Sindbis Virus E2 Glycoprotein Research Articles

Visualization of cell-type dependent effects of anti-E2 antibody and interferon-gamma treatments on localization and expression of Broccoli aptamer-tagged alphavirus RNAs

Sindbis virus (SINV) is an alphavirus that causes age-dependent encephalomyelitis in mice. Within 7–8 days after infection infectious virus is cleared from neurons through the antiviral effects of antibody and interferon-gamma (IFNγ), but RNA persists. To better understand changes in viral RNA associated with immune-mediated clearance we developed recombinant strains of SINV that have genomic and subgenomic viral RNAs tagged with the Broccoli RNA aptamer that binds and activates a conditional fluorophore for live cell imaging of RNA. Treatment of SINV-Broccoli-infected cells with antibody to the SINV E2 glycoprotein had cell type-specific effects. In BHK cells, antibody increased levels of intracellular viral RNA and changed the primary location of genomic RNA from the perinuclear region to the plasma membrane without improving cell viability. In undifferentiated and differentiated AP7 (dAP7) neuronal cells, antibody treatment decreased levels of viral RNA. Occasional dAP7 cells escaped antibody-mediated clearance by not expressing cell surface E2 or binding antibody to the plasma membrane. IFNγ decreased viral RNA levels only in dAP7 cells and synergized with antibody for RNA clearance and improved cell survival. Therefore, analysis of aptamer-tagged SINV RNAs identified cell type- and neuronal maturation-dependent responses to immune mediators of virus clearance.

Sindbis Virus-Pseudotyped Lentiviral Vectors Carrying VEGFR2-Specific Nanobody for Potential Transductional Targeting of Tumor Vasculature

Introduction of selectivity/specificity into viral-based gene delivery systems, such as lentiviral vectors (LVs), is crucial in their systemic administration for cancer gene therapy. The pivotal role of tumor-associated endothelial cells (TAECs) in tumor angiogenesis and overexpression of vascular endothelial growth factor receptor-2 (VEGFR2 or KDR) in TAECs makes them a potent target in cancer treatment. Herein, we report the development of VEGFR2-targeted LVs pseudotyped with chimeric sindbis virus E2 glycoprotein (cSVE2s). For this purpose, either sequence of a VEGFR2-specific nanobody or its natural ligand (VEGF121) was inserted into the binding site of sindbis virus E2 glycoprotein. In silico modeling data suggested that the inserted targeting motifs were exposed in the context of cSVE2s. Western blot analysis of LVs indicated the incorporation of cSVE2s into viral particles. Capture ELISA demonstrated the specificity/functionality of the incorporated cSVE2s. Transduction of 293/KDR (expressing VEGFR2) or 293T cells (negative control) by constructed LVs followed by fluorescent microscopy and flow cytometric analyses indicated selective transduction of 293/KDR cells (30%) by both targeting motifs compared to 293T control cells (1-2%). These results implied similar targeting properties of VEGFR2-specific nanobody compared to the VEGF121 and indicated the potential for transductional targeting of tumor vasculature by the nanobody displaying LVs.

Amino acid substitutions in the E2 glycoprotein of Sindbis-like virus XJ-160 confer the ability to undergo heparan sulfate-dependent infection of mouse embryonic fibroblasts

We have recently demonstrated an essential role of the domain of 145-150 amino acid in the E2 glycoprotein of Sindbis virus in the interaction with cellular heparan sulfate (HS) and in the infection of mouse embryonic fibroblasts (MEF) cells. In this study, we constructed and characterized the mutants of Sindbis-like virus XJ-160 in which Tyr-146 and/or Asn-149 in the E2 glycoprotein had been substituted with His and Arg, respectively. Unlike parental virus XJ-160, mutants with either or both substitutions were able to infect wild-type mouse embryonic fibroblasts (MEF-wt) or MEF-Epi-/- cells which produce mutant HS. Significantly more infectious particles were released from MEF-wt than from MEF-Epi-/- cells. The mutant virus with both substitutions release was inhibited by pre-incubation of virus with heparin or pre-treatment of BHK-21 cells with HS-degrading enzyme. Both XJ-160 and the mutant viruses retained substantial neurovirulence in suckling mice. Our findings provide further support to the importance of positively charged residues in the HS-binding site of E2 in mediating Sindbis virus infection of MEF cells.

Functional characterization of the Sindbis virus E2 glycoprotein by transposon linker-insertion mutagenesis

The glycoprotein envelope of alphaviruses consists of two proteins, E1 and E2. E1 is responsible for fusion and E2 is responsible for receptor binding. An atomic structure is available for E1, but one for E2 has not been reported. In this study, transposon linker-insertion mutagenesis was used to probe the function of different domains of E2. A library of mutants, containing 19 amino acid insertions in the E2 glycoprotein sequence of the prototype alphavirus, Sindbis virus (SINV), was generated. Fifty-seven independent E2 insertions were characterized, of which more than half (67%) gave rise to viable virus. The wild-type-like mutants identify regions that accommodate insertions without perturbing virus production and can be used to insert targeting moieties to direct SINV to specific receptors. The defective and lethal mutants give insight into regions of E2 important for protein stability, transport to the cell membrane, E1–E2 contacts, and receptor binding.

Single and multiple deletions in the transmembrane domain of the Sindbis virus E2 glycoprotein identify a region critical for normal virus growth

Sindbis virus is composed of two nested T = 4 icosahedral protein shells containing 240 copies each of three structural proteins: E1, E2, and Capsid in a 1:1:1 stoichiometric ratio. E2 is a 423 amino acid glycoprotein with a membrane spanning domain 26 amino acids in length and a 33 amino acid cytoplasmic endodomain. The interaction of the endodomain with the nucleocapsid is an essential step in virus maturation and directs the formation of the outer protein shell as envelopment occurs. A previous study had determined that deletions in the transmembrane domain could affect virus assembly and infectivity (Hernandez et al., 2003. J. Virol. 77 (23), 12710–12719). Unexpectedly, a single deletion mutant (from 26 to 25 amino acids) resulted in a 1000-fold decrease in infectious virus production while another deletion of eight amino acids had no affect on infectious virus production. To further investigate the importance of these mutants, other single deletion mutants and another eight amino acid deletion mutant were constructed. We found that deletions located closer to the cytoplasmic (inner leaflet) of the membrane bilayer had a more detrimental effect on virus assembly and infectivity than those located closer to the luminal (outer leaflet) of the membrane bilayer. We also found that selective pressure can restore single amino acid deletions in the transmembrane domain but not necessarily to the wild type sequence. The partial restoration of an eight amino acid deletion (from 18 to 22 amino acids) also partially restored infectious virus production. The amount of infectious virus produced by this revertant was equivalent to that produced for the four amino acid deletion produced by site directed mutagenesis. These results suggest that the position of the deletion and the length of the C terminal region of the E2 transmembrane domain is vital for normal virus production. Deletion mutants resulting in decreased infectivity produce particles that appear to be processed and transported correctly suggesting a role involved in virus entry.

Alkylation of cysteine-containing peptides to mimic palmitoylation

Numerous proteins that are involved in cell signaling and viral replication require post-translational modification by palmitoylation to function properly. The molecular details by which this palmitoyl modification affects protein function remain poorly understood. To facilitate in vitro biochemical and structural studies of the role of palmitoylation on protein function, a method was developed for alkylating peptides with saturated C16 groups at cysteine residues and demonstrated using peptides derived from the palmitoylated region of Sindbis virus E2 glycoprotein. The synthetic approach takes advantage of disulfide chemistry to specifically modify only the cysteine residues within peptides and covalently links C16 groups via disulfide bridges using a new thioalkylating reagent, hexyldexyldithiopyridine. The chemistry presented here takes place in solution under mild conditions without the need for protection of the peptide functional groups. A method for purifying these modified peptides is also described. This protocol can be of general use to investigators studying the role of palmitoylation in biological systems.

A single amino acid change in the E2 glycoprotein of Sindbis virus confers neurovirulence by altering an early step of virus replication.

Amino acid changes in the envelope glycoproteins of Sindbis virus have been linked to neurovirulence; however, the molecular mechanisms by which these amino acid changes alter neurovirulence are not known. Recombinant-virus studies have mapped an important determinant of neurovirulence in adult mice to a single amino acid change, glutamine to histidine, at position 55 of the E2 glycoprotein (P. C. Tucker, E. G. Strauss, R. J. Kuhn, J. H. Strauss, and D. E. Griffin, J. Virol. 67:4605-4610, 1993). To investigate how histidine confers neurovirulence, we examined the various stages of the virus life cycle in neural (N18) and nonneural (BHK) cells. In BHK cells, recombinant viruses 633 (E255Q) and TE (E255H) replicated similarly. In contrast, in N18 neuroblastoma cells, TE established infection more efficiently, replicated faster, and achieved higher rates of virus release than did 633. Viral structural protein synthesis was similar in 633- and TE-infected BHK cells, while in N18 cells, structural protein synthesis was detected only in TE-infected cells at 6 h and remained higher for at least 16 h postinfection. Viral RNA synthesis was initiated more rapidly and was up to fivefold greater in TE- versus 633-infected N18 cells. Taken together with other data demonstrating minimal effects on virus binding and entry (P. C. Tucker, S. H. Lee, N. Bui, D. Martinie, and D. E. Griffin, J. Virol. 71:6106-6112, 1997), these data suggest that E2 position 55 plays an important role at early stages of infection of neural cells, thereby facilitating neurovirulence.

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.

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.

Neurovirulent strains of Alphavirus induce apoptosis in bcl-2-expressing cells: role of a single amino acid change in the E2 glycoprotein.

The isolation and sequence comparison of avirulent and neurovirulent strains of polio virus, alpha virus, herpes virus, immunodeficiency virus, and other viruses have identified genetic changes that are required to cause disease in the nervous system. The molecular mechanisms by which these genetic changes result in neurovirulence are unknown. An avirulent laboratory strain of the Alphavirus Sindbis kills most cultured cell lines not by lethal parasitism, but by inducing apoptosis or programmed cell death. Transfection of cultured cells with the human bcl-2 oncogene can block Sindbis virus-induced apoptosis, resulting in a persistent viral infection resembling that observed in brains of immunodeficient mice. We investigated the possibility that neurovirulent strains of Sindbis virus could overcome the protective effects of bcl-2--a potential mechanism to explain the ability of these strains to cause fatal disease. Strains of Sindbis virus that were lethal for 2- to 4-week-old mice induced apoptotic death in cultured cells despite the presence of bcl-2. Using recombinant viruses, we show that a single amino acid change in the E2 glycoprotein of Sindbis virus confers both neurovirulence and the ability to kill cells expressing bcl-2.

Sindbis virus pathogenesis: phenotypic reversion of an attenuated strain to virulence by second-site intragenic suppressor mutations.

Monoclonal antibodies (MAbs) specific for the E2c neutralizing antigenic site on the Sindbis virus E2 glycoprotein define a pathogenesis domain that affects neonatal mouse virulence. Sequence analysis of E2c MAb escape mutants showed that the domain included E2 amino acids 62, 96 and 159. The pathogenesis domain is also influenced by changes at E2 position 114. Mutation of E2 residues Asn 62 to Asp or Lys 159 to Glu results in suppression of the attenuated phenotype conferred by a mutation from Ser to Arg at E2 position 114. Possible mechanisms of phenotypic suppression within the E2c pathogenesis domain were investigated by using site-directed mutagenesis to determine the effects of specific combinations of positively charged, negatively charged and uncharged amino acid substitutions at E2 positions 62, 114 and 159. Phenotypic reversion to virulence by second-site suppressor mutations at E2 amino acids 62 or 159 was not dependent on ionic interaction with the residue at E2 114. Rather, suppression appeared to be the result of independent virulence effects mediated by specific residues.

Site-directed mutations in the Sindbis virus E2 glycoprotein identify palmitoylation sites and affect virus budding

The assembly and budding of Sindbis virus, a prototypic member of the alphavirus subgroup in the family Togaviridae, requires a specific interaction between the nucleocapsid core and the membrane-embedded glycoproteins E1 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 acylation sites in the E2 glycoprotein as the cysteines near the carboxyl terminus of the protein which is oriented to the cytoplasmic domain of this type 1 transmembrane protein. Additional mutations were made at two prolines within a hydrophobic sequence of E2 that is highly conserved among several alphaviruses, and the mutant viruses were aberrant in assembly and particle formation. These data support earlier studies indicating that the native structure of the cytoplasmic domain of E2 is essential for proper assembly of this enveloped virus.

Inhibition of Enveloped RNA Virus Formation by Peptides Corresponding to Glycoprotein Sequences

Peptides, corresponding to amino acid sequences in the cytoplasmic domains of the transmembranal glycoproteins encoded by Sindbis and vesicular stomatitis viruses, inhibited release of virus particles and infectious virus when added to infected cells for a 2h period during a one-cycle growth curve. Each inhibitory peptide was specific for its intended virus. The shortest peptide with antiviral activity for Sindbis virus contained six amino acids, and a related peptide that was acylated at the amino terminus with octanoic acid was ten-fold more potent as an inhibitor. Neither of these peptides affected the synthesis of viral structural proteins, but a third peptide inhibited proteolytic processing of the Sindbis virus E2 glycoprotein. Inhibition of vesicular stomatitis virus particle release was dose-dependent in the range of 50–400 μg ml−1 for a peptide corresponding to the G glycoprotein cytoplasmic domain. We postulate that these peptides competitively inhibit attachment of the glycoprotein to intracellular virus structures during assembly of the virion. Thus, drugs, based on peptides that mimic the protein-protein interactions required for virus assembly, may have therapeutic potential.

Site-directed mutations in sindbis virus E2 glycoprotein's cytoplasmic domain and the 6K protein lead to similar defects in virus assembly and budding

Site-directed mutagenesis was used to obtain four mutants with amino acid replacements in the cytoplasmic domain of the E2 glycoprotein and three with replacements in the 6K protein of Sindbis virus. All but one of these mutants yielded progeny virus after transfection of chicken embryo fibroblasts with RNA prepared by in vitro transcription of the virus cDNA; however, even this nonproducer mutant made virus structural proteins in the transfected cells. The other six mutants divided into two groups based on growth in chicken embryo fibroblasts. One group of four mutants (two in E2 and two in 6K) was indistinguishable from wild-type in formation of infectious virus in avian cells while the other group, consisting of two mutants, grew significantly slower. All six mutants grew slower than the parental wild-type virus in mosquito cells. In avian cells, all mutants produced extracellular particles at a slower rate than the wild-type and many of the particles contained multiple nucleocapsids, based on electron microscopy and kinetics of thermal inactivation. One of the E2 mutants with a cysteine changed to alanine and the 6K mutant with four cysteines replaced were deficient in covalent-bound palmitic acid. Two mutants with changes near the signalase cleavage sites between E2 and 6K and between 6K and El appeared to be defective in proteolytic processing. Despite individual differences, all of these mutants and the two previously described produced similar phenotypes in which multicored infectious virus particles were released more slowly from mosquito cells than from avian cells.

Attenuating mutations in glycoproteins E1 and E2 of Sindbis virus produce a highly attenuated strain when combined in vitro

Alterations in either the E1 or the E2 glycoprotein of Sindbis virus can affect pathogenesis in animals. Previously, we identified two distinct E1 glycoprotein gene sequences which differed in their effect on pathogenesis. One had an attenuation phenotype following subcutaneous inoculation of neonatal mice (E1 Ala-72, Gly-75, and Ser-237), while the other was virulent (E1 Val-72, Asp-75, and Ala-237). In this study, we examined the basis for this difference in pathogenesis by using a full-length cDNA clone of Sindbis virus from which infectious RNA could be transcribed in vitro. The relative contribution of each E1 residue to the pathogenesis phenotype was determined by using site-directed mutagenesis to alter each codon individually and in combination. Residues 75 and 237, in combination, appeared to be the major E1 determinants affecting pathogenesis. In addition, the effect of directly combining independently attenuating E1 and E2 mutations in the same virus was examined. The attenuating E1 sequences characterized in this study were coupled to a previously characterized attenuating mutation at E2 residue 114. The resulting recombinant virus, constructed in vitro, exhibited an increased attenuation of neurovirulence as compared with recombinant viruses containing either of the attenuating elements alone.

Monoclonal Antibodies to the E1 and E2 Glycoproteins of Sindbis Virus: Definition of Epitopes and Efficiency of Protection from Fatal Encephalitis

Protection of mice from fatal neuroadapted Sindbis virus encephalitis can be accomplished by passive transfer of monoclonal antibodies (MAbs) to either the E1 or E2 glycoprotein of Sindbis virus. Both neutralizing and non-neutralizing MAbs can be protective. To define further the characteristics of MAbs that provide protection from fatal disease, antigenic epitopes on the E1 and E2 glycoproteins were identified using a competitive binding enzyme immunoassay. Four distinct epitopes on E1 and three on E2 were defined. MAbs to all E1 epitopes, both neutralizing (three) and non-neutralizing (one) protected mice from fatal encephalitis. MAbs to the E2 neutralizing epitopes (two) protected mice from fatal encephalitis while those to the non-neutralizing epitope did not. The efficiency of protection from fatal Sindbis virus encephalitis of four neutralizing and non-neutralizing protective anti-E1 and anti-E2 MAbs representing different epitopes was compared. The neutralizing MAbs (against epitopes E2-ab, E2-c and E1-c) gave 50% protection at lower doses (2 to 20 micrograms) than the non-neutralizing MAb representing epitope E1-e (150 micrograms) when given before virus challenge. When given after virus challenge, MAbs to E2-ab and E2-c protected at lower doses (0.03 to 0.3 micrograms) than did either MAbs to E1-c (greater than 100 micrograms) or E1-e (10 micrograms). The MAbs to E1-e, E2-ab and E2-c were required in larger amounts to afford protection before than after challenge, while the opposite was true for MAb to E1-c.

Antibody-mediated activation of sindbis virus

The biological activity of an anti-Sindbis monoclonal antibody (MCAB 49) has been explored. The antibody recognizes an epitope on the E2 glycoprotein of Sindbis virus and, in the presence of complement (C′), neutralizes virus infectivity. In the absence of C′, reaction of the antibody with our laboratory strain of Sindbis, SB, increased the number of plaque-forming units (PFU) detected on baby hamster kidney (BHK) cells rather than neutralizing virus infectivity. The elevated titers of SB approached, but never exceeded, the number of virions calculated from the particle:PFU ratio, indicating that the additional PFU might have resulted from activation of normally noninfectious particles. The apparent activation could not be attributed to disaggregation of SB by MCAB 49 as shown by ultraviolet inactivation experiments with antibody-treated and untreated virus preparations. Fc receptors did not appear to be involved in the antibody-mediated activation. Fab′ and F(ab′ 2 fragments of MCAB 49 also increased the number of observed PFU of SB. Control monoclonal antibodies of the same isotype, but specific for the tobacco etch virus capsid protein, were unable to compete for cellular binding sites with the SB/MCAB 49 complex. Rather, the SB/MCAB 49 complex appeared to utilize the same receptor(s) as SB in that SB and the SB/MCAB 49 complexes competed with each other for binding sites on BHK cells. Binding studies with 32P-labeled SB showed that a higher proportion of MCAB 49 activated virions than untreated virions associated with BHK cells. Moreover, activated virions were much less susceptible to elution. These results suggest that reaction which MCAB 49 may facilitate successful attachment of SB to its receptor, or receptors, on BHK cells.

Alternative forms of a strain-specific neutralizing antigenic site on the Sindbis virus E2 glycoprotein

Experiments with monoclonal antibodies raised against two laboratory strains of Sindbis virus, SB and SIN, suggested the existence of a strain-specific neutralizing antigenic site (E2-b) on the E2 glycoprotein. A comparison of monoclonal antibody binding patterns and E2 glycoprotein gene sequences of six laboratory strains distinguished three different configurations of E2-b that correlated with specific amino acid substitutions at position 216 of the E2 glycoprotein. Further study of neutralization escape mutants selected with E2-b-specific antibodies confirmed that amino acid 216 is a major determinant of the E2-b antigenic site. Eight of nine mutants showed a coding change at position 216. One neutralization escape mutation created a new glycosylation site at position 213 and resulted in an E2 protein with an altered migration rate in SDS-PAGE. The neutralization escape mutants studied included amino acid substitutions not found in the laboratory strains that revealed differing binding requirements for two E2-b-specific monoclonal antibodies. The E2-b site is contrasted with the E2-c neutralizing antigenic site described previously (R. A. Olmsted, W. J. Meyer, and R. E. Johnston, 1986, Virology 148, 245–254) .

Altered E2 glycoprotein of Sindbis virus and its use in complementation studies.

We have detected a Sindbis virus variant that contains a smaller-molecular-weight form of the viral glycoprotein E2. The molecular weight of the PE2 precursor and the glycosylation pattern of the smaller E2 are normal, thus indicating that this E2 is formed by an aberrant proteolytic cleavage. The altered E2 was detected in an RNA+ temperature-sensitive mutant that was defective in proteolytic cleavage, but the abnormal PE2-to-E2 reaction could be separated from the ts mutation and is not itself a temperature-sensitive defect. We used the variant E2 as a marker to monitor the complementation reaction between an RNA+ and an RNA- mutant and discovered that complementation was not reciprocal; the RNA defect was corrected by the RNA+ mutant gene products but the RNA+ defect was not complemented by any RNA- gene products. Other studies have shown that the smaller E2 is not preferentially selected during viral maturation and budding. No significant changes have been detected in the biological activity of virions with this altered E2 protein. Comparison of the electrophoretic migration of the E1 and E2 Sindbis viral glycoproteins in a two-dimensional polyacrylamide slab gel system that was first run in the absence of sulfhydryl-reducing reagent and then with beta-mercaptoethanol indicated that the mobility of E1, but not that of E2, was significantly altered by reduction.