Role of Cell Surface Spikes in Alphavirus Budding

Transcription

1 JOURNAL OF VIROLOGY, Dec. 1992, p X/92/ $02.00/0 Copyright 1992, Americn Society for Microiology Vol. 66, No. 12 Role of Cell Surfce Spikes in Alphvirus Budding HONGXING ZHAO AND HENRIK GAROFF* Deprtment ofmoleculr Biology, Krolinsk Institute, S Huddinge, Sweden Received 29 June 1992/Accepted 8 Septemer 1992 Alphviruses mture y udding t cell surfces. According to previling hypothesis, the virl memrne protein, which is heterodimeric protein unit, is trnsported to the plsm memrne (PM), where it wits inding to the virl nucleocpsid (NC). This hypothesis predicts tht the virl memrne protein heterodimers ccumulte t the cell surfce when expressed in the sence of NCs. We hve tested this prediction y nlyzing the spike protein expression phenotype of Semliki Forest virus (SFV) vrint which contins cpsid gene deletion. We found tht virl memrne protein heterodimers were formed nd trnsported to the cell surfce normlly. However, insted of ccumulting t the PM s expected, the memrne proteins were rpidly degrded. In the cse of the El suunit, degrdtion resulted in the relese of solule El frgment into the medium. The fct tht this pthwy of protein degrdtion is mostly inhiited during wild-type virus infection suggests tht virl memrne proteins re very efficiently cptured y NCs into udding complexes nd tht normlly no sizele pool of free memrne protein complexes exists t the PM. Most if not ll enveloped viruses of niml cells mture y udding t cellulr memrnes. Some viruses, such s influenz virus, vesiculr stomtitis virus, nd vrious retroviruses nd lphviruses use the plsm memrne (PM), wheres others, such s coronviruses nd unyviruses, prefer intrcellulr memrnes tht line the iosynthetic trnsport route of the infected cell (5, 15, 18). According to the previling hypothesis of virus udding, the site of virus mturtion is determined y the locliztion of spike proteins nd the driving force for the udding process y the interction of the trnsmemrne spike proteins with the virus core or nucleocpsid (NC) (5, 15, 18). While there re mny experimentl results supporting udding site-specific locliztion phenotypes of virus spike proteins, until recently there hs een no direct proof for the core-spike interction nd its role in virus udding (15). The successful construction of complete nd infectious DNA clones of some enveloped DNA nd positive-strnded RNA viruses hs now mde it possile to test this spect of the udding model. So fr, it is cler tht hepdnviruses (heptitis B virus) nd lphviruses (Semliki Forest virus [SFV]) conform strictly to the proposed model, wheres retroviruses do not (2, 19). Expression experiments using env gene deletion vrints of vrious retroviruses show clerly tht the gg gene encoding the virl core is sufficient for supporting efficient memrne udding (4, 10, 16). However, prt from the questions out the udding site nd the driving force for virus udding, there re mny other spects of this process tht re importnt nd still unresolved, such s the identities of the regions of interction etween the vrious virl suunits during udding nd how the expression of these inding functions is controlled in time nd spce. We re interested in the udding process of lphviruses nd use SFV s our model. In this investigtion, we hve studied the importnce of the PM pool of virl spike proteins for virus mturtion. As SFV udding is driven y spike protein-nc interctions t the cell surfce, it is resonle to elieve tht spike proteins which hve een trnsported to the cell surfce will form pool of ssemly-competent suunits witing to e recognized y cytoplsmic NCs. The * Corresponding uthor spike protein of SFV is mde s heterodimeric polypeptide complex (p62-el) from common mrna which lso encodes the cpsid (C) protein (7, 20, 21). The p62-el oligomer is trnsported to the cell surfce, ut efore rriving t the PM, it mtures (y limited proteolysis of p62) into n E2-E1 complex which is used for prticle formtion (3). Therefore, one would predict from the SFV udding model descried ove tht expression of spike proteins lone should result in the ccumultion of E2-El heterodimers on the cell surfce. In this study, we hve tested this prediction y nlyzing the expression of the spike protein in n SFV vrint tht lcks the C gene (SFV-spike virus). We find tht spike proteins of this vrint virus re synthesized normlly nd trnsported to the cell surfce. However, in the sence of virus udding, the spike protein heterodimers do not ccumulte on the PM s expected ut re sujected to rpid processing y proteolysis. As this pthwy is voided during wild-type virus udding, it ppers tht during norml infection there is very efficient inding of the spike proteins to the NC very soon fter they hve rrived to the PM or possily even efore this stge. MATERIALS AND METHODS Mterils. Medi nd regents for cell culture were purchsed from GIBCO Lortories Life Technologies Ltd., Pisley, Scotlnd. The monoclonl ntiodies ginst p62/e2 (UM 5.1) nd El (UM 8.139) were used s scites fluid preprtions nd hve een descried previously (1, 20). [35S]methionine ws from Amershm Interntionl, Amershm, United Kingdom. Sulfosuccinimidyl-6-(iotin-mido) hexnote (NHS-LC-iotin) nd sulfosuccinimidyl 2-(iotinmido) ethyl-1,3-dithiopropionte (NHS-SS-iotin) were purchsed from Pierce Chemicl Co., Rockford Ill. Redyto-use streptvidin-grose, phenylmethylsulfonyl fluoride (PMSF), nd soyen trypsin inhiitor were from Sigm Chemicl Co., St. Louis, Mo. Proteinse K ws from Bethesd Reserch Lortories, Githersurg, Md., nd trypsin ws from Boehringer Mnnheim Corp., Indinpolis, Ind. Protein A-Sephrose ws from Phrmci LKB Biotechnology, Uppsl, Sweden. Cells nd viruses. Stocks of wild-type SFV nd [35S]methionine-leled SFV were propgted in BHK-21 cells s Downloded from on Octoer 23, 2018 y guest

2 7090 ZHAO AND GAROFF descried previously (20). Preprtion of recominnt SFV-TR hs een descried elsewhere (11). BHK-21 cells were grown in BHK medium supplemented with 5% fetl clf serum, 10% tryptose phosphte roth, 2 mm glutmine, nd 20 mm N-2-hydroxyethylpiperzine-N'-2-ethnesulfonic cid (HEPES). DNA constructs. Deletion of the C gene from the 5' region of the SFV structurl gene hs een descried previously (6, 19). This process involved synthesis of gene which encompsses, from 5' to 3', n EcoRI site, the C protein inititor codon together with upstrem flnking sequences, nd the coding sequence for the first 40 codons of the p62 gene, including the XhoI site. This DNA frgment ws isolted from plsmid p62 dhfr nd used to replce the 5' prt of the structurl gene region within the EcoRI(7391)-XhoI(8338) frgment of psp6-sfv4 (6). The ltter plsmid represents n SP6-sed trnscription plsmid tht hrors the full-length cdna copy of the SFV genome (11). The new plsmid, psp6-sfv-spike, cn e used for in vitro trnscription of SFV-spike RNA. This RNA is repliction competent when introduced into the cell cytoplsm nd produces truncted sugenome tht is trnslted into memrne polyprotein sequence without preceding C protein sequence s occurs normlly in the cse of the wild-type 26S sugenomic mrna (19). The DNA sucloning steps were done ccording to stndrd techniques (13). Preprtion of SFV-spike virus stock. For RNA trnscription, 5 p,g of psp6-sfv-spike DNA ws used in 50-,u rections s descried previously (11). One-fifth of the trnscription mixture nd the corresponding mount of helper RNA were used for trnsfection of 5 x 106 BHK-21 cells y electroportion for the production of SFV-spike stock virus. In cotrnsfected cells, SFV-spike RNA specifies polymerse production nd will replicte oth SFV-spike RNA s well s helper RNA. The helper RNA is used for synthesis of virl structurl proteins. These pckge SFVspike RNA into virus prticles. However, helper RNA itself is not used for pckging ecuse it lcks functionl encpsidtion signl. Recominnt viruses were isolted from culture medi y centrifugtion t 30,000 rpm in n SW41 Ti rotor t 4 C for 90 min, nd the pellet ws suspended in TNE uffer (50 mm Tris [ph 7.4], 100 mm NCl, 0.5 mm EDTA). One trnsfection yielded totl of 109 SFV-spike infectious virus prticles in the culture medium. Titers were determined y indirect immunofluorescence stining (with nti-el ntiody) of BHK-21 cells infected with dilutions of virus stocks. Pulse-leling of intrcellulr proteins nd immunoprecipittion. BHK-21 cell monolyers in 35-mm-dimeter dishes were infected with 50 PFU of wild-type SFV per ml or 50 infectious units of recominnt SFV (SFV-spike or SFV-TR) per ml. After 60 min of dsorption t 37 C, the medium ws replced with fresh Egle's minimum essentil medium (EMEM). At S h fter infection, the cells were strved with methionine-free EMEM for 30 min nd pulsed with 50,uCi of [35S]methionine in 500,ul of methionine-free medium for 10 min. Cells were wshed twice with 100-fold-excess methionine nd chsed for vrious times with EMEM contining 10 times the norml concentrtion of methionine. Cells were lysed with Nonidet P-40 (NP-40) lysis uffer (1% NP-40, 50 mm Tris [ph 7.4], 150 mm NCl, 2 mm EDTA, 1 jig PMSF of per ml) nd immunoprecipitted with monoclonl ntiodies ginst the El nd E2 suunits s descried previously (20). Finlly, smples were processed for sodium dodecyl sulfte (SDS)-polycrylmide gel electrophoresis (PAGE). J. VIROL. Leling of virl proteins t the cell surfce. Leling conditions were optimized y using [35S]methionine-leled SFV. Trce mounts of leled virus were mixed with 2.5,ug of cold virus nd rected with the iotin regent NHS-LCiotin t different concentrtions in 240,ul of cold phosphteuffered sline contining C2' nd Mg"+ (PBS+) for 30 min t 4 C. Excess PBS+ ws dded, nd the virus ws isolted y centrifugtion in Ti75 rotor (Beckmn) t 35,000 rpm for 45 min t 4 C. After wshing wy of residul regent with PBS+ nd recentrifugtion of the virus, pellets were soluilized in 50,1u of 1% SDS in PBS+. One-fourth of smples were used s virus protein controls for SDS-PAGE. To the rest, 300,ul of NP-40 lysis uffer ws dded together with 40 p,l of 50% (vol/vol) streptvidin-grose slurry, nd the mixture ws incuted with shking t 4 C for either 2 h or overnight. Streptvidin-grose ws then pelleted y centrifugtion in Eppendorf microcentrifuge t 10,000 rpm for 3 min t 4 C, nd superntnt frctions were used for second round streptvidin-grose precipittion under the conditions descried ove. Pellets were wshed twice with wsh uffer A (0.2% NP-40, 10 mm Tris hydrochloride [ph 7.4], 150 mm NCl, 2 mm EDTA), twice with wsh uffer B (0.2% NP-40, 10 mm Tris hydrochloride [ph 7.4], 500 mm NCl, 2 mm EDTA), once with uffer C (10 mm Tris hydrochloride [ph 7.4]), nd finlly once with 1% SDS in uffer C. Adsored proteins were relesed from streptvidin y incution t 95 C in SDS-PAGE smple uffer (0.1 M Tris hydrochloride [ph 8.8], 0.5 M sucrose, 0.02% romphenol lue, 5 mm EDTA [ph 8.8], 10 mg of methionine per ml, 4% SDS) for vrious times. The preliminry surfce leling tests showed tht efficient leling required the use of 1 mg of iotin regent per ml in 30-min incution t 4 C, tht efficient cpturing of iotinylted proteins needed one incution with 40,ul of 50% streptvidin-grose slurry for more thn 2 h, nd tht optiml elution from the ltter ws otined during 10-min incution with SDS-PAGE smple uffer t 95 C. However, these conditions were optiml only for leling of the E2 suunit of the virus spike; the El suunit ws very inefficiently leled y the iotinyltion regent. For leling of virl proteins on the cell surfce fter pulse-leling, the cells were first wshed twice with PBS+ nd then incuted with iotin regent s descried ove. The rection ws locked y two wshes in 50 mm NH4C1 in PBS+ nd then wshed once in PBS+. Cells were lysed s when processed for immunoprecipittion. However, for cpturing iotin-leled proteins, streptvidin-grose ws used s descried ove. Internliztion ssys. To mesure internliztion of E2 suunits, the uptke of pulse-leled E2 suunits from the cell surfce ws monitored fter different times of incution t 37 C. SFV-spike virus-infected cells in severl prllel dishes were pulse-leled for 20 min nd chsed for 60 min. [35S]methionine-leled virl proteins which hd reched the cell surfce were then rected with the iotin regent t 4 C s descried ove. After termintion of the iotinyltion rection, cell surfce proteins were llowed to internlize y incution for different times t 37 C fter the ddition of 2 ml of prewrmed EMEM. Remining virl spike proteins on the cell surfce were removed y digestion with proteinse K (1 mg/ml in PBS+) for 1 h on ice. Proteinse K ws inhiited y ddition of PMSF (0.5 mg/ml), nd cells were processed for streptvidin-grose precipittion. In these internliztion experiments, the humn trnsferrin receptor (htr) molecule ws used s control. htr ws expressed in BHK-21 cells y using the psfvl-tr-derived recominnt Downloded from on Octoer 23, 2018 y guest

3 VOL. 66, 1992 virus SFV-TR (11) nd ws leled with the iotin regent NHS-SS-iotin insted of NHS-LC-iotin, which ws used for the spike proteins. For removl of iotin-leled htr remining on the cell surfce fter37 C incutions, we used trypsin digestion (0.5 mg/ml in PBS+) for 1 h on ice. Trypsin ws inctivted y ddition of soyen trypsin inhiitor (1 mg/ml). Leled htr ws relesed from the streptvidingrose y heting to95 C for 10 min in SDS-PAGE smple uffer contining 50 mm dithiothreitol, then 1/10 volume of 1 M iodocetmide ws dded, nd smples were nlyzed y electrophoresis. Other methods. SDS-polycrylmide gels composed of 10% seprting gel nd 5% stcking gel were prepred nd processed for utordiogrphy s descried previously (12). Rdioctivity in protein nds in the gel ws quntitted s descried elsewhere (20). All vlues otined were normlized to the methionine content of the respective polypeptide chin (7). For trichlorocetic cid (TCA) precipittion, the culture fluid with viruses or virl memrne protein frgments ws precipitted with 10% TCA nd kept on ice for 1 h. Smples were sujected to centrifugtion (14,000 rpm) for 15 min t4 C. Pellets were dissolved in SDS-PAGE smple uffer y soniction for 5 min nd heting t95 C for 5 min. RESULTS Spike protein synthesis nd posttrnsltion modifictions in SFV-spike virus-infected cells. The spike proteins of SFV re normlly trnslted fter the C-coding region from the 26S mrna (7). After synthesis, the C chin is relesed from the nscent chin y utoproteolysis. This process revels the N terminus of the p62 chin, which cts s signl sequence nd directs the riosome to the endoplsmic reticulum memrne for synthesis of the SFV spike proteins. In the SFV-spike virus construction, the preceding C-gene region ws deleted, nd we wished to determine whether this deletion would hve ny effect on the synthesis nd mturtion of the SFV spike proteins. To this end, we used psp6-sfv-spike DNA for the production of n SFV-spike virus stock preprtion, which ws then used to infect BHK-21 cells. Protein synthesis ws nlyzed y pulseleling with [35S]methionine. Figure l shows the spike proteins immunoprecipitted from cell lystes tht hd een chsed for different times. El nd p62 polypeptides were produced efficiently from the SFV-spike genome, nd p62 ws lso efficiently cleved to E2. Figure l shows results for the wild-type virus control. In Fig. lc, the p62 clevge efficiencies of SFV-spike virus nd the wild-type virus pper to e identicl. The migrtion rte of El from the SFV-spike construct decresed slightly t lter chse times in n SDS-polycrylmide gel (Fig. l); this is typicl for the El of wild-type SFV nd hs een shown to result from processing of its sugr unit when routed through the Golgi complex (8). In nother experiment, p62 nd El from oth SFV-spike virus- nd wild-type virus-infected cell smples were nlyzed next to ech other in SDS-polycrylmide gels nd shown to hve the sme moility (dt not shown). Therefore, we conclude tht the spike proteins p62 nd El were correctly produced from the SFV-spike construct. However, it should e noted tht the overll level of spike protein synthesis in SFV-spike virus-infected cells never reched tht produced during wild-type virus infection. This level ws out one-third of tht of the wild-type when 50 infectious units per cell ws used. The reson for this finding is uncler. Another interesting feture of the SFV-spike virus infection is tht newly synthesized spike protein su-. S =,. 5 > r ALPHAVIRUS ASSEMBLY C Dr J C C6C -C.A E-_ -UUM M' :.J ~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ FIG. 1. Synthesis nd posttrnsltionl modifictions of spike proteins produced from SFV-spike virus-infected cells. Monolyers of BHK-21 cells in 35-mm-dimeter dishes were infected with SFV-spike () or control () virus nd pulse-leled. After the indicted chse times, cell lystes were prepred nd spike proteins (p62, E2, nd El) were immunoprecipitted with monoclonl ntiodies ginst p62/e2 nd El suunits. The immunoprecipittes were nlyzed on n SDS-10% polycrylmide gel. Gels were first used for utordiogrphy ( nd ) nd then for quntittion of spike proteins in the respective nds (c). Quntittion of p62 clevge (c) is expressed s the percentge of p62 in the totl of p62 plus E2 in ech smple. Wt, wild-type virus. units, prticulrly the E2 suunits, decresed significntly in quntity during the chse, lthough virus udding ws not tking plce. Formtion of heterodimers. The formtion of p62-el heterodimeric complexes hs een shown to e very efficient process which tkes plce very soon fter completion of trnsltion nd trnsloction of the corresponding polypeptide chins in the endoplsmic reticulum (20, 21). This noncovlent protein complex is not dissocited y NP-40 lysis uffer nd cn e demonstrted y coimmunoprecipittion with monoclonl ntiodies ginst either p62 or El. In the experiment shown in Fig. 2, we used monoclonl ntiody ginst the El suunit to nlyze heterodimeriztion of newly synthesized p62 nd El suunits when expressed from the SFV-spike genome. p62 nd E2 suunits coimmunoprecipitted lmost s efficiently s did those of the wild-type virus-infected cells in smples chsed for 10 to 30 min (Fig. 2 to c). At longer chse time points, less E2 thn El ws precipitted, primrily ecuse of the preferentil loss of the E2 suunit from these cell smples (Fig. l). If one clcultes the proportion of totl p62 nd E2 which coprecipitted with El, then the decrese in coprecipitting E2 mteril is less evident (Fig. 2d). Nevertheless, the E2-El complex seems to e somewht more lile in SFV-spike virus-infected cells thn in wild-type virus-infected cells. We conclude tht spike proteins expressed from the SFV-spike virus formed p62-el complexes s efficiently s in wild-type infection ut tht the mturtion clevge rendered the former complex somewht more lile thn the ltter complex. Appernce of spike proteins on the cell surfce. The fct tht p62 nd El expressed from the SFV-spike virus formed p62-el heterodimers which mtured y clevge to E2-El Downloded from on Octoer 23, 2018 y guest

4 7092 ZHAO AND GAROFF J. VIROL. Chse Time (mirl) p62 $414 8,4 ""* E2 *..4 4* *.- * I E1 &-AI" i "_ c d StPv r1i k s... :.I;%t; II.5f i, i.(.. ~ ~ ~ ~ ~ ~ ~ ~.s I t.lnr FIG. 2. Formtion of p62-el heterodimers. Portions of the lystes of the cell smples descried in the legend to Fig. 1 were used for nti-el immunoprecipittion. Precipittes were nlyzed s descried in the legend to Fig. 1. The coimmunoprecipittion of p62/e2 with El reflects the efficiency of heterodimer formtion. ( nd ) Coimmunoprecipittion rections of wild-type virus-infected () nd SFV-spike virus-infected () cells; (c nd d) quntity of p62/e2 suunits s percentge of El suunits (c) nd quntity of p62/e2 suunits s percentge of totl p62/e2 suunits (d) present in ech cell lyste smple (see Fig. 1). Wt, wild-type virus. complexes s efficiently s did those of the wild-type virus suggested tht SFV-spike virus-derived complexes lso reched the cell surfce with wild-type efficiency nd kinetics. To confirm this conclusion, iotin-streptvidin ws used to precipitte pulse-leled virl spike proteins expressed t the surfce of SFV-spike virus- nd wild-type virus-infected cells. Biotin leling conditions were optimized with [35S] leled SFV s descried in Mterils nd Methods. Under these conditions, E2 suunits were quntittively recovered from soluilized SFV. It should e noted tht iotinyltion of El ws very inefficient, nd hence this protein could not e quntitted y this ssy. To monitor trnsport of newly synthesized spike proteins to the cell surfce in oth SFVspike virus- nd wild-type virus-infected cells, BHK-21 cell monolyers were infected in prllel, leled, nd chsed for vrious times efore iotinyltion t 4 C for 30 min. Cell smples were soluilized with NP-40 lysis uffer, nd iotinleled virus proteins were precipitted with streptvidingrose. Figures 3 nd show SDS-PAGE nlyses of precipitted mteril; Fig. 3c shows the quntity of E2 suunits on the cell surfce. It ws evident tht in oth SFV-spike virus- nd wild-type virus-infected cells, E2 suunits ppered t the cell surfce fter 30-min chse. Mximl ppernce (40% of totl cell-ssocited E2 in wild-type virus nd 30% in SFV-spike virus) ws reched t 60 min of chse. With longer chse times, the mount of E2 expressed on the cell surfce decresed. This decrese ws much fster for SFV-spike virus-infected cells thn for wild-type virus-infected cells. We conclude tht the heterodimeric spike complex of SFV-spike virus-infected cells ws trnsported to the cell surfce with norml kinetics nd lmost norml efficiency. However, E2 ppered to e removed from the cell surfce soon fter its rrivl (tl,2 of out 60 min). Relese of spike protein frgments from the cell surfce into medium. Anlysis of the mount of virl proteins in wildtype virus-infected cells nd medi showed tht the decrese in E2 nd El in cell lystes with time correlted well with the relese of spikes into medi (quntittion not shown). As demonstrted in Fig. 4, spike proteins re initilly relesed t 30 min of chse. The mount of pulse-leled spike proteins in the medium incresed rpidly during the following 30 min, corresponding to out 20% of totl leled El nd E2. During this time, most of the leled spikes rrive t the PM (Fig. 3). The relese of E2 nd El continued linerly up to the lst chse time point determined (180 min). By 3 h of chse, 50% of totl leled spike proteins were found in Chse Ti e rrm rn. E OO A'W # *. c ~~~~~~~~~~ FIG. 3. Cell surfce ppernce of spike proteins. Cels were infected, pulse-leled, nd chsed s descried in legend to Fig. 1. At the indicted chse times, cells were processed for surfce leling with iotin. After soluiliztion with NP-40 lysis uffer, iotinylted proteins were cptured with streptvidin-grose nd nlyzed y SDS-PAGE. Gels were used for utordiogrphy nd then for quntittion of rdioctive E2 protein in the corresponding nds. ( nd ) Leled protein of wild-type virus-infected cells () nd of SFV-spike virus-infected cells (). (c) Quntittion (surfce E2 expressed s percentge of totl p62 plus E2 in ech cell smple). Wt, wild-type virus. Downloded from on Octoer 23, 2018 y guest

5 VOL. 66, 1992 ALPHAVIRUS ASSEMBLY 7093 Chse Time Imin E2 El C W. MUo f, 1w - dels 37 C incution (min) Proteinse TR on - "- M-0 It,* o* #"$ 4 E2 c C 77 Cnse /me Sin FIG. 4. Relese of spike protein frgments into the medium of SFV-spike virus-infected cells. Portions of the medi from the infected nd pulse-leled cells descried in the legend to Fig. 1 were sujected to TCA precipittion nd nlyzed y SDS-PAGE. ( nd ) Proteins in medi from wild-type virus-infected () nd SFV-spike virus-infected () cells; (c) quntittion of virus-ssocited El nd solule El frgment (Els), expressed s percentge of totl El present in cell lyste nd medium smples. Wt, wild-type virus. the medium. SDS-PAGE nlyses shown in Fig. 4 represent totl virl protein precipitted from cell culture medi y TCA. When monoclonl ntiody ginst E2 (or El) ws used to immunoprecipitte protein from medi (in the sence of detergent), it ws evident tht the lrge mjority of spike protein ws present in virion prticles ecuse the precipitte included ll of the relesed C protein (Fig. 5). However, smll frction of leled proteins remined in the superntnt fluid fter precipittion with the nti-e2 monoclonl ntiody. This protein migrted slightly fster thn the El suunit when sujected to SDS-PAGE. It ws identified s solule El frgment y using n nti-el monoclonl ntiody (Fig. 5) nd Triton X-114 cloud point precipittion nlyses (not shown). In Fig. 4c, the relese of solule El frgment is quntified. Aout 5% of leled El ws relesed in this form from wild-type virus-infected cells during 180-min chse. In contrst, the El frgment ws C.ISse T"e E2 El C :nuu m FIG. 5. Relese of solule El frgments from wild-type virusinfected cells. Portions of the medi from the infected nd pulseleled cells descried in the legend to Fig. 1 were first used for nti-e2 monoclonl ntiody immunoprecipittion to cpture virus, nd the superntnt fluid ws used for second immunoprecipittion with the nti-el monoclonl ntiody. () Anti-E2-rective mteril (virus prticles) nlyzed y SDS-PAGE; () the remining nti-el monoclonl ntiody-rective frction, which constitutes the solule El frgment (Els). Els... - _ F FIG. 6. E2 nd endocytosis. BHK-21 cells were infected with SFV-TR virus, which expressed htr, or SFV-spike virus. After leling with [35S]methionine for 20 min nd 60-min chse, surfce proteins of infected cells were leled with iotin on ice. Internliztion ws initited y incution t 37 C for the indicted times. Remining htr molecules t the cell surfce were then removed y trypsin digestion, nd spike proteins were removed y digestion with proteinse K, oth on ice. Cells were soluilized with NP-40 lysis uffer, nd iotin-leled proteins were cptured with streptvidin-grose. ( nd ) SDS-PAGE nlyses of htr () nd of E2 (); (c) the corresponding quntittions. much more undnt in cell culture medi of SFV-spike virus-infected cells, in which no udding tkes plce (Fig. 4 nd c). Relese of the El frgment corresponded with rrivl of the spike suunits t the PM. At 180 min of chse, out 30% of totl leled El ws found in the frgment form in culture medi from SFV-spike virus-infected cells. We conclude tht during infection with wild-type virus, smll frction of El suunits ws relesed s solule El frgments. However, when spike proteins were expressed in the sence of NCs, El ws not removed y virus udding, resulting in clevge of El nd its relese from the cell surfce. The mount of El which ws relesed s solule frgments correlted roughly to the decrese in cell-ssocited El in SFV-spike virus-infected nd chsed cell smples (Fig. l). The ctul clevge event ws proly tking plce t the cell surfce, s no El frgment ws found to e cell ssocited in the pulse-chse experiments. Fte of the E2 suunit. To monitor the fte of E2 expressed from SFV-spike virus, we exmined cell culture medi for the presence of E2 frgments, using the nti-e2 monoclonl ntiody. However, no E2 frgments were precipitted with this ntiody. Therefore, we nlyzed the possiility tht E2 ws routed to the endocytosis pthwy which would led to its degrdtion. For this purpose, severl dishes of 35Sleled SFV-spike virus-infected cells were leled with iotin on ice. To determine whether the surfce-leled E2 ws tken up y endocytosis, cell monolyers were wrmed to 37 C nd incuted for different times. E2 remining t the PM ws removed y digestion with proteinse K, nd internlized mteril ws quntified y streptvidin-grose precipittion (Fig. 6 nd c). There ws very inefficient uptke of E2 from the cell surfce, s proteinse K removed most of the iotin-leled E2 on the cell surfce in ll Downloded from on Octoer 23, 2018 y guest

6 7094 ZHAO AND GAROFF smples. In contrst, the control (htr) underwent very rpid nd efficient endocytosis, s expected (Fig. 6 nd c). We conclude tht E2 ws not tken up y rpid endocytosis s ws htr. One possiility is tht it ws cleved into severl frgments which were relesed into the medium ut could not e detected y our immunoprecipittion nd SDS-PAGE ssys. DISCUSSION In this study, we hve shown tht when spike protein heterodimers were expressed in the sence of C proteins, the heterodimers reched the cell surfce in form which ws unstle nd ws sujected to rpid removl through proteolysis. The El suunit ws relesed into the medium s solule frgment. The fte of the E2 suunit ws not possile to monitor. It could e relesed from the cell surfce s severl smller frgments. In contrst, in the cse of the wild-type virus, heterodimers were incorported into virus prticles nd only minor frction of El (nd proly lso E2) ws sujected to proteolytic removl. As spike proteins re normlly generted from polyprotein structure, the trnsltion of which strts with the C protein, one might rgue tht n intct polyprotein orgniztion is importnt for the genertion of stle spike protein heterodimer. This possiility ppers unlikely for severl resons. First, we hve shown previously tht the p62 signl sequence is s efficiently used even without preceding C protein (7). Second, we hve showed in this study tht n pprently fully norml p62-el heterodimer complex is produced y the SFV-spike construction. Third, we hve lso recently used the SFV-spike construction in complementtion experiments with n SFV-cpsid virus vrint tht expresses NCs ut not spike proteins ecuse the corresponding gene region hs een deleted (19). In these experiments, efficient complementtion took plce nd virus prticle formtion ws regenerted to out 28% of the level of wild-type virus. Therefore, we re confident tht the spike protein complex produced from the SFV-spike construct corresponded to tht of the wild type. It ppers from these results tht during wild-type virus infection, the mjority of virl spike proteins were prevented from entering the degrdtive pthwy y their interction with the virl NCs. However, in the cse of SFV-spike virus, in which no NCs were present, spike proteins were rpidly degrded. As only very few spike suunits were found to e degrded in wild-type virus infection, this finding suggests tht most of the newly synthesized spike proteins re recognized y NCs very soon fter they rrived t the cell surfce or possily even efore they reched the PM. These results re in greement with those of Johnson nd Schlesinger (9). These investigtors used photoleching recovery ssy to study the moility of Sindis virus nd vesiculr stomtitis virus spike proteins on the surfce of infected cells. They found tht the Sindis spike proteins, in contrst to those of vesiculr stomtitis virus, were predominntly present in n immoile frction. They suggested tht most Sindis virus spikes formed n ssocition with NCs efore reching the cell surfce. However, other investigtors hve rgued ginst this possiility nd fvor udding model wherey spikes form lrge pool of ssemly-competent suunits t the PM (14, 17, 18). The most importnt evidence in fvor of the ltter model is the fct tht pulse-leled spike proteins re relesed into virions over very extended period of time (more thn 4 h) (14, 17). However, the evidence presented ove is comptile with udding model in which it is J. VIROL. ssumed tht there is very efficient NC-spike interction t the stge when spikes rrive t the cell surfce ut tht ctul completion of the udding process nd the finl relese of virus prticles is slow. Thus, ccording to this model of lphvirus udding, there is no significnt pool of free nd ssemly-competent spike proteins in the PM of infected cells. An importnt question tht then follows is, y wht mechnism is such n efficient cpturing of spikes y NCs mde possile? This process could e explined y overproduction of C protein in comprison with spike proteins during norml infection (17). However, it is lso possile tht the nswer is more complicted, perhps involving colocliztion of NCs into some specific regions on the cell surfce where spike proteins first pper. ACKNOWLEDGMENTS We thnk Jon Smyth for helping with the English text nd Peter Liljestrom, Johnn Whlerg, nd Mrit Suomlinen for vlule discussions throughout this work. This work ws supported y grnts from the Swedish Medicl Reserch Council (B90-12X A), the Swedish Ntionl Bord for Technicl Development ( P), nd the Swedish Nturl Science Reserch Council (B-BU ). REFERENCES 1. Boere, W. A. M., T. Hrmsen, J. Vinje, B. J. Beniss-Trouw, C. A. KrUeeveld, nd H. Snippe Identifiction of distinct ntigenic determinnts on Semliki Forest virus y using monoclonl ntiodies with different ntivirl ctivities. J. Virol. 52: Bruss, V., nd D. Gnem The role of envelope proteins in heptitis B virus ssemly. Proc. Ntl. Acd. Sci. USA 88: DeCurtis, I., nd K. Simons Dissection of Semliki Forest virus glycoprotein delivery from the trns-golgi network to the cell surfce in permeilized BHK cells. Proc. Ntl. Acd. Sci. USA 85: Delchmre, M., D. Gheysen, D. Thines, C. Thirirt, E. Jcos, E. Verdin, M. Horth, A. Bruny, nd F. Bex The GAG precursor of simin immunodeficiency virus ssemles into virus-like prticles. EMBO J. 8: Duois-Dlcq, M., R. V. Holmes, nd B. Rentier Assemly of enveloped RNA viruses. Springer Verlg, Vienn. 6. Groff, H., D. Huyleroeck, A. Roinson, U. Tillmn, nd P. Liljestrom The signl sequence of the p62 protein of Semliki Forest virus is involved in initition ut not completing chin trnsloction. J. Cell Biol. 111: Groff, H., nd H. Riedel Structure nd ssemly of lphviruses. Curr. Top. Microiol. Immunol. 99: Green, J., G. Griffiths, D. Louvrd, P. Quinn, nd G. Wrren Pssge of virl memrne proteins through the Golgi complex. J. Mol. Biol. 152: Johnson, D. C., nd M. J. Schlesinger Fluorescence photoleching recovery mesurements revel differences in envelopment of Sindis nd vesiculr stomtitis viruses. Cell 23: Krcosts, V., K. Ngshim, M. A. Gond, nd B. Moss Humn immunodeficiency virus-like prticles produced y vccini virus expression vector. Proc. Ntl. Acd. Sci. USA 86: Liljestr6m, P., nd H. Groff A new genertion of niml cell expression vectors sed on the Semliki Forest virus replicon. Bio/Technology 9: Loigs, M., H. Zho, nd H. Groff Function of Semliki Forest virus E3 peptide in virus ssemly: replcement of E3 with n rtificil signl peptide olishes spike heterodimeriztion nd surfce expression of El. J. Virol. 64: Mnitis, T., E. F. Fritsch, nd J. Smrook Moleculr cloning: lortory mnul. Cold Spring Hror Lortory, Cold Spring Hror, N.Y. 14. Myne, J. T., C. M. Rice, E. G. Struss, M. W. Hunkpiller, nd Downloded from on Octoer 23, 2018 y guest

7 VOL. 66, 1992 J. H. Struss Biochemicl studies of the mturtion of the smll Sindis virus glycoprotein E3. Virology 134: Pettersson, R. F Protein locliztion nd virus ssemly t intrcellulr memrnes. Curr. Top. Microiol. Immunol. 170: Rhee, S. S., H. Hui, nd E. Hunter Pressemled cpsids of type D retroviruses contin signl sufficient for trgeting specificlly to the plsm memrne. J. Virol. 64: Scheele, C. M., nd E. R. Pfefferkorn Kinetics of incorportion of structurl proteins into Sindis virions. J. Virol. 3: ALPHAVIRUS ASSEM13LY Simons, K., nd H. Groff The udding mechnisms of enveloped niml viruses. J. Gen. Virol. 50: Suomlinen, M., P. Liljestr6m, nd H. Grof Spike protein-nucleocpsid interctions drive the udding of lphviruses. J. Virol. 66: Whlerg, J. M., W. A. Boere, nd H. Groff The heterodimeric ssocition etween the memrne proteins of Semliki Forest virus chnges its sensitivity to mildly cidic ph during virus mturtion. J. Virol. 63: Ziemiecki, A., H. Groff, nd K. Simons Formtion of the Semliki forest virus memrne glycoprotein complexes in the infected cell. J. Gen. Virol. 50: Downloded from on Octoer 23, 2018 y guest