Sindbis virus mutations which coordinately affect glycoprotein processing, penetration, and virulence in mice

Rapid penetration of baby hamster kidney cells was used as a selective pressure for the isolation of pathogenesis mutants of the S.A.AR86 strain of Sindbis virus. Unlike most Sindbis virus strains, S.A.AR86 is virulent in adult as well as neonatal mice. Two classes of mutants were defined. One class was attenuated in adult mice inoculated intracerebrally as well as in neonatal mice inoculated either intracerebrally or subcutaneously. Sequence analysis of the glycoprotein genes of the parent virus and three such mutant strains revealed a single point mutation which resulted in an amino acid change at position 1 in the E2 glycoprotein. The change from a serine in S.A.AR86 to an asparagine in the mutants created a new site for N-linked glycosylation which appeared to be utilized. This mutation did not retard release of infectious particles; however, mutant virions contained the E2 precursor protein (PE2) rather than the E2 glycoprotein itself. The mutants also lost the ability to bind two E2-specific monoclonal antibodies, R6 and R13. A second class of mutants was attenuated in neonatal mice upon subcutaneous inoculation but remained virulent in adults and in neonates when inoculated intracerebrally. Sequence analysis of three such strains revealed the substitution of an arginine residue for a serine at position 114 in the E2 glycoprotein. Reactivity with monoclonal antibodies R6 and R13 was reduced, yet members of this mutant class were more susceptible than S.A.AR86 to neutralization by these antibodies.     

Single amino acid insertions at the junction of the sindbis virus E2 transmembrane domain and endodomain disrupt virus envelopment and alter infectivity

The final steps in the envelopment of Sindbis virus involve specific interactions of the E2 endodomain with the virus nucleocapsid. Deleting E2 K at position 391 (E2 DeltaK391) resulted in the disruption of virus assembly in mammalian cells but not insect cells (host range mutant). This suggested unique interactions of the E2 DeltaK391 endodomain with the different biochemical environments of the mammalian and insect cell lipid bilayers. To further investigate the role of the amino acid residues located at or around position E2 391 and constraints on the length of the endodomain on virus assembly, amino acid insertions/substitutions at the transmembrane/endodomain junction were constructed. An additional K was inserted at amino acid position 392 (KK391/392), a K-->F substitution at position 391 was constructed (F391), and an additional F was inserted at 392 (FF391/392). These changes should lengthen the endodomain in the KK391/392 insertion mutant or shorten the endodomain in the FF391/392 mutant. The mutant FF391/392 grown in BHK cells formed virus particles containing extruded material not found on wild-type virus. This characteristic was not seen in FF391/392 virus grown in insect cells. The mutant KK391/392 grown in BHK cells was defective in the final membrane fission reaction, producing multicored or conjoined virus particles. The production of these aberrant particles was ameliorated when the KK391/392 mutant was grown in insect cells. These data indicate that there is a critical minimal spanning distance from the E2 membrane proximal amino acid at position 391 and the conserved E2 Y400 residue. The observed phenotypes of these mutants also invoke an important role of the specific host membrane lipid composition on virus architecture and infectivity.     

Mutations in the endodomain of Sindbis virus glycoprotein E2 define sequences critical for virus assembly

Envelopment of Sindbis virus at the plasma membrane is a multistep process in which an initial step is the association of the E2 protein via a cytoplasmic endodomain with the preassembled nucleocapsid. Sindbis virus is vectored in nature by blood-sucking insects and grows efficiently in a number of avian and mammalian vertebrate hosts. The assembly of Sindbis virus, therefore, must occur in two very different host cell environments. Mammalian cells contain cholesterol which insect membranes lack. This difference in membrane composition may be critical in determining what requirements are placed on the E2 tail for virus assembly. To examine the interaction between the E2 tail and the nucleocapsid in Sindbis virus, we have produced substitutions and deletions in a region of the E2 tail (E2 amino acids 408 to 415) that is initially integrated into the endoplasmic reticulum. This sequence was identified as being critical for nucleocapsid binding in an in vitro peptide protection assay. The effects of these mutations on virus assembly and function were determined in both vertebrate and invertebrate cells. Amino acid substitutions (at positions E2: 408, 410, 411, and 413) reduced infectious virus production in a position-dependent fashion but were not efficient in disrupting assembly in mammalian cells. Deletions in the E2 endodomain (delta406-407, delta409-411, and delta414-417) resulted in the failure to assemble virions in mammalian cells. Electron microscopy of BHK cells transfected with these mutants revealed assembly of nucleocapsids that failed to attach to membranes. However, introduction of these deletion mutants into insect cells resulted in the assembly of virus-like particles but no assayable infectivity. These data help define protein interactions critical for virus assembly and suggest a fundamental difference between Sindbis virus assembly in mammalian and insect cells.     

Sindbis virus attachment: isolation and characterization of mutants with impaired binding to vertebrate cells.

Sindbis virus can infect a broad range of insect and vertebrate cell types. The ability to restrict tissue tropism and target virus infection to specific cell types would expand the usefulness of engineered alphaviruses as gene expression vectors. In this study, virus pools derived from libraries of full-length Sindbis virus cDNA clones containing random insertion mutations in the PE2 or E1 virion glycoprotein gene were screened for mutants defective for binding to vertebrate cells. Binding-competent mutants were depleted by serial adsorption to chicken embryo fibroblast (CEF) monolayers at 4 degrees C, and the remaining population was amplified by immune-enhanced infection of P388D1 cells. From the PE2 libraries, 12 candidate mutants showing reduced cytopathic effects on CEF monolayers were isolated and three representative mutants, NB1, NB2, and NB12, were characterized in detail. Insertion mutations for NB1 and NB12 were found near the PE2 cleavage site, whereas the insertion in NB2 occurred between residues 69 and 74 of E2. Although virion assembly and release occurred normally for all three mutants, PE2 cleavage was completely (NB1) or partially (NB12) blocked for the mutants with insertions near the PE2 cleavage site. Both NB1 and NB2 were defective for binding to CEF and BHK-21 cells. Mild trypsin digestion of isolated NB1 virions resulted in PE2 cleavage and partially restored binding to CEF. Besides defective binding, NB1 also exhibited slower CEF penetration kinetics. Consistent with previous work, these results implicate PE2 cleavage and domains in the N-terminal portion of E2 as important determinants of alphavirus binding and penetration. Binding-defective mutants such as NB2, which exhibit normal particle assembly, release, and penetration, may be useful for future efforts to target Sindbis virus infection.     

An Amino Acid Substitution in the Coding Region of the E2 Glycoprotein Adapts Ross River Virus To Utilize Heparan Sulfate as an Attachment Moiety

Passage of Ross River virus strain NB5092 in avian cells has been previously shown to select for virus variants that have enhanced replication in these cells. Sequencing of these variants identified two independent sites that might be responsible for the phenotype. We now demonstrate, using a molecular cDNA clone of the wild-type T48 strain, that an amino acid substitution at residue 218 in the E2 glycoprotein can account for the phenotype. Substitutions that replaced the wild-type asparagine with basic residues had enhanced replication in avian cells while acidic or neutral residues had little or no observable effect. Ross River virus mutants that had increased replication in avian cells also grew better in BHK cells than the wild-type virus, whereas the remaining mutants were unaffected in growth. Replication in both BHK and avian cells of Ross River virus mutants N218K and N218R was inhibited by the presence of heparin or by the pretreatment of the cells with heparinase. Binding of the mutants, but not of the wild type, to a heparin-Sepharose column produced binding comparable to that of Sindbis virus, which has previously been shown to bind heparin. Replication of these mutants was also adversely affected when they were grown in a CHO cell line that was deficient in heparan sulfate production. These results demonstrate that amino acid 218 of the E2 glycoprotein can be modified to create an heparan sulfate binding site and this modification expands the host range of Ross River virus in cultured cells to cells of avian origin.     

Amino acid changes in the Sindbis virus E2 glycoprotein that increase neurovirulence improve entry into neuroblastoma cells.

Sindbis virus (SV) is an alphavirus that causes encephalitis in mice and results in age-dependent mortality. The outcome is dependent on the virus strain. Residues at 55 and 172 in the E2 glycoprotein determine the neurovirulence for mice of different ages and the efficiency of replication in the nervous system and neuronal cells. To determine the effects of these two residues on the initial steps in replication, we studied viruses with a histidine or glutamine at E2 position 55 and a glycine or an arginine at position 172, E2[H55G172], E2[Q55G172], E2[H55R172], and E2[Q55R172]. The production of virus was detected earlier for viruses with a histidine at E2 position 55 in BHK-21 cells (4 to 6 versus 6 to 8 h) and for E2[H55G172] in N18 cells (6 versus 8 to 10 h). As shown previously, viruses with a glycine at E2 position 172 bound more efficiently to N18 cells and a histidine at E2 position 55 further improved binding only slightly. Viruses with E2[H55] exhibited more rapid internalization and degradation of viral proteins in both BHK-21 and N18 cells. Incubation of E2[H55G172] and E2[Q55G172] at various pHs and temperatures did not reveal differences in virion stability. These data suggest that the amino acids at E2 positions 172 and 55 affect both adsorption and penetration of SV and that these early steps in the replicative pathway contribute to increased neurovirulence.     

Non-histone proteins and long-range organization of HeLa interphase DNA

We have studied the association of the Sindbis virus glycoproteins in mature virions and infected cells. The glycoproteins were cross-linked with bifunctional amino-reactive reagents (11 Å cross-linking distance), some of which could be subsequently cleaved by reduction. Using monospecific rabbit antisera against each viral envelope glycoprotein it was found that >90% of the cross-linked glycoprotein dimers from intact virions or virions solubilized with Triton X100 prior to cross-linking were heterodimers of E1 and E2. The pattern of cross-linked glycoproteins from intact virions as well as infected cells suggested that three E1-E2 dimers may be associated to form a hexameric subunit. Cross-linking of pulselabeled infected monolayers showed that PE2 was cross-linked to E1 less efficiently than was E2, suggesting that if PE2 and E1 are associated as a complex in infected cells then their conformation with respect to reactive amino groups is distinct from that of the mature virion glycoproteins. ts mutants of Sindbis virus in the complementation groups corresponding to E1 and PE2 fail to cleave PE2 at the non-permissive temperature (40 °C). In monolayers infected with these mutants or a heat-resistant variant of Sindbis virus, the glycoprotein precursors synthesized at the elevated temperature were readily cross-linked into large aggregates, indicating a temperature-sensitive tendency for the proteins to aggregate.     

Molecular Determinants of Mouse Neurovirulence and Mosquito Infection for Western Equine Encephalitis Virus

Western equine encephalitis virus (WEEV) is a naturally occurring recombinant virus derived from ancestral Sindbis and Eastern equine encephalitis viruses. We previously showed that infection by WEEV isolates McMillan (McM) and IMP-181 (IMP) results in high (∼90–100%) and low (0%) mortality, respectively, in outbred CD-1 mice when virus is delivered by either subcutaneous or aerosol routes. However, relatively little is known about specific virulence determinants of WEEV. We previously observed that IMP infected Culex tarsalis mosquitoes at a high rate (app. 80%) following ingestion of an infected bloodmeal but these mosquitoes were infected by McM at a much lower rate (10%). To understand the viral role in these phenotypic differences, we characterized the pathogenic phenotypes of McM/IMP chimeras. Chimeras encoding the E2 of McM on an IMP backbone (or the reciprocal) had the most significant effect on infection phenotypes in mice or mosquitoes. Furthermore, exchanging the arginine, present on IMP E2 glycoprotein at position 214, for the glutamine present at the same position on McM, ablated mouse mortality. Curiously, the reciprocal exchange did not confer mouse virulence to the IMP virus. Mosquito infectivity was also determined and significantly, one of the important loci was the same as the mouse virulence determinant identified above. Replacing either IMP E2 amino acid 181 or 214 with the corresponding McM amino acid lowered mosquito infection rates to McM-like levels. As with the mouse neurovirulence, reciprocal exchange of amino acids did not confer mosquito infectivity. The identification of WEEV E2 amino acid 214 as necessary for both IMP mosquito infectivity and McM mouse virulence indicates that they are mutually exclusive phenotypes and suggests an explanation for the lack of human or equine WEE cases even in the presence of active transmission.     

Polycaryocyte formation mediated by Sindbis virus glycoproteins.

The process of cell fusion mediated by Sindbis virus membrane proteins synthesized after infection was examined. At the times after infection at which virus proteins were detectable on the cell surface, Sindbis virus-infected BHK-21 cells were found to express a fusion function after brief treatment at acid pH. In studies employing wild-type virus and temperature-sensitive mutants and testing drug or protease inhibition of virus production, we made the following observations on Sindbis virus-mediated fusion from within. (i) Fusion requires the synthesis of virus glycoproteins and their transport to the cell surface. (ii) Modification of the cell plasma membrane by polypeptides PE2 and E1 alone is not sufficient for expression of the fusion function. (iii) The proteolytic conversion of plasma membrane-associated PE2 to E2 is not essential for fusion. (iv) Glycosylation of virus plasma membrane proteins is essential for fusion. (v) The lesions of Sindbis virus temperature-sensitive mutants do not affect their ability to fuse cells.     

Large-plaque mutants of Sindbis virus show reduced binding to heparan sulfate, heightened viremia, and slower clearance from the circulation

Laboratory strains of Sindbis virus must bind to the negatively charged glycosaminoglycan heparan sulfate in order to efficiently infect cultured cells. During infection of mice, however, we have frequently observed the development of large-plaque viral mutants with a reduced ability to bind to heparan sulfate. Sequencing of these mutants revealed changes of positively charged amino acids in putative heparin-binding domains of the E2 glycoprotein. Recombinant viruses were constructed with these changes as single amino acid substitutions in a strain Toto 1101 background. All exhibited decreased binding to heparan sulfate and had larger plaques than Toto 1101. When injected subcutaneously into neonatal mice, large-plaque viruses produced higher-titer viremia and often caused higher mortality. Because circulating heparin-binding proteins are known to be rapidly sequestered by tissue heparan sulfate, we measured the kinetics of viral clearance following intravenous injection. Much of the parental small-plaque Toto 1101 strain of Sindbis virus was cleared from the circulation by the liver within minutes, in contrast to recombinant large-plaque viruses, which had longer circulating half-lives. These findings indicate that a decreased ability to bind to heparan sulfate allows more efficient viral production in vivo, which may in turn lead to increased mortality. Because Sindbis virus is only one of a growing number of viruses from many families which have been shown to bind to heparan sulfate, these results may be generally applicable to the pathogenesis of such viruses.     

Identification of antigenically important domains in the glycoproteins of Sindbis virus by analysis of antibody escape variants.

To study important epitopes on glycoprotein E2 of Sindbis virus, eight variants selected to be singly or multiply resistant to six neutralizing monoclonal antibodies reactive against E2, as well as four revertants which had regained sensitivity to neutralization, were sequenced throughout the E2 region. To study antigenic determinants in glycoprotein E1, four variants selected for resistance to a neutralizing monoclonal antibody reactive with E1 were sequenced throughout the E2 and E1 regions. All of the salient changes in E2 occurred within a relatively small region between amino acids 181 and 216, a domain that encompasses a glycosylation site at residue 196 and that is rich in charged amino acids. Almost all variants had a change in charge, suggesting that the charged nature of this domain is important for interaction with antibodies. Variants independently isolated for resistance to the same antibody were usually altered in the same amino acid, and reversion to sensitivity occurred at the sites of the original mutations, but did not always restore the parental amino acid. The characteristics of this region suggest that this domain is found on the surface of E2 and constitutes a prominent antigenic domain that interacts directly with neutralizing antibodies. Previous studies have shown that this domain is also important for penetration of cells and for virulence of the virus. Resistance to the single E1-specific neutralizing monoclonal antibody resulted from changes of Gly-132 of E1 to either Arg or Glu. Analogous to the findings with E2, these changes result in a change in charge and are found near a glycosylation site at residue 139. This domain of E1 may therefore be found near the 181 to 216 domain of E2 on the surface of the E1-E2 heterodimer; together, they could form a domain important in virus penetration and neutralization.     

Alphavirus-induced apoptosis in mouse brains correlates with neurovirulence.

Sindbis virus induces apoptotic cell death in cultured cell lines, raising the possibility that apoptosis of infected neurons and other target cells in vivo may contribute to the resulting disease and mortality. To investigate the role of apoptosis in Sindbis virus pathogenesis, infected mouse brains were assayed by the in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling technique and for DNA ladder formation. Infection with recombinant Sindbis virus strain 633 resulted in widespread apoptosis in newborn mouse brains and spinal cords, but few apoptotic cells were observed following infection of 2-week-old animals. This finding correlates with the age-dependent mortality observed in mice. The more neurovirulent virus TE, which differs from 633 by a single amino acid in the E2 glycoprotein, induced significant apoptosis in brains and spinal cords of 2-week-old animals, consistent with its ability to cause fatal disease in older animals. Double-labeling experiments demonstrated that the apoptotic cells were also infected with Sindbis virus. Thus, Sindbis virus-induced apoptosis appears to be a result of virus infection and is likely to reflect pathogenic mechanisms for other viruses.     

Mechanism of altered Sindbis virus neurovirulence associated with a single-amino-acid change in the E2 Glycoprotein.

The mechanism by which amino acid changes in the E1 and E2 surface glycoproteins of Sindbis virus affect neurovirulence is unknown. We have studied two recombinant viruses which differ in virulence. One (TE) contains Gly and the other (TES) contains Arg at position 172 in E2. TE causes more rapid death than TES in newborn mice. Both viruses replicate similarly in nonneuronal cells, but TE replicates more rapidly in the brains of newborn mice and in neuroblastoma cells. TE also induces earlier viral RNA synthesis in neuroblastoma cells. 35S-labeled TE binds more efficiently to brain and neuroblastoma cells, but not to nonneuronal cells, than TES. We propose that a region of the E2 glycoprotein affected by the amino acid occupying position 172 is important for binding to an alphavirus receptor on neurons and influences neurovirulence by this mechanism.     

The amino-terminal residue of Sindbis virus glycoprotein E2 influences virus maturation, specific infectivity for BHK cells, and virulence in mice.

The E2 glycoprotein of Sindbis virus is synthesized as a precursor, PE2, which is cleaved by furin or a furin-like host cell protease at a late stage of maturation. The four-residue PE2 cleavage signal conforms to the basic amino acid-X-basic-basic motif which is present in many other viral and cellular glycoproteins which are processed by the cellular enzyme(s). In this report, we present evidence that the amino acid which immediately follows the signal, the N-terminal residue of E2, can influence protease recognition, binding, and/or cleavage of PE2. Constructs encoding nine different amino acids at E2 position 1 (E2 1) were produced by site-directed mutagenesis of the full-length cDNA clone of our laboratory strain of Sindbis virus AR339 (pTRSB). Viruses derived from clones encoding Arg (TRSB), Asp, Ser, Phe, His, and Asn in a nonglycosylated form at E2 1 contained predominantly E2. Viruses encoding Ile, Leu, or Val at E2 1 contained the uncleaved form of PE2. The specific infectivity of TRSB (E2 Arg-1) for baby hamster kidney (BHK-21) cells was from 5- to greater than 100-fold higher than those of isogenic constructs with other residues at E2 1, suggesting that E2 Arg-1 represents a BHK-21 cell adaptive mutation in our laboratory strain. In newborn CD-1 mice, TRSB was more virulent than the PE2-containing viruses but less virulent than other PE2-cleaving viruses with alternative amino acids at E2 1. These results indicate that in TRSB, E2 Arg-1 increased the efficiency of virus-cell interactions in cultured BHK-21 cells but simultaneously decreased the ability of virus to mediate in vivo virus-cell interactions critical for the induction of disease. This suggests that the N terminus of E2 may participate in or be associated with virion domains which mediate these viral functions.     

Molecular analysis of neurovirulent strains of Sindbis virus that evolve during persistent infection of scid mice.

To understand the role of tissue-specific adaptation and antibody-induced selectional pressures in the evolution of neurovirulent viruses, we analyzed three strains of Sindbis virus isolated from the brains of persistently infected scid mice and four strains of Sindbis virus isolated from the brains of scid mice with viral reactivation following immune serum treatment. For each viral isolate, we tested neurovirulence in weanling BALB/c mice and sequenced regions of the E2 and E1 envelope glycoprotein genes that are known to contain important determinants of Sindbis virus neurovirulence. One strain isolated from a persistently infected scid mouse and two strains isolated from scid mice with viral reactivation were neurovirulent, resulting in mortality in 80 to 100% of weanling BALB/c mice. All three neurovirulent strains contained an A-->U change at nucleotide 8795, which predicts a Gln-->His substitution at E2 amino acid position 55. No nucleotide changes were detected in the other sequenced regions of the E2 and E1 envelope glycoprotein genes or in the avirulent isolates. Our findings indicate that tissue-specific adaptations, rather than antibody-induced selectional pressures, are a critical determinant of the evolution of neurovirulent strains of Sindbis virus and provide evidence that E2 His-55 is an important neuroadaptive mutation that confers neurovirulence properties on Sindbis virus.     

Deduced consensus sequence of Sindbis virus strain AR339: mutations contained in laboratory strains which affect cell culture and in vivo phenotypes.

The consensus sequence of the Sindbis virus AR339 isolate, the prototype alphavirus, has been deduced. THe results presented here suggest (i) that a substantial proportion of the sequence divergence evident between the consensus sequence and sequences of laboratory strains of AR339 has resulted from selection for efficient growth in cell culture, (ii) that many of these changes affect the virulence of the virus in animal models, and (iii) that such modified genetic backgrounds present in laboratory strains can exert a significant influence on genetic studies of virus pathogenesis and host range. A laboratory strain of Sindbis virus AR339 was sequenced and cloned as a cDNA (pTRSB) from which infectious virus (TRSB) could be derived. The consensus sequence was deduced from the complete sequences of pTRSB and HRsp (E. G. Strauss, C. M. Rice, and J. H. Strauss, Virology 133:92-110, 1984), from partial sequences of the glycoprotein genes of three other AR339 laboratory strains, and by comparison with the sequences of the glycoprotein genes of three other AR339 sequence. HRsp differed form the consensus sequence by eight coding changes, and TRSB differed by three coding changes. In the 5' untranslated region, HRsp differed from the consensus sequence at nucleotide (nt) 5. These differences were likely the result of cell culture passage of the original AR339 isolate. At three of the difference loci (one in TRSB and two in HRsp), selection of cell-culture-adaptive mutations was documented with Sindbis virus or other alphaviruses. Selection in cell culture often results in attenuation of virulence in animals. Considering the TRSB and HRsp sequences together, one noncoding difference from the consensus (an A-for-G substitution in the 5' untranslated region at nt 5) and six coding differences in the glycoprotein genes (at E2 amino acids 1, 3, 70, and 172 and at E1 amino acids 72 and 237) were at loci which, either individually or in combination, significantly affected alphavirus virulence in mice. Although the levels of virulence of isogenic strains containing either nt 5 A or nt 5 G did not differ significantly in neonatal mice, the presence of nt 5 A greatly enhanced the effect of a second attenuating mutation in the E2 gene. These results suggest that minimal differences in the "wild type" genetic background into which an additional mutation is introduced can have a dramatic effect on apparent virulence and pathogenesis phenotypes. A cDNA clone of the consensus AR339 sequence, a sequence devoid of occult attenuating mutations introduced by cell culture passage, will allow the molecular genetic examination of cell culture and in vivo phenotypes of a virus which may best reflect the sequence of Sindbis virus AR339 at the time of its isolation.     

Sindbis virus membrane fusion is mediated by reduction of glycoprotein disulfide bridges at the cell surface.

We have examined the role of thiol-disulfide exchange reactions during the penetration of cells by Sindbis virus. The protein-protein association that form the rigid icosahedral lattice of the Sindbis virus envelope have been shown to be stabilized by disulfide bridges, and reduction of these critical disulfide bridges during cell penetration may be the mechanism by which the rigid protein lattice is disrupted prior to fusion (R. Anthony and D. T. Brown, J. Virol. 65:1187-1194, 1991; R. Anthony, A. Paredes, and D. T. Brown, Virology 190:330-336, 1992). Reduction of disulfide bridges occurs at near neutral pHs via thiol-disulfide exchange reactions, and these reactions can be blocked by covalent modification of the thiol involved. In this study, the effects of the reducing agent 2-mercaptoethanol on Sindbis virus-mediated cell-cell fusion from without and the effects of the membrane-impermeable thiol-alkylating reagent 5,5'-dithiobis(2-nitrobenzoic acid) on Sindbis virus penetration were determined. The presence of exogenous reducing agent was found to induce fusion from without under conditions unfavorable to both typical Sindbis virus-mediated fusion from without and cysteine-mediated thiol-disulfide exchange reactions. In addition, the thiol-alkylating reagent was found to inhibit Sindbis virus entry when present during infection. These results are consistent with a model for Sindbis virus entry in which reduction of critical disulfide bridges at the cell surface disrupts the rigid protein-protein associations of the envelope, allowing membrane fusion and release of the viral genome into the cell.     

Restricted replication of two alphaviruses in ricin-resistant mouse L cells with altered glycosyltransferase activities.

Two mouse L cell variant lines (CL 3 and CL 6) selected for resistance to the toxic plant lectin ricin were restricted in their ability to replicate the two alphaviruses Sindbis virus and Semliki Forest virus. CL 3 cells have been shown to exhibit increased CMP-sialic acid:glycoprotein sialyltransferase and GM3 synthetase activities, whereas CL 6 cells have been shown to contain decreased UDPgalactose:glycoprotein galactosyltransferase and UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase activities. The adsorption of Sindbis virus to CL 6 cells was considerably reduced, suggesting that the loss or inaccessibility of the receptors for Sindbis virus accounted for a major defect in virus production in these cells. In contrast, CL 3 synthesized Sindbis viral RNA and proteins but were unable to convert the precursor glycoprotein PE2 to the structural protein E2. The cleavage of PE2 to E2 was also blocked in both CL 3 and CL 6 cells infected with Semliki Forest virus.