The Structure of Barmah Forest Virus as Revealed by Cryo-Electron Microscopy at a 6-Angstrom Resolution Has Detailed Transmembrane Protein Architecture and Interactions

Kostyuchenko, V.A.; Jakana, Joanita; Liu, Xiangan; Haddow, Andrew D.; Aung, Myint Thazin; Weaver, Scott C.; Chiu, Wah; Lok, Shee-Mei

doi:10.1128/jvi.05015-11

Cited by 57 publications

(57 citation statements)

References 39 publications

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“…To date, characterizations of alphaviral particles have not identified any overt differences in morphology between particles derived from the two hosts (27)(28)(29). Differences in particle composition between alphaviral particles generated in mammalian and mosquito hosts have been described.…”

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confidence: 99%

Encapsidation of Host-Derived Factors Correlates with Enhanced Infectivity of Sindbis Virus

Sokoloski

Snyder²,

Liu

et al. 2013

J Virol

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The genus Alphavirus consists of a group of enveloped, single-stranded RNA viruses, many of which are transmitted by arthropods to a wide range of vertebrate host species. Here we report that Sindbis virus (SINV) produced from a representative mammalian cell line consists of at least two unique particle subpopulations, separable on the basis of virion density. In contrast, mosquito-derived SINV consists of a homogeneous population of particles. Our findings indicate that the denser particle subpopulation, SINV Heavy , is more infectious on a per-particle basis than SINV Light . SINV produced in mosquito cell lines (SINV C6/36 ) exhibited particle-to-PFU ratios similar to those observed for SINV Heavy . In mammalian cells, viral RNA was synthesized and accumulated more rapidly following infection with SINV Heavy or SINV C6/36 than following infection with SINV Light , due partly to enhanced translation of viral genomic RNA early in infection. Analysis of the individual particle subpopulations indicated that SINV Heavy and SINV C6/36 contain host-derived factors whose presence correlates with the enhanced translation, RNA synthesis, and infectivity observed for these particles. Members of the genus Alphavirus, of the family Togaviridae, are a group of enveloped positive-sense RNA viruses with a wide host range. For the mosquito-borne species, the virus is maintained in the enzootic cycle through transmission between a sylvatic reservoir and the mosquito host (1). The maintenance of this cycle directly affects the genetic fitness of the mosquito-borne alphaviruses. Prolonged disruption of this cycle leads to deleterious effects on viral transmission as the virus becomes adapted to a single host (2-5). Spillover from the enzootic cycle often results in the tangential infection of both humans and equines, which can result in significant outbreaks of disease. The outcome of alphaviral infection is dependent on the host system (6-12). Infection of mosquito cells does not result in the shutoff of host macromolecular synthesis and often culminates in persistent infection for the majority of mosquito cell lines (12-15). Nevertheless, cell death as a result of infection has been reported for several members of the genus in whole mosquitoes (16)(17)(18)(19)(20)(21). In contrast, infection of mammalian cells induces the shutoff of host macromolecular synthesis, resulting in a predominantly cytolytic infection. In vertebrates, the immune response to infection generally results in virus clearance. This is initiated by the recognition of viral doublestranded RNA and a rapid type I interferon (IFN-␣/␤) response (22)(23)(24)(25).Previously, we reported that the infectivity of Sindbis virus (SINV), as measured by the ratio of particles to infectious units, depends on the host cell line from which it is derived (26). SINV derived from mammalian cell lines exhibited a higher particle-to-PFU ratio, on average, than SINV generated from mosquito cell lines. This was due largely to differences in the quantity of total virus particles pr...

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confidence: 99%

Encapsidation of Host-Derived Factors Correlates with Enhanced Infectivity of Sindbis Virus

Sokoloski

Snyder²,

Liu

et al. 2013

J Virol

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“…The structures of a number of alphaviruses have been determined to resolutions better than 10 Å, including the structure of VEEV, which has been determined to a 4.8-Å resolution (6)(7)(8)(9). Alphaviruses are icosahedral with quasi-Tϭ4 symmetry and contain a positive-sense single-stranded RNA (ssRNA) genome.…”

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Locking and Blocking the Viral Landscape of an Alphavirus with Neutralizing Antibodies

Porta

Jose

Roehrig

et al. 2014

J Virol

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Alphaviruses are serious, sometimes lethal human pathogens that belong to the family Togaviridae. The structures of human Venezuelan equine encephalitis virus (VEEV), an alphavirus, in complex with two strongly neutralizing antibody Fab fragments (F5 and 3B4C-4) have been determined using a combination of cryo-electron microscopy and homology modeling. We characterize these monoclonal antibody Fab fragments, which are known to abrogate VEEV infectivity by binding to the E2 (envelope) surface glycoprotein. Both of these antibody Fab fragments cross-link the surface E2 glycoproteins and therefore probably inhibit infectivity by blocking the conformational changes that are required for making the virus fusogenic. The F5 Fab fragment cross-links E2 proteins within one trimeric spike, whereas the 3B4C-4 Fab fragment cross-links E2 proteins from neighboring spikes. Furthermore, F5 probably blocks the receptor-binding site, whereas 3B4C-4 sterically hinders the exposure of the fusion loop at the end of the E2 B-domain. IMPORTANCEAlphaviral infections are transmitted mainly by mosquitoes. Venezuelan equine encephalitis virus (VEEV) is an alphavirus with a wide distribution across the globe. No effective vaccines exist for alphaviral infections. Therefore, a better understanding of VEEV and its associated neutralizing antibodies will help with the development of effective drugs and vaccines.

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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.…”

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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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The Structure of Barmah Forest Virus as Revealed by Cryo-Electron Microscopy at a 6-Angstrom Resolution Has Detailed Transmembrane Protein Architecture and Interactions

Cited by 57 publications

References 39 publications

Encapsidation of Host-Derived Factors Correlates with Enhanced Infectivity of Sindbis Virus

Encapsidation of Host-Derived Factors Correlates with Enhanced Infectivity of Sindbis Virus

Locking and Blocking the Viral Landscape of an Alphavirus with Neutralizing Antibodies

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

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