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.