scholarly journals Differential Roles of Two Conserved Glycine Residues in the Fusion Peptide of Semliki Forest Virus

Virology ◽  
2001 ◽  
Vol 279 (1) ◽  
pp. 146-160 ◽  
Author(s):  
Swati Ghosh Shome ◽  
Margaret Kielian
Keyword(s):  
Semliki Forest Virus ◽  
Fusion Peptide

scholarly journals Novel Mutations That Control the Sphingolipid and Cholesterol Dependence of the Semliki Forest Virus Fusion Protein

2002 ◽  
Vol 76 (24) ◽  
pp. 12712-12722 ◽  
Author(s):  
Prodyot K. Chatterjee ◽  
Christina H. Eng ◽  
Margaret Kielian
Keyword(s):  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Fusion Peptide ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Novel Mutations ◽  
E1 Protein ◽  
Target Membrane

ABSTRACT The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction mediated by the E1 membrane protein. Efficient SFV-membrane fusion requires the presence of cholesterol and sphingolipid in the target membrane. Here we report on two mutants, srf-4 and srf-5, selected for growth in cholesterol-depleted cells. Like the previously isolated srf-3 mutant (E1 proline 226 to serine), the phenotypes of the srf-4 and srf-5 mutants were conferred by single-amino-acid changes in the E1 protein: leucine 44 to phenylalanine and valine 178 to alanine, respectively. Like srf-3, srf-4 and srf-5 show striking increases in the cholesterol independence of growth, infection, membrane fusion, and exit. Unexpectedly, and unlike srf-3, srf-4 and srf-5 showed highly efficient fusion with sphingolipid-free membranes in both lipid- and content-mixing assays. Both srf-4 and srf-5 formed E1 homotrimers of decreased stability compared to the homotrimers of the wild type and the srf-3 mutant. All three srf mutations lie in the same domain of E1, but the srf-4 and srf-5 mutations are spatially separated from srf-3. When expressed together, the three mutations could interact to produce increased sterol independence and to cause temperature-sensitive E1 transport. Thus, the srf-4 and srf-5 mutations identify novel regions of E1 that are distinct from the fusion peptide and srf-3 region and modulate the requirements for both sphingolipid and cholesterol in virus-membrane fusion.

scholarly journals An Epitope of the Semliki Forest Virus Fusion Protein Exposed during Virus-Membrane Fusion

1999 ◽  
Vol 73 (12) ◽  
pp. 10029-10039 ◽  
Author(s):  
Anna Ahn ◽  
Matthew R. Klimjack ◽  
Prodyot K. Chatterjee ◽  
Margaret Kielian
Keyword(s):  
Membrane Fusion ◽  
Binding Site ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Ph Dependence ◽  
Fusion Peptide ◽  
Endocytic Pathway ◽  
Spike Protein ◽  
Viral Fusion ◽  
Wide Range

ABSTRACT Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by acidic pH in the endocytic pathway. Fusion is mediated by the spike protein E1 subunit, an integral membrane protein that contains the viral fusion peptide and forms a stable homotrimer during fusion. We have characterized four monoclonal antibodies (MAbs) specific for the acid conformation of E1. These MAbs did not inhibit fusion, suggesting that they bind to an E1 region different from the fusion peptide. Competition analyses demonstrated that all four MAbs bound to spatially related sites on acid-treated virions or isolated spike proteins. To map the binding site, we selected for virus mutants resistant to one of the MAbs, E1a-1. One virus isolate, SFV 4-2, showed reduced binding of three acid-specific MAbs including E1a-1, while its binding of one acid-specific MAb as well as non-acid-specific MAbs to E1 and E2 was unchanged. The SFV 4-2 mutant was fully infectious, formed the E1 homotrimer, and had the wild-type pH dependence of infection. Sequence analysis demonstrated that the relevant mutation in SFV 4-2 was a change of E1 glycine 157 to arginine (G157R). Decreased binding of MAb E1a-1 was observed under a wide range of assay conditions, strongly suggesting that the E1 G157R mutation directly affects the MAb binding site. These data thus localize an E1 region that is normally hidden in the neutral pH structure and becomes exposed as part of the reorganization of the spike protein to its fusion-active conformation.

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

2002 ◽  
Vol 76 (7) ◽  
pp. 3267-3275 ◽  
Author(s):  
Anna Ahn ◽  
Don L. Gibbons ◽  
Margaret Kielian
Keyword(s):  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Strong Association ◽  
Fusion Peptide ◽  
Low Ph ◽  
Membrane Domains ◽  
E1 Protein ◽  
Target Membrane ◽  
Virus Mutant ◽  
Detergent Resistant Membrane

ABSTRACT 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 β-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.

scholarly journals Prefusion Rearrangements Resulting in Fusion Peptide Exposure in Semliki Forest Virus

2002 ◽  
Vol 278 (9) ◽  
pp. 7189-7198 ◽  
Author(s):  
Lena Hammar ◽  
Sevak Markarian ◽  
Lars Haag ◽  
Hilkka Lankinen ◽  
Aimo Salmi ◽  
...  
Keyword(s):  
Semliki Forest Virus ◽  
Fusion Peptide

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

2002 ◽  
Vol 76 (3) ◽  
pp. 1194-1205 ◽  
Author(s):  
Don L. Gibbons ◽  
Margaret Kielian
Keyword(s):  
Conformational Changes ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Fusion Peptide ◽  
Structural Basis ◽  
Reducing Conditions ◽  
Target Membrane ◽  
Membrane Bound ◽  
Harsh Conditions

ABSTRACT Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered 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.

scholarly journals Multistep Regulation of Membrane Insertion of the Fusion Peptide of Semliki Forest Virus

2004 ◽  
Vol 78 (7) ◽  
pp. 3312-3318 ◽  
Author(s):  
Don L. Gibbons ◽  
Anna Ahn ◽  
Maofu Liao ◽  
Lena Hammar ◽  
R. Holland Cheng ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Fusion Peptide ◽  
Low Ph ◽  
Membrane Insertion ◽  
Neutral Ph ◽  
Influenza Virus Hemagglutinin ◽  
Target Membrane ◽  
Membrane Fusion Proteins

ABSTRACT A prevailing model for virus membrane fusion proteins has been that the hydrophobic fusion peptide is hidden in the prefusion conformation, becomes exposed once the fusion reaction is triggered, and then either inserts into target membranes or is rapidly inactivated. This model is in general agreement with the structure and mechanism of class I fusion proteins, such as the influenza virus hemagglutinin. We here describe studies of the class II fusion protein E1 from the alphavirus Semliki Forest virus (SFV). SFV fusion is triggered by low pH, which releases E1 from its heterodimeric interaction with the E2 protein and induces the formation of a stable E1 homotrimer. The exposure and target membrane interaction of the E1 fusion peptide (residues 83 to 100) were followed using a monoclonal antibody (MAb E1f) mapping to E1 residues 85 to 95. In agreement with the known structure of SFV and other alphaviruses, the fusion peptide was shielded in native SFV particles and exposed when E1-E2 dimer dissociation was triggered by acidic pH. In contrast, the fusion peptide on purified E1 ectodomains (E1*) was fully accessible at neutral pH. Functional assays showed that MAb E1f binding at neutral pH prevented subsequent low-pH-triggered E1* interaction with target membranes and trimerization. E1* was not inactivated by low pH when treated either in the absence of target membranes or in the presence of fusion-inactive cholesterol-deficient liposomes. Thus, the membrane insertion of the E1 fusion peptide is regulated by additional low-pH-dependent steps after exposure, perhaps involving an E1-cholesterol interaction.

Destabilization of a model membrane by a predicted fusion peptide of fertilin α

1998 ◽  
Vol 95 (2) ◽  
pp. 467-473 ◽  
Author(s):  
A. Schanck ◽  
R. Brasseur ◽  
J. Peuvot
Keyword(s):  
Fusion Peptide ◽  
Model Membrane
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