A Single Point Mutation Controls the Cholesterol Dependence of Semliki Forest Virus Entry and Exit

Vashishtha, Malini; Phalen, Thomas; Marquardt, Marianne T.; Ryu, Jae S.; Ng, Alice C.; Kielian, Margaret

doi:10.1083/jcb.140.1.91

Cited by 131 publications

(193 citation statements)

References 45 publications

Supporting

Mentioning

179

Contrasting

Order By: Relevance

“…This might indicate a perturbation of the membrane and result in fragmentation of the particles, especially at higher CD concentrations. Instability of SFV particles has been observed before, when virions were produced in cholesterol-free insect cells (30). However, clearly cholesterol in influenza viruses is in a detergent-resistant state, whereas this is not the case for VSV and SFV.…”

Section: Glycoproteins In the Influenza Virus Envelope But Not In Vsmentioning

confidence: 93%

Influenza Viruses Select Ordered Lipid Domains during Budding from the Plasma Membrane

Scheiffele¹,

Rietveld²,

Wilk

et al. 1999

Journal of Biological Chemistry

402

386

View full text Add to dashboard Cite

Section: Glycoproteins In the Influenza Virus Envelope But Not In Vsmentioning

confidence: 93%

Influenza Viruses Select Ordered Lipid Domains during Budding from the Plasma Membrane

Scheiffele¹,

Rietveld²,

Wilk

et al. 1999

Journal of Biological Chemistry

402

386

View full text Add to dashboard Cite

“…BHK-21 cells were propagated in complete BHK medium (Dulbecco's modified Eagle's medium containing 5% fetal calf serum, 10% tryptose phosphate broth, 100 U penicillin/ml, and 100 g streptomycin/ml) at 37 or 28°C. Cholesterol-containing C6/36 mosquito cells and cholesterol-depleted C6/36 cells were cultivated as described for previous studies (45).…”

Section: Methodsmentioning

confidence: 99%

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

Roman-Sosa

Kielian

2011

J Virol

Self Cite

View full text Add to dashboard Cite

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...

show abstract

“…srf-3 (sterol requirement in function), an SFV mutant with a decreased requirement for cholesterol in fusion, is less dependent on cholesterol for the conformational changes in E1 (8). The decrease in srf-3's cholesterol requirement is due to a single point mutation on the E1 protein, P2263S (50), which lies outside of the putative fusion peptide.…”

mentioning

confidence: 99%

The Fusion Peptide of Semliki Forest Virus Associates with Sterol-Rich Membrane Domains

Ahn

Gibbons

Kielian

2002

J Virol

Self Cite

115

View full text Add to dashboard Cite

Semliki Forest virus (SFV) is an enveloped alphavirus whose membrane fusion is triggered by low pH and promoted by cholesterol and sphingolipid in the target membrane. Fusion is mediated by E1, a viral membrane protein containing the putative fusion peptide. Virus mutant studies indicate that SFV's cholesterol dependence is controlled by regions of E1 outside of the fusion peptide. Both E1 and E1*, a soluble ectodomain form of E1, interact with membranes in a reaction dependent on low pH, cholesterol, and sphingolipid and form highly stable homotrimers. Here we have used detergent extraction and gradient floatation experiments to demonstrate that E1* associated selectively with detergent-resistant membrane domains (DRMs or rafts). In contrast, reconstituted full-length E1 protein or influenza virus fusion peptide was not associated with DRMs. Methyl ␤-cyclodextrin quantitatively extracted both cholesterol and E1* from membranes in the absence of detergent, suggesting a strong association of E1* with sterol. Monoclonal antibody studies demonstrated that raft association was mediated by the proposed E1 fusion peptide. Thus, although other regions of E1 are implicated in the control of virus cholesterol dependence, once the SFV fusion peptide inserts in the target membrane it has a high affinity for membrane domains enriched in cholesterol and sphingolipid.

show abstract

A Single Point Mutation Controls the Cholesterol Dependence of Semliki Forest Virus Entry and Exit

Cited by 131 publications

References 45 publications

Influenza Viruses Select Ordered Lipid Domains during Budding from the Plasma Membrane

Influenza Viruses Select Ordered Lipid Domains during Budding from the Plasma Membrane

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

The Fusion Peptide of Semliki Forest Virus Associates with Sterol-Rich Membrane Domains

Contact Info

Product

Resources

About