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.     

Structures of immature flavivirus particles

Structures of prM-containing dengue and yellow fever virus particles were determined to 16 and 25 A resolution, respectively, by cryoelectron microscopy and image reconstruction techniques. The closely similar structures show 60 icosahedrally organized trimeric spikes on the particle surface. Each spike consists of three prM:E heterodimers, where E is an envelope glycoprotein and prM is the precursor to the membrane protein M. The pre-peptide components of the prM proteins in each spike cover the fusion peptides at the distal ends of the E glycoproteins in a manner similar to the organization of the glycoproteins in the alphavirus spikes. Each heterodimer is associated with an E and a prM transmembrane density. These transmembrane densities represent either an EE or prMprM antiparallel coiled coil by which each protein spans the membrane twice, leaving the C-terminus of each protein on the exterior of the viral membrane, consistent with the predicted membrane-spanning domains of the unprocessed polyprotein.     

A Chimeric Vesiculo/Alphavirus Is an Effective Alphavirus Vaccine

While a large number of mosquito-transmitted alphaviruses are known to cause serious human diseases, there are no licensed vaccines that protect against alphavirus infections. The alphavirus chikungunya virus (CHIKV) has caused multiple recent outbreaks of chikungunya fever. This virus has the potential to cause a worldwide epidemic and has generated strong interest in development of a prophylactic CHIKV vaccine. We report here on the development of a potent experimental vaccine for CHIKV based on a chimeric vesicular stomatitis virus (VSV) expressing the entire CHIKV envelope polyprotein (E3-E2-6K-E1) in place of the VSV glycoprotein (G). These VSVΔG-CHIKV chimeras incorporated functional CHIKV glycoproteins into the viral envelope in place of VSV G. The chimeric viruses were attenuated for growth in tissue culture but could be propagated to high titers without VSV G complementation. They also generated robust neutralizing antibody and cellular immune responses to CHIKV in mice after a single dose and protected mice against CHIKV infection. VSVΔG-alphavirus chimeras could have general applicability as alphavirus vaccines.     

Fusion function of the Semliki Forest virus spike is activated by proteolytic cleavage of the envelope glycoprotein precursor p62.

The precursor protein p62 of the prototype alphavirus Semliki Forest virus (SFV) undergoes during transport to the cell surface a proteolytic cleavage to form the mature envelope glycoprotein E2. To investigate the biological significance of this cleavage event, single amino acid substitutions were introduced at the cleavages site through mutagenesis of cDNA corresponding to the structural region of the SFV genome. The phenotypes of the cleavage site mutants were studied in BHK cells by using recombinant vaccinia virus vectors. Nonconservative substitutions completely abolished p62 cleavage. Uncleaved p62 was transported with normal kinetics to the cell surface, where it became accessible to low concentrations of exogenous trypsin. The proteolytic cleavage of envelope glycoprotein precursors has been shown to activate the membrane fusion potential of viral spikes in several virus families. Here we demonstrate that the fusion function of the SFV spike is activated by the cleavage of p62. Cleavage-deficient p62 expressed at the cell surface did not function in low-pH-triggered (pH 5.5) cell-cell membrane fusion; however, cleavage of the mutated p62 with exogenous trypsin restored the fusion function. We discuss a model for SFV assembly and fusion where p62 cleavage plays a crucial role in the stability of the multimeric association of the viral envelope glycoproteins.     

The structure of barmah forest virus as revealed by cryo-electron microscopy at a 6-angstrom resolution has detailed transmembrane protein architecture and interactions

Barmah Forest virus (BFV) is a mosquito-borne alphavirus that infects humans. A 6-Å-resolution cryo-electron microscopy three-dimensional structure of BFV exhibits a typical alphavirus organization, with RNA-containing nucleocapsid surrounded by a bilipid membrane anchored with the surface proteins E1 and E2. The map allows details of the transmembrane regions of E1 and E2 to be seen. The C-terminal end of the E2 transmembrane helix binds to the capsid protein. Following the E2 transmembrane helix, a short α-helical endodomain lies on the inner surface of the lipid envelope. The E2 endodomain interacts with E1 transmembrane helix from a neighboring E1-E2 trimeric spike, thereby acting as a spacer and a linker between spikes. In agreement with previous mutagenesis studies, the endodomain plays an important role in recruiting other E1-E2 spikes to the budding site during virus assembly. The E2 endodomain may thus serve as a target for antiviral drug design.     

Characterization of an endoplasmic reticulum retention signal in the rubella virus E1 glycoprotein.

Rubella virus contains three structural proteins, capsid, E2, and E1. E2 and E1 are type I membrane glycoproteins that form a heterodimer in the endoplasmic reticulum (ER) before they are transported to and retained in the Golgi complex, where virus assembly occurs. The bulk of unassembled E2 and E1 subunits are not transported to the Golgi complex. We have recently shown that E2 contains a Golgi-targeting signal that mediates retention of the E2-E1 complex (T. C. Hobman, L. Woodward, and M. G. Farquhar, Mol. Biol. Cell 6:7-20, 1995). The focus of this study was to determine if E1 glycoprotein also contains intracellular targeting information. We constructed a series of chimeric reporter proteins by fusing domains from E1 to the ectodomains of two other type I membrane proteins which are normally transported to the cell surface, vesicular stomatitis virus G protein (G) and CD8. Fusion of the E1 transmembrane and cytoplasmic regions, but not analogous domains from two control membrane proteins, to the ectodomains of G and CD8 proteins caused the resulting chimeras to be retained in the ER. Association of the ER-retained chimeras with known ER chaperone proteins was not detected. ER localization required both the transmembrane and cytoplasmic regions of E1, since neither of these domains alone was sufficient to retain the reporter proteins. Increasing the length of the E1 cytoplasmic domain by 10 amino acids completely abrogated ER retention. This finding also indicated that the chimeras were not retained as a result of misfolding. In summary, we have identified a new type of ER retention signal that may function to prevent unassembled E1 subunits and/or immature E2-E1 dimers from reaching the Golgi complex, where they could interfere with viral assembly. Accordingly, assembly of E2 and E1 would mask the signal, thereby allowing transport of the heterodimer from the ER.     

Spike protein-nucleocapsid interactions drive the budding of alphaviruses.

Semliki Forest virus (SFV) particles are released from infected cells by budding of nucleocapsids through plasma membrane regions that are modified by virus spike proteins. The budding process was studied with recombinant SFV genomes which lacked the nucleocapsid protein gene or, alternatively, the spike genes. No subviral particles were released from cells which expressed only the nucleocapsid protein or the spike proteins. Virus release was found to be strictly dependent on the coexpression of the nucleocapsid and the spike proteins. These results provide direct proof for the hypothesis that the alphavirus budding is driven by nucleocapsid-spike interactions. The importance of the viral 42S RNA for virus assembly and budding was investigated by using the heterologous vaccinia virus-T7 expression system for the synthesis of the SFV structural proteins. The results demonstrate that the viral genome is not absolutely required for formation of budding competent nucleocapsids, since small amounts of viruslike particles were assembled in the absence of 42S RNA.     

Characterization of Hepatitis C Virus Intra- and Intergenotypic Chimeras Reveals a Role of the Glycoproteins in Virus Envelopment

Hepatitis C virus (HCV) is highly variable and associated with chronic liver disease. Viral isolates are grouped into seven genotypes (GTs). Accumulating evidence indicates that viral determinants in the core to NS2 proteins modulate the efficiency of virus production. However, the role of the glycoproteins E1 and E2 in this process is currently poorly defined. Therefore, we constructed chimeric viral genomes to explore the role of E1 and E2 in HCV assembly. Comparison of the kinetics and efficiency of particle production by intragenotypic chimeras highlighted core and p7 as crucial determinants for efficient virion release. Glycoprotein sequences, however, had only a minimal impact on this process. In contrast, in the context of intergenotypic HCV chimeras, HCV assembly was profoundly influenced by glycoprotein genes. On the one hand, insertion of GT1a-derived (H77) E1-E2 sequences into a chimeric GT2a virus (Jc1) strongly suppressed virus production. On the other hand, replacement of H77 glycoproteins within the GT1a-GT2a chimeric genome H77/C3 by GT2a-derived (Jc1) E1-E2 increased infectious particle production. Thus, within intergenotypic chimeras, glycoprotein features strongly modulate virus production. Replacement of Jc1 glycoprotein genes by H77-derived E1-E2 did not grossly affect subcellular localization of core, E2, and NS2. However, it caused an accumulation of nonenveloped core protein and increased abundance of nonenveloped core protein structures with slow sedimentation. These findings reveal an important role for the HCV glycoproteins E1 and E2 in membrane envelopment, which likely depends on a genotype-specific interplay with additional viral factors.     

Proteins of Vesicular Stomatitis Virus and of Phenotypically Mixed Vesicular Stomatitis Virus-Simian Virus 5 Virions

The identity of the glycoprotein of vesicular stomatitis virus (VSV) as the spike protein has been confirmed by the removal of the spikes with a protease from Streptomyces griseus, leaving bullet-shaped particles bounded by a smooth membrane. This treatment removes the glycoprotein but does not affect the other virion proteins, apparently because they are protected from the enzyme by the lipids in the viral membrane. The proteins of phenotypically mixed, bullet-shaped virions produced by cells mixedly infected with VSV and the parainfluenza virus simian virus 5 (SV5) have been analyzed by polyacrylamide gel electrophoresis. These virions contain all the VSV proteins plus the two SV5 spike proteins, both of which are glycoproteins. The finding of the SV5 spike glycoproteins on virions with the typical morphology of VSV indicates that there is not a stringent requirement that only the VSV glycoprotein can be used to form the bullet-shaped virion. On the other hand, the SV5 nucleocapsid protein and the major non-spike protein of the SV5 envelope were not detected in the phenotypically mixed virions, and this suggests that a specific interaction between the VSV nucleocapsid and regions of the cell membrane which contain the nonglycosylated VSV envelope protein is necessary for assembly of the bullet-shaped virion.     

Semliki forest virus budding: assay, mechanisms, and cholesterol requirement

All enveloped viruses must bud through a cellular membrane in order to acquire their lipid bilayer, but little is known about this important stage in virus biogenesis. We have developed a quantitative biochemical assay to monitor the budding of Semliki Forest virus (SFV), an enveloped alphavirus that buds from the plasma membrane in a reaction requiring both viral spike proteins and nucleocapsid. The assay was based on cell surface biotinylation of newly synthesized virus spike proteins and retrieval of biotinylated virions using streptavidin-conjugated magnetic particles. Budding of biotin-tagged SFV was continuous for at least 2 h, independent of microfilaments and microtubules, strongly temperature dependent, and relatively independent of continued exocytic transport. Studies of cell surface spike proteins at early times of infection showed that these spikes did not efficiently bud into virus particles and were rapidly degraded. In contrast, at later times of infection, spike protein degradation was markedly reduced and efficient budding was then observed. The previously described cholesterol requirement in SFV exit was shown to be due to a block in budding in the absence of cholesterol and correlated with the continued degradation of spike proteins at all times of virus infection in sterol-deficient cells.     

Role of Conserved Cysteines in the Alphavirus E3 Protein

Alphavirus particles are covered by 80 glycoprotein spikes that are essential for viral entry. Spikes consist of the E2 receptor binding protein and the E1 fusion protein. Spike assembly occurs in the endoplasmic reticulum, where E1 associates with pE2, a precursor containing E3 and E2 proteins. E3 is a small, cysteine-rich, extracellular glycoprotein that mediates proper folding of pE2 and its subsequent association with E1. In addition, cleavage of E3 from the assembled spike is required to make the virus particles efficiently fusion competent. We have found that the E3 protein in Sindbis virus contains one disulfide bond between residues Cys19 and Cys25. Replacing either of these two critical cysteines resulted in mutants with attenuated titers. Replacing both cysteines with either alanine or serine resulted in double mutants that were lethal. Insertion of additional cysteines based on E3 proteins from other alphaviruses resulted in either sequential or nested disulfide bond patterns. E3 sequences that formed sequential disulfides yielded virus with near-wild-type titers, while those that contained nested disulfide bonds had attenuated activity. Our data indicate that the role of the cysteine residues in E3 is not primarily structural. We hypothesize that E3 has an enzymatic or functional role in virus assembly, and these possibilities are further discussed.Alphaviruses are members of the Togaviradae family and are single-stranded, positive-sense RNA, enveloped viruses (17). The lipid membranes of the viruses have 80 glycoprotein spikes which are required for viral entry. Each spike is comprised of three copies of a heterodimer which consists of the E2 and E1 proteins (22, 54). E2 and E1 are glycoproteins with a single transmembrane helix that traverses the host-derived lipid bilayer. E2 interacts with the nucleocapsid core at the C terminus (12, 16, 27, 43) and contains the receptor binding site at the N terminus (5, 21, 45). E1 is the viral fusion protein responsible for mediating fusion between the virus membrane and the host cell membrane during an infection (13, 39, 47). Specific interactions in both the ectodomain and transmembrane regions are critical for heterodimer formation (30, 35, 46, 54). The assembly of each heterodimer, its subsequent assembly into a spike, and the interaction of the cytoplasmic tail of the spike with the nucleocapsid core are all essential for the efficient production of infectious particles.Glycoprotein spike assembly requires four structural proteins, E3, E2, 6K, and E1, which are expressed as a single polyprotein. E3 is a small, 64-amino-acid protein (Sindbis virus [SINV] numbering) and contains a signal sequence that translocates the protein into the endoplasmic reticulum (ER) (3, 4, 15). Early in translation, glycosylation of N14 (SINV numbering) occurs and this promotes E3''s release from the ER membrane into the lumen. As a result, the signal sequence is not cleaved from the E3 protein (14). Cellular enzymes cleave the polyprotein to yield pE2 (an uncleaved protein consisting of E3 and E2), 6K, and E1 (23, 55) proteins. In the ER, E1 is found in several conformations, only one of which will form a functional heterodimer with pE2, allowing its transport to the Golgi apparatus (1, 2, 6, 7, 36). After pE2-E1 heterodimerization, self-association between three heterodimers occurs and each individual spike is formed (25, 26, 36). As observed with Semliki Forest virus, disulfide bonds reshuffle within pE2 during protein folding (34), possibly forming intermolecular disulfide bonds between E3 and E2 residues. However, no intermolecular disulfide bonds between pE2 and E1 have been identified (34). Once the viral spikes have been assembled, they are transported to the plasma membrane (11) and are thus exposed to subcellular changes of pH, from pH 7.2 in the ER to pH 5.7 in the vesicles constitutively transporting the spikes to the plasma membrane. In the trans-Golgi network, the E3 protein is cleaved from pE2 by the cellular protein furin (18, 44, 55). E3 remains noncovalently attached to the released virus particle, while in other species E3 is found in the medium of virus-infected cells (32, 49).E3 is required for efficient particle assembly, both in mediating spike folding and in spike activation for viral entry. When an ER signal sequence was substituted for the E3 protein, heterodimerization of pE2 and E1 was abolished (26). Furthermore, when E2 and E1 were expressed individually, low levels of E2 were transported to the cell surface while E1 remained in the ER, suggesting that heterodimerization with pE2 is necessary for E1 to be transported to the cell surface (24, 26, 46). These results are consistent with E3 playing a critical role in mediating the folding of pE2 and the association of pE2 and E1 proteins during spike assembly (7, 38). In viruses where the furin cleavage site was mutated, the virus particles were correctly assembled but severely reduced in infectivity, presumably because the fusion protein was unable to dissociate from pE2 and initiate fusion (44, 55).A comparison of an amino acid sequence alignment of E3 proteins from different alphaviruses (Fig. ​(Fig.1)1) shows that the E3 protein is a small protein with four conserved cysteine (Cys) residues. A subset of E3 proteins contains an additional two Cys residues in a narrow cysteine/proline-rich region, PPCXPCC (Fig. ​(Fig.1).1). We have purified recombinant E3 protein from SINV and have determined that a disulfide bond is present and, furthermore, that these Cys residues are important in virus assembly. Within the alphavirus E3 proteins, we have identified a region that is important for mediating spike transport to the plasma membrane and thus is critical for spike assembly.Open in a separate windowFIG. 1.E3 amino acid sequence alignment from a representative group of alphaviruses. The cysteines marked with asterisks are conserved in all alphavirus species. The ⋄ indicates the conserved but nonessential glycosylation site. The PPCXPCC motif present in ∼50% of alphaviruses is underlined. SFV, Semliki Forest virus; RRV, Ross River virus; BFV, Barmah Forest virus; EEE, eastern equine encephalitis virus; ONN, O''nyong nyong virus; IGB, Igbo Ora virus; OCK, Ockelbo virus; WEE, western equine encephalitis virus; AUR, Aura virus; VEE, Venezuelan equine encephalitis virus.     

The cytoplasmic domain of alphavirus E2 glycoprotein contains a short linear recognition signal required for viral budding.

Intracellular alphavirus nucleocapsids express a binding site for the cytoplasmic domain of the viral E2 spike glycoprotein. This binding site is recognized by the anti-idiotype monoclonal antibody, F13. The monoclonal anti-anti-idiotype antibody, raised against F13 and designated 3G10, recognizes the carboxy-terminal eight residues of the E2 cytoplasmic domain in Semliki Forest virus (SFV), identifying this as the signal for nucleocapsid interaction. F13 binding to cells infected with SFV or a second alphavirus, Sindbis virus, is inhibited by a synthetic peptide corresponding to the entire 31 residue cytoplasmic domain (E2c), and also by a synthetic peptide corresponding to the eight residue epitope recognized by 3G10. Both E2c and the eight residue peptide inhibited viral budding in microinjection experiments and when conjugated to colloidal gold are bound specifically to nucleocapsids in infected cells. These results identify a short linear signal in the E2 cytoplasmic domain required for the interaction with nucleocapsids which leads to budding of at least two alphaviruses from infected cells.     

Function of Semliki Forest virus E3 peptide in virus assembly: replacement of E3 with an artificial signal peptide abolishes spike heterodimerization and surface expression of E1.

The Semliki Forest virus spike glycoproteins E1 and p62 form a heterodimeric complex in the endoplasmic reticulum (ER) and are transported as such to the cell surface. In the mature virus particle, the heterodimeric association of E1 and E2 (the cleavage product of p62) is maintained, but as a more labile and acid-sensitive oligomer than the E1-p62 complex. The E3 peptide forms the N-terminal part of the p62 precursor and carries the signal for the translocation of p62 into the lumen of the ER. The question of whether E3 is also important in the formation and stabilization of the E1-p62 heterodimer has been addressed here with the aid of an E3 deletion mutant cDNA. In this construct, the entire E3 was replaced with a cleavable, artificial signal sequence which preserved the membrane topology of an authentic E2. The E3 deletion, when expressed via a recombinant vaccinia virus, abolished heterodimerization of the spike proteins. It also resulted in the complete retention of E1 in the ER and almost total inhibition of E2 transport to the plasma membrane. The oligomerization and transport defect of E1 expressed from the E3 deletion mutant could be complemented with a wild-type p62 provided from a separate coding unit in double infections. These results point to a central role of E3 in complex formation and transport of the viral structural components to the site of budding. In conjunction with earlier work (M. Lobigs and H. Garoff, J. Virol. 64:1233-1240, 1990; J. Wahlberg, W. A. M. Boere, and H. Garoff, J. Virol. 63:4991-4997, 1989), the data support a model of spike protein oligomerization control of Semliki Forest virus assembly and disassembly which may be mediated by the presence of E3 in the uncleaved p62 precursor and release of E3 after cleavage.     

Sindbis virus ts103 has a mutation in glycoprotein E2 that leads to defective assembly of virions.

Sindbis virus mutant ts103 is aberrant in the assembly of virus particles. During virus budding, proper nucleocapsid-glycoprotein interactions fail to occur such that particles containing many nucleocapsids are formed, and the final yield of virus is low. We have determined that a mutation in the external domain of glycoprotein E2, Ala-344----Val, is the change that leads to this phenotype. Mapping was done by making recombinant viruses between ts103 and a parental strain of the virus, using a full-length cDNA clone of Sindbis virus from which infectious RNA can be transcribed, together with sequence analysis of the region of the genome shown in this way to contain the ts103 lesion. A partial revertant of ts103, called ts103R, was also mapped and sequenced and found to be a second-site revertant in which a change in glycoprotein E1 from lysine to methionine at position 227 partially suppresses the phenotypic effects of the change at E2 position 344. An analysis of revertants from ts103 mutants in which the Ala----Val change had been transferred into a defined background showed that pseudorevertants were more likely to arise than were true revertants and that the ts103 change itself reverted very infrequently. The assembly defect in ts103 appeared to result from weakened interactions between the virus membrane glycoproteins or between these glycoproteins and the nucleocapsid during budding. Both the E2 mutation leading to the defect in virus assembly and the suppressor mutation in glycoprotein E1 are in the domains external to the lipid bilayer and thus in domains that cannot interact directly with the nucleocapsid. This suggests that in ts103, either the E1-E2 heterodimers or the trimeric spikes (consisting of three E1-E2 heterodimers) are unstable or have an aberrant configuration, and thus do not interact properly with the nucleocapsid, or cannot assembly correctly to form the proper icosahedral array on the surface of the virus.     

Association of sindbis virus capsid protein with phospholipid membranes and the E2 glycoprotein: implications for alphavirus assembly

A late stage in assembly of alphaviruses within infected cells is thought to be directed by interactions between the nucleocapsid and the cytoplasmic domain of the E2 protein, a component of the viral E1/E2 glycoprotein complex that is embedded in the plasma membrane. Recognition between the nucleocapsid protein and the E2 protein was explored in solution using NMR spectroscopy, as well as in binding assays using a model phospholipid membrane system that incorporated a variety of Sindbis virus E2 cytoplasmic domain (cdE2) and capsid protein constructs. In these binding assays, synthetic cdE2 peptides were reconstituted into phospholipid vesicles to simulate the presentation of cdE2 on the inner leaflet of the plasma membrane. Results from these binding assays showed a direct interaction between a peptide containing the C-terminal 16 amino acids of the cdE2 sequence and a Sindbis virus capsid protein construct containing amino acids 19-264. Additional experiments that probed the sequence specificity of this cdE2-capsid interaction are also described. Further binding assays demonstrated an interaction between the 19-264 capsid protein and artificial vesicles containing neutral or negatively charged phospholipids, while capsid protein constructs with N-terminal truncations displayed either little or no affinity for such vesicles. The membrane-binding property of the capsid protein suggests that the membrane may play an active role in alphavirus assembly. The results are consistent with an assembly process involving an initial membrane association, whereby an association with E2 glycoprotein further enhances capsid binding to facilitate membrane envelopment of the nucleocapsid for budding. Collectively, these experiments elucidate certain requirements for the binding of Sindbis virus capsid protein to the cytoplasmic domain of the E2 glycoprotein, a critical event in the alphavirus maturation pathway.     

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.     

Two small virus-specific polypeptides are produced during infection with Sindbis virus.

We have identified and characterized two small virus-specific polypeptides which are produced during infection of cells with Sindbis virus, but which are not incorporated into the mature virion. The larger of these is a glycoprotein with an approximate molecular weight of 9,800 and is found predominantly in the medium of infected cells. Three independent lines of evidence demonstrate conclusively that this 9,800-dalton glycoprotein is produced during the proteolytic conversion of the precursor polypeptide, PE2, to the virion glycoprotein E2. This small glycoprotein is therefore analogous to the virion glycoprotein E3 of the very closely related alphavirus, Semliki Forest virus. The 9,800-dalton glycoprotein of Sindbis virus, unlike the E3 glycoprotein of Semliki Forest virus, is not, however, present in the viral particle. The other virus-specific polypeptide is 4,200 daltons in size, does not appear to be a glycoprotein, and is neither incorporated into the mature virus nor released into the culture medium. The gene for this small polypeptide is present in the viral 26S mRNA (the mRNA which encodes all the viral structural polypeptides) and appears to be located in the portion of the mRNA which encodes the two viral glycoproteins. The possibility that this 4,200-dalton polypeptide functions as a signal peptide during the synthesis of the viral membrane glycoproteins is discussed.     

The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine

To identify genetic determinants of classical swine fever virus (CSFV) virulence and host range, chimeras of the highly pathogenic Brescia strain and the attenuated vaccine strain CS were constructed and evaluated for viral virulence in swine. Upon initial screening, only chimeras 138.8v and 337.14v, the only chimeras containing the E2 glycoprotein of CS, were attenuated in swine despite exhibiting unaltered growth characteristics in primary porcine macrophage cell cultures. Additional viral chimeras were constructed to confirm the role of E2 in virulence. Chimeric virus 319.1v, which contained only the CS E2 glycoprotein in the Brescia background, was markedly attenuated in pigs, exhibiting significantly decreased virus replication in tonsils, a transient viremia, limited generalization of infection, and decreased virus shedding. Chimeras encoding all Brescia structural proteins in a CS genetic background remained attenuated, indicating that additional mutations outside the structural region are important for CS vaccine virus attenuation. These results demonstrate that CS E2 alone is sufficient for attenuating Brescia, indicating a significant role for the CSFV E2 glycoprotein in swine virulence.     

Spike protein VP4 assembly with maturing rotavirus requires a postendoplasmic reticulum event in polarized caco-2 cells

Rotavirus assembly is a multistep process that requires the successive association of four major structural proteins in three concentric layers. It has been assumed until now that VP4, the most external viral protein that forms the spikes of mature virions, associates with double-layer particles within the endoplasmic reticulum (ER) in conjunction with VP7 and with the help of a nonstructural protein, NSP4. VP7 and NSP4 are two glycosylated proteins. However, we recently described a strong association of VP4 with raft-type membrane microdomains, a result that makes the ER a highly questionable site for the final assembly of rotavirus, since rafts are thought to be absent from this compartment. In this study, we used tunicamycin (TM), a drug known to block the first step of protein N glycosylation, as a tool to dissect rotavirus assembly. We show that, as expected, TM blocks viral protein glycosylation and also decreases virus infectivity. In the meantime, viral particles were blocked as enveloped particles in the ER. Interestingly, TM does not prevent the targeting of VP4 to the cell surface nor its association with raft membranes, whereas the infectivity associated with the raft fractions strongly decreased. VP4 does not colocalize with the ER marker protein disulfide-isomerase even when viral particles were blocked by TM in this compartment. These results strongly support a primary role for raft membranes in rotavirus final assembly and the fact that VP4 assembly with the rest of the particle is an extrareticular event.