Mutating conserved cysteines in the alphavirus e2 glycoprotein causes virus-specific assembly defects

There are 80 trimeric, glycoprotein spikes that cover the surface of an alphavirus particle. The spikes, which are composed of three E2 and E1 glycoprotein heterodimers, are responsible for receptor binding and mediating fusion between the viral and host-cell membranes during entry. In addition, the cytoplasmic domain of E2 interacts with the nucleocapsid core during the last stages of particle assembly, possibly to aid in particle stability. During assembly, the spikes are nonfusogenic until the E3 glycoprotein is cleaved from E2 in the trans-Golgi network. Thus, a mutation in E2 potentially has effects on virus entry, spike assembly, or spike maturation. E2 is a highly conserved, cysteine-rich transmembrane glycoprotein. We made single cysteine-to-serine mutations within two distinct regions of the E2 ectodomain in both Sindbis virus and Ross River virus. Each of the E2 Cys mutants produced fewer infectious particles than wild-type virus. Further characterization of the mutant viruses revealed differences in particle morphology, fusion activity, and polyprotein cleavage between Sindbis and Ross River virus mutants, despite the mutations being made at corresponding positions in E2. The nonconserved assembly defects suggest that E2 folding and function is species dependent, possibly due to interactions with a virus-specific chaperone.     

The cholesterol requirement for sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence

Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped alphaviruses that enter cells via low-pH-triggered fusion in the endocytic pathway and exit by budding from the plasma membrane. Previous studies with cholesterol-depleted insect cells have shown that SFV requires cholesterol in the cell membrane for both virus fusion and efficient exit of progeny virus. An SFV mutant, srf-3, shows efficient fusion and exit in the absence of cholesterol due to a single point mutation in the E1 spike subunit, proline 226 to serine. We have here characterized the role of cholesterol in the entry and exit of SIN, an alphavirus quite distantly related to SFV. Growth, primary infection, fusion, and exit of SIN were all dramatically inhibited in cholesterol-depleted cells compared to control cells. Based on sequence differences within the E1 226 region between SFV, srf-3, and SIN, we constructed six SIN mutants with alterations within this region and characterized their cholesterol dependence. A SIN mutant, SGM, that had the srf-3 amino acid sequence from E1 position 224 to 235 showed increases of approximately 100-fold in infection and approximately 250-fold in fusion with cholesterol-depleted cells compared with infection and fusion of wild-type SIN. Pulse-chase analysis demonstrated that SGM exit from cholesterol-depleted cells was markedly more efficient than that of wild-type SIN. Thus, similar to SFV, SIN was cholesterol dependent for both virus entry and exit, and the cholesterol dependence of both steps could be modulated by sequences within the E1 226 region.     

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.     

Role of glycoprotein PE2 in formation and maturation of the Sindbis virus spike.

Sindbis virus envelope assembly is a multistep process resulting in the maturation of a rigid, highly ordered T=4 icosahedral protein lattice containing 80 spikes composed of trimers of E1-E2 heterodimers. Intramolecular disulfide bonds within E1 stabilize E1-E1 associations required for envelope formation and maintenance of the envelope's structural integrity. The structural integrity of the envelope protein lattice is resistant to reduction by dithiothreitol (DTT), indicating that E1 disulfides which stabilize structural domains become inaccessible to DTT at some point during virus maturation. The development of E1 resistance to DTT occurs prior to the completion of E1 folding and is temporally correlated with spike assembly in the endoplasmic reticulum. From these data we have predicted that in the final stages of spike assembly, E1 intramolecular disulfides, which stabilize the structural integrity of the envelope protein lattice, are buried within the spike and become inaccessible to the reductive activity of DTT. The spike is formed prior to the completion of E1 folding, and we have suggested that PE2 (the precursor to E2) may play a critical role in E1 folding after PE2-E1 oligomer formation has occurred. In this study we have investigated the role of PE2 in E1 folding, oligomer formation, and development of E1 resistance to both protease digestion and reduction by DTT by using a Sindbis virus replicon (SINrep/E1) which allows for the expression of E1 in the presence of truncated PE2. Through pulse-chase analysis of both Sindbis virus- and SINrep/E1-infected cells, we have determined that the folding of E1 into a trypsin-resistant conformation and into its most compact and stable form is not dependent upon association of E1 with PE2. However, E1 association with PE2 is required for oligomer formation, the export of E1 from the endoplasmic reticulum, and E1 acquisition of resistance to DTT.     

The surface conformation of Sindbis virus glycoproteins E1 and E2 at neutral and low pH, as determined by mass spectrometry-based mapping

Sindbis virus contains two membrane glycoproteins, E1 and E2, which are organized into 80 trimers of heterodimers (spikes). These trimers form a precise T=4 icosahedral protein lattice on the surface of the virus. Very little is known about the organization of the E1 and E2 glycoproteins within the spike trimer. To gain a better understanding of how the proteins E1 and E2 are arranged in the virus membrane, we have used the techniques of limited proteolysis and amino acid chemical modification in combination with mass spectrometry. We have determined that at neutral pH the E1 protein regions that are accessible to proteases include domains 1-21 (region encompassing amino acids 1 to 21), 161-176, and 212-220, while the E2 regions that are accessible include domains 31-84, 134-148, 158-186, 231-260, 299-314, and 324-337. When Sindbis virus is exposed to low pH, E2 amino acid domains 99-102 and 262-309 became exposed while other domains became inaccessible. Many new E1 regions became accessible after exposure to low pH, including region 86-91, which is in the putative fusion domain of E1 of Semliki Forest virus (SFV) (M. C. Kielian et al., J. Cell Biol. 134:863-872, 1996). E1 273-287 and region 145-158 were also exposed at low pH. These data support a model for the structure of the alphavirus spike in which the E1 glycoproteins are centrally located as trimers which are surrounded and protected by the E2 glycoprotein. These data improve our understanding of the structure of the virus membrane and have implications for understanding the protein conformational changes which accompany the process of virus-cell membrane fusion.     

Structural rearrangement of infecting Sindbis virions at the cell surface: mapping of newly accessible epitopes.

Sindbis virus glycoproteins E1 and E2 undergo a conformational alteration during early virus-cell interaction at the cell surface (D. Flynn, W. J. Meyer, J. M. MacKenzie, Jr., and R. E. Johnston, J. Virol. 64:3643-3653, 1990). Certain epitopes normally internal on native virus become accessible to monoclonal antibody (MAb) binding after attachment but before internalization of virus particles. These newly exposed epitopes, termed transitional epitopes, may be part of functionally important domains made accessible at the surface of the altered virus to facilitate entry into cells. Heating Sindbis virions at 51 degrees C for a short time induced a similar, although not identical, exposition of transitional epitopes on the E1 and E2 glycoproteins (W. J. Meyer, S. Gidwitz, V. K. Ayers, R. J. Schoepp, and R. E. Johnston, J. Virol. 66:3504-3513, 1992). In the current report, we have identified several of the transitional epitopes that become exposed as a consequence of early virus-cell interactions. Transitional epitope MAbs that bound to rearranged, heated virions and virus-cell complexes were used in antibody competition binding assays on heated Sindbis virions to map the spatial relationships between native, external, neutralizing antigenic sites and newly exposed transitional epitopes. Because the heated, rearranged particles retained their infectivity, MAbs that bound to transitional epitopes also were used to isolate MAb neutralization escape mutants. Sequencing the glycoprotein genes of the escape mutants identified specific E1 and E2 loci where mutation prevented MAb binding to transitional epitopes. One of the transitional epitopes identified (E2 residues 200 to 202) lies in the E2 190-216 region, which harbors two major neutralization sites, E2a and E2b, and an N-linked glycosylation site at E2 196. The glycosylation signal was eliminated by site-directed mutagenesis of a full-length cDNA clone of the Sindbis virus genome. The absence of a carbohydrate moiety did not expose the transitional epitopes mapped to this locus, suggesting that on native virions, the inaccessibility of the E2 200-202 determinant was inherent in the structure of the glycoprotein spike.     

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.     

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.     

Disulfide bridge-mediated folding of Sindbis virus glycoproteins.

The Sindbis virus envelope is composed of 80 E1-E2 (envelope glycoprotein) heterotrimers organized into an icosahedral protein lattice with T=4 symmetry. The structural integrity of the envelope protein lattice is maintained by E1-E1 interactions which are stabilized by intramolecular disulfide bonds. Structural domains of the envelope proteins sustain the envelope's icosahedral lattice, while functional domains are responsible for virus attachment and membrane fusion. We have previously shown that within the mature Sindbis virus particle, the structural domains of the envelope proteins are significantly more resistant to the membrane-permeative, sulfhydryl-reducing agent dithiothreitol (DTT) than are the functional domains (R. P. Anthony, A. M. Paredes, and D. T. Brown, Virology 190:330-336, 1992). We have used DTT to probe the accessibility of intramolecular disulfides within PE2 (the precursor to E2) and E1, as these proteins fold and are assembled into the spike heterotrimer. We have determined through pulse-chase analysis that intramolecular disulfide bonds within PE2 are always sensitive to DTT when the glycoproteins are in the endoplasmic reticulum. The reduction of these disulfides results in the disruption of PE2-E1 associations. E1 acquires increased resistance to DTT as it folds through a series of disulfide intermediates (E1alpha, -beta, and -gamma) prior to assuming its native and most compact conformation (E1epsilon). The transition from a DTT-sensitive form into a form which exhibits increased resistance to DTT occurs after E1 has folded into its E1beta conformation and correlates temporally with the dissociation of BiP-E1 complexes and the formation of PE2-E1 heterotrimers. We propose that the disulfide bonds within E1 which stabilize the protein domains required for maintaining the structural integrity of the envelope protein lattice form early within the folding pathway of E1 and become inaccessible to DTT once the heterotrimer has formed.     

The formation of intramolecular disulfide bridges is required for induction of the Sindbis virus mutant ts23 phenotype.

The Sindbis virus envelope protein spike is a hetero-oligomeric complex composed of a trimer of glycoprotein E1-E2 heterodimers. Spike assembly is a multistep process which occurs in the endoplasmic reticulum (ER) and is required for the export of E1 from the ER. PE2 (precursor to E2), however, can transit through the secretory pathway and be expressed at the cell surface in the absence of E1. Although oligomer formation does not appear to be required for the export of PE2, there is evidence that defects in E1 folding can affect PE2 transit from the ER. Temperature-sensitive mutant ts23 of Sindbis virus contains two amino acid substitutions in E1, while PE2 and capsid protein have the wild-type sequence; however, at the nonpermissive temperature, both E1 and PE2 are retained within the ER and can be isolated in protein aggregates with the molecular chaperone GRP78-BiP. We previously demonstrated that the temperature sensitivity for ts23 was lost as oligomer formation took place at the permissive temperature, suggesting that temperature sensitivity is initiated early in the process of viral spike assembly (M. Carleton and D. T. Brown, J. Virol. 70:952-959, 1996). Experiments described herein investigated the defects in envelope protein maturation that occur in ts23-infected cells and which result in retention of both envelope proteins in the ER. The data demonstrate that in ts23-infected cells incubated at the nonpermissive temperature, E1 folding is disrupted early after synthesis, resulting in the rapid incorporation of both E1 and PE2 into disulfide-stabilized aggregates. Furthermore, the aberrant E1 conformation which is responsible for induction of the ts phenotype requires the formation of intramolecular disulfide bridges formed prior to E1 association with PE2 and the completion of E1 folding.     

PE2 cleavage mutants of Sindbis virus: correlation between viral infectivity and pH-dependent membrane fusion activation of the spike heterodimer

The spike glycoprotein E2 of Sindbis virus (SIN) is synthesized in the infected cell as a PE2 precursor protein, which matures through cleavage by a cellular furin-like protease. Previous work has shown that SIN mutants impaired in PE2 cleavage are noninfectious on BHK-21 cells, the block in infection being localized at a step after virus-receptor interaction but prior to RNA replication. Here, we studied the membrane fusion properties of SIN PE2 cleavage mutants and observed that these viruses are impaired in their ability to form an E1 homotrimer and to fuse with liposomes at a mildly acidic pH. The block in spike rearrangement and fusion could be overridden by exposure of the mutant viruses to very low pH (<4.5). Cleavage mutants with second-site resuscitating mutations in PE2 were highly infectious for BHK-21 cells. The ability of these viruses to form E1 homotrimers and to fuse at a mildly acidic pH was completely restored despite a sustained lack of PE2 cleavage.     

The Transmembrane Domains of Sindbis Virus Envelope Glycoproteins Induce Cell Death

Sindbis virus, the prototype alphavirus, kills cells by inducing apoptosis. To investigate potential mechanisms by which Sindbis virus induces apoptosis, we examined whether specific viral gene products were able to induce cell death. Genes encoding the three structural proteins—capsid, the precursor E1 (6K plus E1), and the precursor E2 (P62 or E3 plus E2)—were cotransfected with a β-galactosidase reporter plasmid in transient-transfection assays in rat prostate adenocarcinoma AT3 cells. Cell death, as determined by measuring the loss of blue cells, was observed in AT3 cells transfected with 6K plus E1 and with P62 but not in cells transfected with capsid. Deletion mutagenesis of P62 indicated that large regions of the cytoplasmic domain and extracellular domain were not essential for the induction of cell death. However, constructs containing the minimal E3 signal sequence fused to the E2 transmembrane domain and the minimal E3 signal sequence fused to the E1 transmembrane domain induced death as efficiently as full-length P62 and 6K plus E1, whereas no cell death was observed after transfection with a control construct containing the E3 signal sequence linked to the transmembrane domain of murine CD4. These data demonstrate that intracellular expression of the transmembrane domains of the Sindbis virus envelope glycoproteins can kill AT3 cells.     

Antibody-selected variation and reversion in Sindbis virus neutralization epitopes.

Sindbis virus variants evidencing a complex and bidirectional tendency toward spontaneous antigenic change were isolated and characterized. Variants were selected on the basis of their escape from neutralization by individual monoclonal antibodies to either of the two envelope glycoproteins, E2 and E1. Multisite variants, including one altered in three neutralization sites, were obtained by selecting mutants consecutively in the presence of different neutralizing monoclonal antibodies. Two phenotypic revertants, each of which reacquired prototype antigenicity, were back-selected on the basis of their reactivity with a neutralizing monoclonal antibody. An incidental oligonucleotide marker distinguished these and the variant from which they arose from parental Sindbis virus and other mutants, thereby confirming that the revertants were true progeny of the antigenic variant. Prototype Sindbis virus and variants derived from it were compared on the basis of their reactivities with each of a panel of monoclonal antibodies; patterns revealed a minimum of five independently mutable Sindbis virus neutralization epitopes, segregating as three antigenic sites (two E2 and one E1).     

Interaction of Sindbis virus glycoproteins during morphogenesis.

In cells infected with the Sindbis temperature-sensitive mutants ts-23 and ts-10 (complementation group D), which contain a defect in the envelope glycoprotein E1, the precursor polypeptide PE2 is not cleaved to the envelope glycoprotein E2 at the nonpermissive temperature. This defect is phenotypically identical to the defect observed in the complementation group E mutant, ts-20. The lesion in ts-23 is reversible upon shift to permissive temperature, whereas that of ts-10 is not. Antiserum against whole virus, E1, or E2 also prevents the cleavage of PE2 in cells infected with wild-type Sindbis virus. Because the cleavage of PE2 is inhibited by the lesion in mutants that are genotypically distinct and by anti-E1 or -E2 serum, it appears that PE2 and E1 exist as a complex in the membrane of the infected cell.     

Maturation Defects in Temperature-sensitive Mutants of Sindbis Virus

Temperature-sensitive mutants of Sindbis virus, which synthesize viral ribonucleic acid (RNA) but not mature virus at the nonpermissible temperature, were selected for the study of viral maturation. Of these, three mutants which complement each other genetically were used. Two major proteins, the nucleocapsid and membrane proteins, located, respectively, in the viral nucleoid and membrane, were found in intact virions. In cells infected with wild-type Sindbis virus, four distinct types of viral RNA with sedimentation coefficients of 40S, 26S, 20S, and 15S were detected in constant distribution. The 20S RNA was ribonuclease-resistant, whereas the other types were ribonuclease-sensitive. The 40S RNA, identical to that obtained from the virion, was found associated with nucleocapsid protein as a subviral particle, which was assumed to be the nucleoid. Viral materials from cells infected with the mutants under nonpermissive conditions were compared with those from cells infected with wild-type virus, in terms of (i) the distribution of the different types of RNA, (ii) the association of infectious viral RNA into subviral particles, and (iii) the ability of infected cells to hemadsorb goose erythrocytes. According to these criteria, each of the three mutants demonstrated different maturation defects. Defective nucleocapsid proteins and membrane proteins may each account for one of the above mutants. The thrid mutant may have defects in a minor structural protein or possibly a maturation protein which is involved in the assembly of Sindbis virus.     

Placement of the structural proteins in Sindbis virus

The structure of the lipid-enveloped Sindbis virus has been determined by fitting atomic resolution crystallographic structures of component proteins into an 11-A resolution cryoelectron microscopy map. The virus has T=4 quasisymmetry elements that are accurately maintained between the external glycoproteins, the transmembrane helical region, and the internal nucleocapsid core. The crystal structure of the E1 glycoprotein was fitted into the cryoelectron microscopy density, in part by using the known carbohydrate positions as restraints. A difference map showed that the E2 glycoprotein was shaped similarly to E1, suggesting a possible common evolutionary origin for these two glycoproteins. The structure shows that the E2 glycoprotein would have to move away from the center of the trimeric spike in order to expose enough viral membrane surface to permit fusion with the cellular membrane during the initial stages of host infection. The well-resolved E1-E2 transmembrane regions form alpha-helical coiled coils that were consistent with T=4 symmetry. The known structure of the capsid protein was fitted into the density corresponding to the nucleocapsid, revising the structure published earlier.     

Deacylation of the transmembrane domains of Sindbis virus envelope glycoproteins E1 and E2 does not affect low-pH-induced viral membrane fusion activity

The envelope glycoproteins E1 and E2 of Sindbis virus are palmitoylated at cysteine residues within their transmembrane domains (E1 at position 430, and E2 at positions 388 and 390). Here, we investigated the in vitro membrane fusion activity of Sindbis virus variants (derived from the Toto 1101 infectious clone), in which the E1 C430 and/or E2 C388/390 residues had been substituted for alanines. Both the E1 and E2 mutant viruses, as well as a triple mutant virus, fused with liposomes in a strictly low-pH-dependent manner, the fusion characteristics being indistinguishable from those of the parent Toto 1101 virus. These results demonstrate that acylation of the transmembrane domain of Sindbis virus E1 and E2 is not required for expression of viral membrane fusion activity.