Membrane fusion of Semliki Forest virus in a model system: correlation between fusion kinetics and structural changes in the envelope glycoprotein.

Bron, Romke; Wahlberg, J M; Garoff, Henrik; Wilschut, Jan

doi:10.1002/j.1460-2075.1993.tb05703.x

Cited by 166 publications

(264 citation statements)

References 56 publications

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“…1A). Lipid mixing occurred rapidly when the control sample was adjusted to pH 5.5 (curve a), in keeping with the reported results of previous studies (2,5). Exogenous SFV DIII caused a dose-dependent inhibition of virus fusion (curves c to f).…”

Section: Resultssupporting

confidence: 89%

“…Brief low-pH treatment of SFV at 4°C in the presence of target membranes causes the dissociation of the E1/E2 dimer without triggering the fusion of the virus membrane (2,17). Under these conditions, the trypsin-and-SDS-resistant E1 homotrimer does not form, in keeping with the absence of the final postfusion trimer in the cold-arrested intermediate.…”

Section: Resultsmentioning

confidence: 99%

“…Virus fusion with target liposomes was monitored using a lipid mixing assay that follows the decrease in the excimer signal of pyrene-labeled virus (2,5). Pyrene-labeled virus was mixed with liposomes at a final liposomal lipid concentration of 200 M and the indicated final concentration of SFV or DV DIII protein.…”

Section: Methodsmentioning

confidence: 99%

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The Interaction of Alphavirus E1 Protein with Exogenous Domain III Defines Stages in Virus-Membrane Fusion

Roman-Sosa

Kielian

2011

J Virol

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Alphaviruses such as Semliki Forest virus (SFV) are enveloped viruses that infect cells through a low-pHtriggered membrane fusion reaction mediated by the transmembrane fusion protein E1. E1 drives fusion by insertion of its hydrophobic fusion loop into the cell membrane and refolding to a stable trimeric hairpin. In this postfusion conformation, the immunoglobulin-like domain III (DIII) and the stem region pack against the central core of the trimer. Membrane fusion and infection can be specifically inhibited by exogenous DIII, which binds to an intermediate in the E1 refolding pathway. Here we characterized the properties of the E1 target for interaction with exogenous DIII. The earliest target for DIII binding was an extended membraneinserted E1 trimer, which was not detectable by assays for the stable postfusion hairpin. DIII binding provided a tool to detect this extended trimer and to define a series of SFV fusion-block mutants. DIII binding studies showed that the mutants were blocked in distinct steps in fusion protein refolding. Our results suggested that formation of the initial extended trimer was reversible and that it was stabilized by the progressive fold-back of the DIII and stem regions.The alphaviruses are members of a genus of small spherical enveloped viruses with positive-sense RNA genomes (reviewed in reference 23). Alphaviruses include a number of medically important pathogens such as Eastern equine encephalitis virus and the emerging pathogen chikungunya virus, which has caused recent epidemics in India (10, 41, 43). Although human infections by pathogenic alphaviruses are increasing, to date there are no vaccines or antiviral therapies available for use in treatment of patients. Well-characterized alphaviruses such as Semliki Forest virus (SFV) and Sindbis virus have been used extensively to study the structure, entry, replication, and biogenesis of this important group of viruses (23).The alphavirus particle contains an inner core of the viral RNA in a complex with the capsid protein (23). This is surrounded by a lipid membrane containing the transmembrane E2 and E1 proteins, organized as trimers of E2 and E1 (E2/E1) heterodimers and arranged with T ϭ 4 icosahedral symmetry. Alphaviruses infect host cells by binding to receptors at the plasma membrane followed by uptake via clathrin-mediated endocytosis (reviewed in reference 18). The low-pH environment of the endosome then triggers the fusion of the viral and endosome membranes to deliver the nucleocapsid into the cytosol. Endocytic uptake and virus infection are blocked by expression of dominant-negative versions of host proteins involved in endocytosis (e.g., see references 7 and 42), whereas fusion and virus infection are inhibited by neutralizing the low pH of endocytic vesicles (e.g., see references 9 and 16). During entry, the E2 protein binds the virus receptor(s) while E1 mediates membrane fusion.The structures of the E2/E1 heterodimer and the prefusion and postfusion structures of the E1 protein provide important information ab...

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Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

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The Interaction of Alphavirus E1 Protein with Exogenous Domain III Defines Stages in Virus-Membrane Fusion

Roman-Sosa

Kielian

2011

J Virol

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“…E1-E2-E3 complex to form homotrimers or even higher homooligomers Bron et al, 1993). E1 dissociation was blocked in a mutant deficient in cleavage of p62, the precursor of E2-E3 (Salminen et al, 1992).…”

Section: Do Alphavirus Proteins Have Channel Activities?mentioning

confidence: 99%

Entry and uncoating of enveloped viruses

Lanzrein

Schlegel

Kempf

1994

Biochemical Journal

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“…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)(6)(7)(8)(9).…”

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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Membrane fusion of Semliki Forest virus in a model system: correlation between fusion kinetics and structural changes in the envelope glycoprotein.

Cited by 166 publications

References 56 publications

The Interaction of Alphavirus E1 Protein with Exogenous Domain III Defines Stages in Virus-Membrane Fusion

The Interaction of Alphavirus E1 Protein with Exogenous Domain III Defines Stages in Virus-Membrane Fusion

Entry and uncoating of enveloped viruses

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

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