Sequence of the region coding for virion proteins C and E2 and the carboxy terminus of the nonstructural proteins of rubella virus: comparison with alphaviruses

The sequence of the 3' 4508 nucleotides (nt) of the genomic RNA of the Therien strain of rubella virus (RV) was determined for cDNA clones. The sequence contains a 3189-nt open reading frame (ORF) which codes for the structural proteins C, E2 and E1. C is predicted to have a length of 300 amino acids (aa). The N-terminal half of the C protein is highly basic and hydrophilic in nature, and is putatively the region of the protein which interacts with the virion RNA. At the C terminus of the C protein is a stretch of 20 hydrophobic aa which also serves as the signal sequence for E2, indicating that the cleavage of C from the polyprotein precursor may be catalyzed by signalase in the lumen of the endoplasmic reticulum. E2 is 282 aa in length and contains four potential N-linked glycosylation sites and a putative transmembrane domain near its C terminus. The sequence of E1 has been previously described [Frey et al., Virology 154 (1986) 228-232]. No homology could be detected between the amino acid sequence of the RV structural proteins and the amino acid sequence of the alphavirus structural proteins. From the position of a region of 30 nt in the RV genomic sequence which exhibited significant homology with the sequence in the alphavirus genome at which subgenomic RNA synthesis is initiated, the RV subgenomic RNA is predicted to be 3346 nt in length and the nontranslated region from the 5' end of the subgenomic RNA to the structural protein ORF is predicted to be 98 nt. In a different translation frame beginning at the 5' end of the RV nt sequence reported here is a 1407 nt ORF which is the C terminal region of the nonstructural protein ORF. This ORF overlaps the structural protein ORF by 149 nt. A low level of homology could be detected between the predicted amino acid sequence of the C-terminus of the RV nonstructural protein ORF and the replicase proteins of several positive RNA viruses of animals and plants, including nsp4 of the alphaviruses, the protein encoded by the C-terminal region of the alphavirus nonstructural ORF. However, the overall homology between RV and the alphaviruses in this region of the genome was only 18%, indicating that these two genera of the Togavirus family are only distantly related. Intriguingly, there is a 2844-nt ORF present in the negative polarity orientation of the RV sequence which could encode a 928-aa polyprotein.     

Subgenomic mRNA of Aura alphavirus is packaged into virions.

Purified virions of Aura virus, a South American alphavirus related to Sindbis virus, were found to contain two RNA species, one of 12 kb and the other of 4.2 kb. Northern (RNA) blot analysis, primer extension analysis, and limited sequencing showed that the 12-kb RNA was the viral genomic RNA, whereas the 4.2-kb RNA present in virus preparations was identical to the 26S subgenomic RNA present in infected cells. The subgenomic RNA is the messenger for translation of the viral structural proteins, and its synthesis is absolutely required for replication of the virus. Although 26S RNA is present in the cytosol of all cells infected by alphaviruses, this is the first report of incorporation of the subgenomic RNA into alphavirus particles. Packaging of the Aura virus subgenomic mRNA occurred following infection of mosquito (Aedes albopictus C6/36), hamster (BHK-21), or monkey (Vero) cells. Quantitation of the amounts of genomic and subgenomic RNA both in virions and in infected cells showed that the ratio of genomic to subgenomic RNA was 3- to 10-fold higher in Aura virions than in infected cells. Thus, although the subgenomic RNA is packaged efficiently, the genomic RNA has a selective advantage during packaging. In contrast, in parallel experiments with Sindbis virus, packaging of subgenomic RNA was not detectable. We also found that subgenomic RNA was present in about threefold-greater amounts relative to genomic RNA in cells infected by Aura virus than in cells infected by Sindbis virus. Packaging of the Aura virus subgenomic RNA, but not those of other alphaviruses, suggests that Aura virus 26S RNA contains a packaging signal for incorporation into virions. The importance of the packaging of this RNA into virions in the natural history of the virus remains to be determined.     

Heterologous interference in Aedes albopictus cells infected with alphaviruses.

Maximum amounts of 42S and 26S single-stranded viral RNA and viral structural proteins were synthesized in Aedes albopictus cells at 24 h after Sindbis virus infection. Thereafter, viral RNA and protein syntheses were inhibited. By 3 days postinfection, only small quantities of 42S RNA and no detectable 26S RNA or structural proteins were synthesized in infected cells. Superinfection of A. albopictus cells 3 days after Sindbis virus infection with Sindbis, Semliki Forest, Una, or Chikungunya alphavirus did not lead to the synthesis of intracellular 26S viral RNA. In contrast, infection with snowshoe hare virus, a bunyavirus, induced the synthesis of snowshoe hare virus RNA in both A. Ablpictus cells 3 days after Sindbis virus infection and previously uninfected mosquito cells. These results suggested that at 3 days after infection with Sindbis virus, mosquito cells restricted the replication of both homologous and heterologous alphaviruses but remained susceptible to infection with a bunyavirus. In superinfection experiments the the alphaviruses were differentiated on the basis of plaque morphology and the electrophoretic mobility of their intracellular 26S viral RNA species. Thus, it was shown that within 1 h after infection with eigher Sindbis or Chikungunya virus, A. albopictus cells were resistant to superinfection with Sindbis, Chikungunya, Una, and Semliki Forest viruses. Infected cultures were resistant to superinfection with the homologous virus indefinitely, but maximum resistance to superinfection with heterologous alphaviruses lasted for approximately 8 days. After that time, infected cultures supported the replication of heterologous alphaviruses to the same extent as did persistently infected cultures established months previously. However, the titer of heterologous alphavirus produced after superinfection of persistently infected cultures was 10- to 50-fold less than that produced by an equal number of previously uninfected A. albopictus cells. Only a small proportion (8 to 10%) of the cells in a persistently infected culture was capable of supporting the replication of a heterologous alphavirus.     

New subtype of salmonid alphavirus (SAV), Togaviridae, from Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss in Norway

In Europe, 2 closely related alphaviruses (Togaviridae) are regarded as the causative agents of sleeping disease (SD) and salmon pancreas disease (SPD): SD virus (SDV) has been isolated from rainbow trout Oncorhynchus mykiss in France and the UK, while SPD virus (SPDV) has been isolated from salmon Salmo salar in Ireland and the UK. Farmed salmonids in western Norway also suffer from a disease called pancreas disease (PD), and this disease is also believed to be caused by an alphavirus. However, this virus has not yet been characterised at the molecular level. We have cultured a Norwegian salmonid alphavirus from moribund fishes diagnosed with cardiac myopathy syndrome (CMS) and fishes diagnosed with PD. The virus has also been found in salmon suffering from haemorrhagic smolt syndrome in the fresh water phase. The genomic organisation of the Norwegian salmonid alphavirus is identical to that in SPDV and SDV, and the nucleotide sequence similarity to the other 2 alphaviruses is 91.6 and 92.9%, respectively. Based on the pathological changes, host species and the nucleotide sequence, we suggest naming this virus Norwegian salmonid alphavirus (NSAV). Together with SPDV and SDV it constitutes a third subtype of salmonid alphavirus (SAV) species within the genus Alphavirus, family Togaviridae.     

A 6K-Deletion Variant of Salmonid Alphavirus Is Non-Viable but Can Be Rescued through RNA Recombination

Pancreas disease (PD) of Atlantic salmon is an emerging disease caused by Salmonid alphavirus (SAV) which mainly affects salmonid aquaculture in Western Europe. Although genome structure of SAV has been characterized and each individual viral protein has been identified, the role of 6K protein in viral replication and infectivity remains undefined. The 6K protein of alphaviruses is a small and hydrophobic protein which is involved in membrane permeabilization, protein processing and virus budding. Because these common features are shared across many viral species, they have been named viroporins. In the present study, we applied reverse genetics to generate SAV3 6K-deleted (Δ6K) variant and investigate the role of 6K protein. Our findings show that the 6K-deletion variant of salmonid alphavirus is non-viable. Despite viral proteins of Δ6K variant are detected in the cytoplasm by immunostaining, they are not found on the cell surface. Further, analysis of viral proteins produced in Δ6K cDNA clone transfected cells using radioimmunoprecipitation (RIPA) and western blot showed a protein band of larger size than E2 of wild-type SAV3. When Δ6K cDNA was co-transfected with SAV3 helper cDNA encoding the whole structural genes including 6K, the infectivity was rescued. The development of CPE after co-transfection and resolved genome sequence of rescued virus confirmed full-length viral genome being generated through RNA recombination. The discovery of the important role of the 6K protein in virus production provides a new possibility for the development of antiviral intervention which is highly needed to control SAV infection in salmonids.     

Rubella virus 40S genome RNA specifies a 24S subgenomic mRNA that codes for a precursor to structural proteins.

We have analyzed the structure of the rubella virus genome RNA and the virus-specific RNA species synthesized in B-Vero cells infected with rubella virus. A single-stranded, capped, and polyadenylated RNA species sedimenting at 40S in a sucrose gradient was released from purified virions treated with sodium dodecyl sulfate. This RNA species migrated with an Mr of about 3.8 X 10(6) in an agarose gel after denaturation with glyoxal and dimethyl sulfoxide. Infected cells labeled with [3H]uridine in the presence of actinomycin D contained, in addition to the 40S RNA, a single-stranded polyadenylated 24S RNA species as shown by sucrose gradient analysis. In a Northern blot analysis, this RNA hybridized to a cDNA probe derived from the 3' portion of the genomic 40S RNA. In vitro translation of the 24S RNA species yielded a 110,000-dalton polypeptide, in addition to some smaller products which were immunoprecipitated with an antiserum prepared against the structural proteins E1, E2a, E2b, and C. Since the sum of the molecular weights of the nonglycosylated envelope proteins and the capsid protein has been estimated to be about 116,000 (C. Oker-Blom et al., J. Virol. 46:964-973, 1983), these results suggest that the 24S RNA species represents a subgenomic mRNA coding for a precursor (p110) to the structural proteins of rubella virus. Thus, the strategy of gene expression of rubella virus appears to be similar to that of the alphaviruses.     

Ribosomal protein S6 associates with alphavirus nonstructural protein 2 and mediates expression from alphavirus messages

Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.     

Conservation of a packaging signal and the viral genome RNA packaging mechanism in alphavirus evolution

Alphaviruses are a group of small, enveloped viruses which are widely distributed on all continents. In infected cells, alphaviruses display remarkable specificity in RNA packaging by encapsidating only their genomic RNA while avoiding packaging of the more abundant viral subgenomic (SG), cellular messenger and transfer RNAs into released virions. In this work, we demonstrate that in spite of evolution in geographically isolated areas and accumulation of considerable diversity in the nonstructural and structural genes, many alphaviruses belonging to different serocomplexes harbor RNA packaging signals (PSs) which contain the same structural and functional elements. Their characteristic features are as follows. (i) Sindbis, eastern, western, and Venezuelan equine encephalitis and most likely many other alphaviruses, except those belonging to the Semliki Forest virus (SFV) clade, have PSs which can be recognized by the capsid proteins of heterologous alphaviruses. (ii) The PS consists of 4 to 6 stem-loop RNA structures bearing conserved GGG sequences located at the base of the loop. These short motifs are integral elements of the PS and can function even in the artificially designed PS. (iii) Mutagenesis of the entire PS or simply the GGG sequences has strong negative effects on viral genome packaging and leads to release of viral particles containing mostly SG RNAs. (iv) Packaging of RNA appears to be determined to some extent by the number of GGG-containing stem-loops, and more than one stem-loop is required for efficient RNA encapsidation. (v) Viruses of the SFV clade are the exception to the general rule. They contain PSs in the nsP2 gene, but their capsid protein retains the ability to use the nsP1-specific PS of other alphaviruses. These new discoveries regarding alphavirus PS structure and function provide an opportunity for the development of virus variants, which are irreversibly attenuated in terms of production of infectious virus but release high levels of genome-free virions.     

Comparison of two aquatic alphaviruses,salmon pancreas disease virus and sleeping disease virus,by using genome sequence analysis,monoclonal reactivity,and cross-infection

Cell culture isolates of salmon pancreas disease virus (SPDV) of farmed Atlantic salmon and sleeping disease virus (SDV) of rainbow trout were compared. Excluding the poly(A) tracts, the genomic nucleotide sequences of SPDV and SDV RNAs include 11,919 and 11,900 nucleotides, respectively. Phylogenetic analysis places SPDV and SDV between the New World viruses of Venezuelan equine encephalitis virus and Eastern equine encephalitis virus and the Old World viruses of Aura virus and Sindbis virus. When compared to each other, SPDV and SDV show 91.1% nucleotide sequence identity over their complete genomes, with 95 and 93.6% amino acid identities over their nonstructural and structural proteins, respectively. Notable differences between the two viruses include a 24-nucleotide insertion in the C terminus of nsP3 protein of SPDV and amino acid sequence variation at the C termini of the capsid and E1 proteins. Experimental infections of Atlantic salmon and rainbow trout with SPDV and SDV confirmed that the disease lesions induced by SPDV and SDV were similar in nature. Although infections with SPDV and SDV produced similar levels of histopathology in rainbow trout, SDV induced significantly less severe lesions in salmon than did SPDV. Virus neutralization tests performed with sera from experimentally infected salmon indicated that SPDV and SDV belonged to the same serotype; however, antigenic variation was detected among SDV and geographically different SPDV isolates by using monoclonal antibodies. Although SPDV and SDV exhibit minor biological differences, we conclude on the basis of the close genetic similarity that SPDV and SDV are closely related isolates of the same virus species for which the name Salmonid alphavirus is proposed.     

Functional defects of RNA-negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses.

Defects in RNA and protein synthesis of seven Sindbis virus and seven Semliki Forest virus RNA-negative, temperature-sensitive mutants were studied after shift to the restrictive temperature (39 degrees C) in the middle of the growth cycle. Only one of the mutants, Ts-6 of Sindbis virus, a representative of complementation group F, was clearly unable to continue RNA synthesis at 39 degrees C, apparently due to temperature-sensitive polymerase. The defect was reversible and affected the synthesis of both 42S and 26S RNA equally, suggesting that the same polymerase component(s) is required for the synthesis of both RNA species. One of the three Sindbis virus mutants of complementation group A, Ts-4, and one RNA +/- mutant of Semliki Forest virus, ts-10, showed a polymerase defect even at the permissive temperature. Seven of the 14 RNA-negative mutants showed a preferential reduction in 26S RNA synthesis. The 26S RNA-defective mutants of Sindbis virus were from two different complementation groups, A and G, indicating that functions of two viral nonstructural proteins ("A" and "G") are required in the regulation of the synthesis of 26S RNA. Since the synthesis of 42S RNA continued, these functions of proteins A and G are not needed for the polymerization of RNA late in infection. The RNA-negative phenotype of 26S RNA-deficient mutants implies that proteins regulating the synthesis of this subgenomic RNA must have another function vital for RNA synthesis early in infection or in the assembly of functional polymerase. Several of the mutants having a specific defect in the synthesis of 26S RNA showed an accumulation of a large nonstructural precursor protein with a molecular weight of about 200,000. One even larger protein was demonstrated in both Semliki Forest virus- and Sindbis virus-infected cells which probably represents the entire nonstructural polyprotein.     

Subgenomic Reporter RNA System for Detection of Alphavirus Infection in Mosquitoes

Current methods for detecting real-time alphavirus (Family Togaviridae) infection in mosquitoes require the use of recombinant viruses engineered to express a visibly detectable reporter protein. These altered viruses expressing fluorescent proteins, usually from a duplicated viral subgenomic reporter, are effective at marking infection but tend to be attenuated due to the modification of the genome. Additionally, field strains of viruses cannot be visualized using this approach unless infectious clones can be developed to insert a reporter protein. To circumvent these issues, we have developed an insect cell-based system for detecting wild-type sindbis virus infection that uses a virus inducible promoter to express a fluorescent reporter gene only upon active virus infection. We have developed an insect expression system that produces sindbis virus minigenomes containing a subgenomic promoter sequence, which produces a translatable RNA species only when infectious virus is present and providing viral replication proteins. This subgenomic reporter RNA system is able to detect wild-type Sindbis infection in cultured mosquito cells. The detection system is relatively species specific and only detects closely related viruses, but can detect low levels of alphavirus specific replication early during infection. A chikungunya virus detection system was also developed that specifically detects chikungunya virus infection. Transgenic Aedes aegypti mosquito families were established that constitutively express the sindbis virus reporter RNA and were found to only express fluorescent proteins during virus infection. This virus inducible reporter system demonstrates a novel approach for detecting non-recombinant virus infection in mosquito cell culture and in live transgenic mosquitoes.     

Neoplastic transformation by E26 leukemia virus is mediated by a single protein containing domains of gag and myb genes.

The genome of avian leukemia virus E26 shares homology with v-myb, the oncogene of avian myeloblastosis virus, and encodes a protein with an Mr of 135,000. Analyses of tryptic oligopeptides show that this protein is related to the proteins encoded by gag (Pr76gag) as well as v-myb (p45v-myb[AMV] ) and c-myb (p75c-myb). We found no evidence for the existence of additional myb-related proteins or subgenomic species of myb-related RNA in myeloblasts transformed by strain E26.     

Genome-Wide RNAi Screen Identifies Novel Host Proteins Required for Alphavirus Entry

The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ), a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.     

Structure of the Recombinant Alphavirus Western Equine Encephalitis Virus Revealed by Cryoelectron Microscopy

Western equine encephalitis virus (WEEV; Togaviridae, Alphavirus) is an enveloped RNA virus that is typically transmitted to vertebrate hosts by infected mosquitoes. WEEV is an important cause of viral encephalitis in humans and horses in the Americas, and infection results in a range of disease, from mild flu-like illnesses to encephalitis, coma, and death. In addition to spreading via mosquito vectors, human WEEV infections can potentially occur directly via aerosol transmission. Due to its aerosol infectivity and virulence, WEEV is thus classified as a biological safety level 3 (BSL-3) agent. Because of its highly infectious nature and containment requirements, it has not been possible to investigate WEEV''s structure or assembly mechanism using standard structural biology techniques. Thus, to image WEEV and other BSL-3 agents, we have constructed a first-of-its-kind BSL-3 cryoelectron microscopy (cryoEM) containment facility. cryoEM images of WEEV were used to determine the first three-dimensional structure of this important human pathogen. The overall organization of WEEV is similar to those of other alphaviruses, consistent with the high sequence similarity among alphavirus structural proteins. Surprisingly, the nucleocapsid of WEEV, a New World virus, is more similar to the Old World alphavirus Sindbis virus than to other New World alphaviruses.The alphaviruses comprise a genus of single-stranded, plus-sense, enveloped RNA viruses that, together with rubella virus, comprise the family Togaviridae. The current classification of the genus Alphavirus includes 29 different species, with multiple subtypes and/or varieties represented within some species (30). These species can be grouped into 8 different complexes based on antigenic and/or genetic similarities (20). Most viruses from the New World are found in the Eastern, Venezuelan, and Western equine encephalitis (EEE, VEE, and WEE, respectively) complexes and cause encephalitis in humans and a variety of domesticated animals. Old World alphaviruses, on the other hand, typically cause only an arthralgia and rash syndrome that is rarely life threatening (5, 24). Among the New World alphaviruses, EEE, VEE, and WEE viruses (EEEV, VEEV, and WEEV, respectively) are potential biological weapons as well as naturally emerging pathogens and are therefore included on the category B Priority Pathogens list of the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (http://www.niaid.nih.gov/topics/biodefenserelated/biodefense/research/pages/cata.aspx).Alphaviruses replicate in the cytoplasm of infected cells after entry via receptor-mediated endocytosis (8). Following internalization, fusion of the viral envelope with the endocytic membrane is mediated by a low-pH-induced conformational change that exposes a fusion peptide found in the E1 envelope glycoprotein. The nucleocapsid then disassembles upon interactions with ribosomes, and an open reading frame (ORF) found in the 5′ two-thirds of the genome is translated. The resultant polyprotein is cleaved into 4 nonstructural proteins (nsP1 to -4) that mediate viral RNA replication, RNA capping, and polyprotein processing (Fig. ​(Fig.1).1). The structural proteins, including the two envelope glycoproteins E2 and E1 as well as the capsid protein, are encoded in a second ORF that is translated from a subgenomic message often referred to as 26S RNA. Following auto-cleavage of the capsid protein in the cytoplasm, the remaining polyprotein is inserted into the endoplasmic reticulum, where it is cleaved by host cell proteases and then processed through the secretory pathway, where the glycosylation of E2 and E1 occurs. Virion maturation occurs after E2/E1 heterodimers are inserted into the plasma membrane and 240 copies of the capsid protein interact with one copy of the genomic RNA to form nucleocapsids. These nucleocapsids then interact with a cytoplasmic domain of the E2 protein to initiate budding. The mature virion thus includes 240 copies of the capsid protein and 240 E2/E1 heterodimers arranged as trimeric spikes on the surface of the virus (8).Open in a separate windowFIG. 1.Diagram of the alphavirus genome, showing the 5′ cap, 5′ untranslated region, nonstructural polyprotein open reading frame, and major functions of the individual proteins, subgenomic promoter, structural polyprotein open reading frame, 3′ untranslated region, and poly(A) tail.The structures of several different alphaviruses, including Sindbis virus (SINV) (13), Ross River virus (RRV) (3, 35), Semliki Forest virus (SFV), (11), and VEEV (16), have been solved to subnanometer resolution using cryoelectron microscopy (cryoEM), and the X-ray crystallographic structure of the E1 protein from Semliki Forest virus has been determined to atomic resolution (9). The alphaviruses are ca. 700 Å in diameter, with 80 trimeric spikes on their surfaces. By fitting the E1 crystal structure into cryoEM reconstruction maps of whole viruses, the orientations of both envelope proteins within the spikes have been estimated (36). The E1 and E2 proteins are similar in shape, and the E2 proteins extend to the tips of the spikes, where most glycosylation and antibody-binding sites have been mapped (13). The underlying T=4 icosahedral capsid is constructed from regularly ordered capsomers arranged as hexons and pentons. These pentons and hexons consist of capsid protein monomers that apparently represent only the C-terminal half of the protein. Crystal structures of alphavirus capsid proteins also indicate that only the C terminus, including the protease domain, is ordered (25). cryoEM reconstructions of VEEV nucleocapsids isolated from virions have a less ordered structure, with density redistributed from the 3-fold to the 5-fold axis, suggesting that the envelope and/or the envelope glycoproteins constrain and stabilize the nucleocapsid in a compressed structure (15). Additionally, the VEEV nucleocapsids within viruses differ from those of Old World alphaviruses, with a counterclockwise rotation of the pentameric and hexameric capsomers in VEEV (16). Similar differences were observed in the capsid of Aura virus (AURAV), another New World alphavirus (34).In addition to being an important human and equine pathogen, WEEV is one of three alphaviruses that descended from a recombinant ancestor (6, 31). This ancestor derived its nonstructural and capsid protein genes from an ancestral EEEV strain, whereas its envelope glycoprotein genes were provided from an ancestral SINV. The recombination event was apparently followed by compensatory mutations in the cytoplasmic domain of the E2 protein that restored efficient interactions with the EEEV-like capsid protein (6). If this interpretation of the WEEV ancestral recombination event is correct, its nucleocapsids, constructed from capsid proteins derived from the New World EEEV ancestor, would be expected be more similar to those of the New World VEEV than to those of the Old World SINV, RRV, and SFV. To test this hypothesis and to investigate other structural features of interest related to its recombinant history and pathogenicity, we determined the structure of WEEV to a 13-Å resolution using cryoEM image reconstruction.     

An attenuating mutation in nsP1 of the Sindbis-group virus S.A.AR86 accelerates nonstructural protein processing and up-regulates viral 26S RNA synthesis

The Sindbis-group alphavirus S.A.AR86 encodes a threonine at nonstructural protein 1 (nsP1) 538 that is associated with neurovirulence in adult mice. Mutation of the nsP1 538 Thr to the consensus Ile found in nonneurovirulent Sindbis-group alphaviruses attenuates S.A.AR86 for adult mouse neurovirulence, while introduction of Thr at position 538 in a nonneurovirulent Sindbis virus background confers increased neurovirulence (M. T. Heise et al., J. Virol. 74:4207-4213, 2000). Since changes in the viral nonstructural region are likely to affect viral replication, studies were performed to evaluate the effect of Thr or Ile at nsP1 538 on viral growth, nonstructural protein processing, and RNA synthesis. Multistep growth curves in Neuro2A and BHK-21 cells revealed that the attenuated s51 (nsP1 538 Ile) virus had a slight, but reproducible growth advantage over the wild-type s55 (nsP1 538 Thr) virus. nsP1 538 lies within the cleavage recognition domain between nsP1 and nsP2, and the presence of the attenuating Ile at nsP1 538 accelerated the processing of S.A.AR86 nonstructural proteins both in vitro and in infected cells. Since nonstructural protein processing is known to regulate alphavirus RNA synthesis, experiments were performed to evaluate the effect of Ile or Thr at nsP1 538 on viral RNA synthesis. A combination of S.A.AR86-derived reporter assays and RNase protection assays determined that the presence of Ile at nsP1 538 led to earlier expression from the viral 26S promoter without affecting viral minus- or plus-strand synthesis. These results suggest that slower nonstructural protein processing and delayed 26S RNA synthesis in wild-type S.A.AR86 infections may contribute to the adult mouse neurovirulence phenotype of S.A.AR86.