4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus

Zhang, Rui; Hryc, Corey F.; Yao, Cong; Liu, Xiangan; Jakana, Joanita; Gorchakov, Rodion; Baker, Matthew L.; Weaver, Scott C.; Chiu, Wah

doi:10.1038/emboj.2011.261

Cited by 186 publications

(268 citation statements)

References 67 publications

(84 reference statements)

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“…Semliki Forest virus and VEEV are also reported to incorporate E3 into virions (35,36). We are attempting to produce higherresolution EILV cryoEM maps to confirm this interpretation.…”

Section: Discussionmentioning

confidence: 97%

Eilat virus, a unique alphavirus with host range restricted to insects by RNA replication

Nasar

Palacios

Gorchakov

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

168

189

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Most alphaviruses and many other arboviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates including birds, rodents, equids, humans, and nonhuman primates. Consequently, they can be propagated in most vertebrate and insect cell cultures. This ability of arboviruses to infect arthropods and vertebrates is usually essential for their maintenance in nature. However, several flaviviruses have recently been described that infect mosquitoes but not vertebrates, although the mechanism of their host restriction has not been determined. Here we describe a unique alphavirus, Eilat virus (EILV), isolated from a pool of Anopheles coustani mosquitoes from the Negev desert of Israel. Phylogenetic analyses placed EILV as a sister to the Western equine encephalitis antigenic complex within the main clade of mosquito-borne alphaviruses. Electron microscopy revealed that, like other alphaviruses, EILV virions were spherical, 70 nm in diameter, and budded from the plasma membrane of mosquito cells in culture. EILV readily infected a variety of insect cells with little overt cytopathic effect. However, in contrast to typical mosquito-borne alphaviruses, EILV could not infect mammalian or avian cell lines, and viral as well as RNA replication could not be detected at 37°C or 28°C. Evolutionarily, these findings suggest that EILV lost its ability to infect vertebrate cells. Thus, EILV seems to be mosquito-specific and represents a previously undescribed complex within the genus Alphavirus. Reverse genetic studies of EILV may facilitate the discovery of determinants of alphavirus host range that mediate disease emergence.evolution | Togavirus

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“…Semliki Forest virus and VEEV are also reported to incorporate E3 into virions (35,36). We are attempting to produce higherresolution EILV cryoEM maps to confirm this interpretation.…”

Section: Discussionmentioning

confidence: 97%

Eilat virus, a unique alphavirus with host range restricted to insects by RNA replication

Nasar

Palacios

Gorchakov

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

168

189

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“…At neutral pH, an alphavirus particle has a T = 4 icosahedral structure that consists of a nucleocapsid core, a viral membrane, and a glycoprotein shell (10)(11)(12)(13). The nucleocapsid core includes an RNA genome and 240 copies of the capsid protein.…”

mentioning

confidence: 99%

Characterization of an early-stage fusion intermediate of Sindbis virus using cryoelectron microscopy

Cao

Zhang

2013

Proc. Natl. Acad. Sci. U.S.A.

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The sequential steps in the alphavirus membrane fusion pathway have been postulated based on the prefusion and postfusion crystal structures of the viral fusion protein E1 in conjunction with biochemical studies. However, the molecular structures of the hypothesized fusion intermediates have remained obscure due to difficulties inherent in the dynamic nature of the process. We developed an experimental system that uses liposomes as the target membrane to capture Sindbis virus, a prototypical alphavirus, in its membrane-binding form at pH 6.4. Cryoelectron micrograph analyses and 3D reconstructions showed that the virus retains its overall icosahedral structure at this mildly acidic pH, except in the membrane-binding region, where monomeric E1 associates with the target membrane and the E2 glycoprotein retains its original trimeric organization. The remaining E2 trimers may hinder E1 homotrimerization and are a potential target for antiviral drugs.enveloped viruses | class II viral fusion protein | viral entry | protein organization | image processing A lphaviruses, a genus of the Togaviridae family, have been an important experimental paradigm for studying virus membrane fusion for 3 decades (1, 2). Enveloped alphaviruses, such as Sindbis virus (SINV), Semliki Forest virus (SFV), and Chikungunya virus (CHIKV), enter host cells via receptor-mediated endocytosis followed by low-pH-triggered membrane fusion within the endosome (3, 4). Liposome-based model systems involving low-pH treatment have provided a convenient in vitro method for studying alphavirus membrane fusion and have greatly advanced our understanding of the molecular mechanism underlying this dynamic process (5-9).At neutral pH, an alphavirus particle has a T = 4 icosahedral structure that consists of a nucleocapsid core, a viral membrane, and a glycoprotein shell (10-13). The nucleocapsid core includes an RNA genome and 240 copies of the capsid protein. The exterior glycoprotein shell features 80 spikes projecting from the viral membrane. Each spike is composed of three copies of the E1-E2 glycoprotein heterodimer in a right-handed arrangement. Both E1 and E2 span the viral membrane. The cytoplasmic tail of E2 interacts with the viral capsid, whereas the ectodomain of E2 makes up the central and outermost portions of each spike. The ectodomain of E1 is oriented almost tangentially to the viral membrane, forming a region called the "skirt" within the glycoprotein shell (11,14).To date, structural insights into alphavirus membrane fusion have been shaped largely by X-ray crystallographic studies of the E1 and E2 molecules in their prefusion states (15-17) as well as the E1 postfusion trimer (18). At its outermost tip, E1 contains a hydrophobic fusion loop that is sequestered by the companion E2 protein at neutral pH. Once low pH triggers dissociation of the E1-E2 heterodimer, the E1 fusion loop inserts itself into the endosomal membrane. Thereafter, the E1 protein forms a homotrimer that mediates fusion of the viral and target membranes through a foldi...

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“…Cryo-electron microscopy (cryo-EM) reconstructions of alphaviruses show the arrangement of the structural proteins in the alphavirus particle (3,18,27,31,44,51,52,54). SINV has an external diameter of 700 Å and contains 240 copies each of the E2 (423 amino acid residues), E1 (439 amino acid residues), and capsid protein (CP; 64 amino acid residues), all of which are arranged with icosahedral Tϭ4 quasisymmetry (2).…”

mentioning

confidence: 99%

“…The palmitoylated cysteines in cdE2 are essential for the membrane anchoring and correct orientation of cdE2 on the CP (51). The binding of cdE2 into the hydrophobic pocket of the CP is aided by the palmitoylated cysteines 416 and 417.…”

mentioning

confidence: 99%

Interactions of the Cytoplasmic Domain of Sindbis Virus E2 with Nucleocapsid Cores Promote Alphavirus Budding

Jose¹,

Przybyla²,

Edwards³

et al. 2012

J Virol

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Alphavirus budding from the plasma membrane occurs through the specific interaction of the nucleocapsid core with the cytoplasmic domain of the E2 glycoprotein (cdE2). Structural studies of the Sindbis virus capsid protein (CP) have suggested that these critical interactions are mediated by the binding of cdE2 into a hydrophobic pocket in the CP. Several molecular genetic studies have implicated amino acids Y400 and L402 in cdE2 as important for the budding of alphaviruses. In this study, we characterized the role of cdE2 residues in structural polyprotein processing, glycoprotein transport, and capsid interactions. Along with hydrophobic residues, charged residues in the N terminus of cdE2 were critical for the effective interaction of cores with cdE2, a process required for virus budding. Mutations in the C-terminal signal sequence region of cdE2 affected E2 protein transport to the plasma membrane, while nonbudding mutants that were defective in cdE2-CP interaction accumulated E2 on the plasma membrane. The interaction of cdE2 with cytoplasmic cores purified from infected cells and in vitro-assembled corelike particles suggests that cdE2 interacts with assembled cores to mediate budding. We hypothesize that these cdE2 interactions induce a change in the organization of the nucleocapsid core upon binding leading to particle budding and priming of the nucleocapsid cores for disassembly that is required for virus infection.

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4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus

Cited by 186 publications

References 67 publications

Eilat virus, a unique alphavirus with host range restricted to insects by RNA replication

Eilat virus, a unique alphavirus with host range restricted to insects by RNA replication

Characterization of an early-stage fusion intermediate of Sindbis virus using cryoelectron microscopy

Interactions of the Cytoplasmic Domain of Sindbis Virus E2 with Nucleocapsid Cores Promote Alphavirus Budding

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