Membrane fusion mutants of Semliki Forest virus.

Kielian, Margaret; Keränen, Sirkka; Kääriäinen, Leevi; Helenius, Ari

doi:10.1083/jcb.98.1.139

Cited by 100 publications

(94 citation statements)

References 49 publications

(83 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In brief, BHK cells were cultured for 25 h in medium containing 1-pyrenehexadecanoic acid (Invitrogen, Carlsbad, CA) (10 g/ml). The cells were then infected at multiplicity of infection (MOI) of 5 PFU/cell for 20 h. The labeled virus was purified by banding on a discontinuous sucrose gradient (22) and stored in aliquots at Ϫ80°C until use. Radiolabeled WT SFV was prepared by infecting BHK cells at high MOI and overnight incubation in methionine-free medium containing Express labeling mix (Perkin Elmer Life and Analytical Sciences) (83 Ci/ml).…”

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

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

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

View full text Add to dashboard Cite

show abstract

“…To block penetration, monensin or ammonium chloride was added to 20 pM and 25 mM, respectively. One set of dishes was prepulsed with the drugs for 30 min in ME:M-BSA medium at slightly alkaline pH to ensure maximal efficacy of the drugs (Kielian et al, 1986). After 120 min, the inoculum was removed, and warm medium containing drugs was added for an additional 30 min.…”

Section: Methodsmentioning

confidence: 99%

Stepwise dismantling of adenovirus 2 during entry into cells

Greber

Willetts

Webster

et al. 1993

Cell

743

726

View full text Add to dashboard Cite

“…The SFV used in these experiments was a well-characterized plaque-purified isolate propagated in BHK-21 cells (16). The virus was radiolabeled with [ 35 S]methionine and purified as previously described (28) or propagated in the absence of label and purified by banding on tartrate gradients (21).…”

Section: Methodsmentioning

confidence: 99%

Molecular Dissection of the Semliki Forest Virus Homotrimer Reveals Two Functionally Distinct Regions of the Fusion Protein

Gibbons

Kielian

2002

J Virol

View full text Add to dashboard Cite

Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reactiontriggered by the acidic pH of endosomes. In response to low pH, the E1 proteins on the virus membrane undergo a series of conformational changes, resulting in the formation of a stable E1 homotrimer. Little is known about the structural basis of either the E1 conformational changes or the resulting homotrimer or about the mechanism of action of the homotrimer in fusion. Here, the E1 homotrimer was formed in vitro from either virus or soluble E1 ectodomain and then probed by various perturbants, proteases, or glycosidase. The preformed homotrimer was extremely stable to moderately harsh conditions and proteases. By contrast, mild reducing conditions selectively disrupted the N-terminal region of trimeric E1, making it accessible to proteolytic cleavage and producing E1 fragments that retained trimer interactions. Trypsin digestion produced a fragment missing a portion of the N terminus just proximal to the putative fusion peptide. Digestion with elastase produced several fragments with cleavage sites between residues 78 and 102, resulting in the loss of the putative fusion peptide and the release of membrane-bound E1 ectodomain as a soluble trimer. Elastase also cleaved the homotrimer within an E1 loop located near the fusion peptide in the native E1 structure. Mass spectrometry was used to map the C termini of several differentially produced and fully functional E1 ectodomains. Together, our data identify two separate regions of the SFV E1 ectodomain, one responsible for target membrane association and one necessary for trimer interactions.Enveloped viruses must circumvent the double barrier of their own membranes and the membrane of the host cell in order to deliver their genomes into the cytoplasm and cause infection. They bypass both membrane barriers simultaneously by means of a membrane fusion event, either at the cell surface or in the endocytic pathway (18). The well-characterized alphavirus Semliki Forest virus (SFV) is a positive-strand RNA virus that enters cells by receptor-mediated endocytosis and then fuses with the endosome membrane to infect the cell (see references 23 and 47 for reviews). SFV fusion is mediated by the viral E1 protein, a type I membrane glycoprotein of about 50 kDa. Upon exposure to the low pH of the endosomes, E1 dissociates from its stable dimeric interaction with the companion E2 protein. E1 then undergoes a series of conformational changes resulting in the exposure of previously hidden acid-conformation-specific epitopes and the formation of a highly stable E1 homotrimer. The protein associates with the target membrane, presumably via the putative fusion peptide (approximately residues 83 to 100), and the dual interaction of E1 with the target and virus membranes drives the fusion reaction. SFV fusion is strongly dependent on the presence of cholesterol and sphingolipid in the target membrane (reviewed in reference 24). Although treatment of virus at low pH in the absence of...

show abstract

Membrane fusion mutants of Semliki Forest virus.

Cited by 100 publications

References 49 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

Stepwise dismantling of adenovirus 2 during entry into cells

Molecular Dissection of the Semliki Forest Virus Homotrimer Reveals Two Functionally Distinct Regions of the Fusion Protein

Contact Info

Product

Resources

About