, con-comitantly, there was an accumulation of virus-specificproteins intracellularly in the mutant-infected cells. The viralparticles secreted from mutant virus-infected cells wereexamined by electron microscopy. An electron micrographof a thin section from cells infected with the mutant A401I isshown in Fig. 6. In contrast to the single-cored virusesassembled and secreted from cells infected with wild-typevirus, this mutant produced larger, aberrant, multicoredvirions. Similar kinds of aberrant particles were observed forthe other five mutants studied in this work, as well as thosedescribed in previous studies from this laboratory (4, 5).These multicored particles apparently were infectious sincethe amounts of infectious virus formed from most of themutant-infected cells were almost equivalent to those fromwild-type-infected cells. In addition, the infectious virusreleased from mutant-infected cells was considerably morethermostable than wild-type virus; for example, when heatedat 56°C for 20 min, wild-type virus titers dropped by 3 to 4log units but titers of all mutant viruses dropped by less than1 to 2 log units. A kinetic analysis of these virus titer datashowed that thermal inactivation of the mutant viruses wasconsistent with a multihit mechanism and supported our

hypothesis that the multicored particles were infectious(data not shown). The kinetics of thermal inactivation ofwild-type virus obeyed a single-hit kinetic curve (4).

DISCUSSION

The data presented in this study established that the twocysteines close to the carboxyl terminus of the E2 proteinwere sites of palmitoylation. These amino acids are con-

served among five strains of alphaviruses sequenced thus far(3, 6, 7, 9, 12), indicating that they are critical for proteinfunction. From our earlier work, we showed that a cysteine

at position 396 was also palmitoylated (5). These three sitesaccount for the three palmitic acids that were reported to beposttranslationally added to the E2 glycoprotein (14, 22).What might be the function of this protein modification?

We postulate that fatty acylation may accompany the reori-entation of the carboxyl terminus of the E2 protein from itsinitial insertion into the endoplasmic reticulum bilayer andanchor the 35-amino-acid polypeptide to the cytoplasmicface of the membrane. The fatty acids would be expected topartition in the bilayer, and the three acylation sites-onenear the transmembrane portion and other two near thecarboxyl terminus-would thus act to constrain the polypep-tide conformation (Fig. 7). In Fig. 7, the stretch of hydro-phobic acids between the anchored sites is postulated toform a ligand recognized by the nucleocapsid during virusassembly but might also partition into the cytoplasmic faceof the bilayer and affect lipid-protein structures.The replacement of cysteines also strongly inhibited virus

particle secretion from insect cells, and the mutation atcysteine 417 showed a similar phenotype on CEFs andBHK21 cells. This defect could be attributed to a block inthe proteolytic processing by signalase, which separates p62from 6K during translation of the polyprotein. We do nothave a good explanation for this effect -the cysteines are toofar from the cleavage site of the protease to directly alter thesubstrate site of this enzyme. Perhaps the conformation ofthe polypeptide has been altered, either by loss of thecysteine or by the presence of the alanine, so as to mask theprotease site or to prevent the reinsertion of the polypeptideinto the membrane. In our earlier study, we found that themutation S420C, which is also in the carboxyl portion of theE2 sequence, affected the p62-6K signalase cleavage (5).Thus, the sequence in this part of the E2 sequence is quitesensitive to cotranslational proteolytic events.The four other site-directed mutations, which are within a

conserved hydrophobic portion of the E2 cytoplasmic do-main, affected virus budding in a manner much like thatpreviously detected with site-directed mutations in the cyto-plasmic domain of E2 (5). We had suggested that thesechanges slightly alter the conformation of this portion of theprotein such that interactions between glycoprotein andnucleocapsids are weakened. As a result, the envelopmentof membrane around the core, which drives the buddingprocess, is aberrant and produces the multicored, largermembranous virions observed in the electron micrographs.In our earlier studies we also showed that these particles areinfectious (4, 5).The slower growth of all of these mutants in the insect cell

line is a phenotypic property that has proved useful forisolating revertants. The analysis of revertants may provideimportant data, leading to a more complete molecular de-scription of the assembly process for this enveloped virus.

ACKNOWLEDGMENTS

We thank Marilyn Aach Levy, Department of Cell Biology andPhysiology, Washington University School of Medicine, for prepar-ing the electron micrographs and Rebecca Moran for technicalassistance with tissue culture experiments.

This study was supported by grant Al 19494 from the U.S. PublicHealth Service.

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