Mutations in the putative fusion peptide of Semliki Forest virus affect spike protein oligomerization and virus assembly.

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.

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Specific Recognition of a Stem-Loop RNA Structure by the Alphavirus Capsid Protein

Viruses ◽

10.3390/v13081517 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1517

Author(s):

Rebecca S. Brown ◽

Lisa Kim ◽

Margaret Kielian

Keyword(s):

Capsid Protein ◽

Rna Structure ◽

Semliki Forest Virus ◽

Virus Assembly ◽

Specific Binding ◽

Genomic Rna ◽

Stem Loop ◽

Stem Loop Structure ◽

Labeled Rna

Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus.

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Multiple capsid protein binding sites mediate selective packaging of the alphavirus genomic RNA

Nature Communications ◽

10.1038/s41467-020-18447-z ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Rebecca S. Brown ◽

Dimitrios G. Anastasakis ◽

Markus Hafner ◽

Margaret Kielian

Keyword(s):

Capsid Protein ◽

Binding Sites ◽

Semliki Forest Virus ◽

Virus Assembly ◽

Genomic Rna ◽

Packaging Signal ◽

Genome Packaging ◽

Genome Recognition ◽

Infected Cells

Abstract The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, here we comprehensively define binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. Instead, we identify multiple Cp binding sites that are enriched on gRNA-specific regions and promote infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrate that budding causes discrete changes in Cp-gRNA interactions. Notably, Cp’s top binding site is maintained throughout virus assembly, and specifically binds and assembles with Cp into core-like particles in vitro. Together our data suggest a model for selective alphavirus genome recognition and assembly.

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

Virology ◽

10.1006/viro.2000.0688 ◽

2001 ◽

Vol 279 (1) ◽

pp. 146-160 ◽

Cited By ~ 8

Author(s):

Swati Ghosh Shome ◽

Margaret Kielian

Keyword(s):

Semliki Forest Virus ◽

Fusion Peptide

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

Journal of Virology ◽

10.1128/jvi.76.24.12712-12722.2002 ◽

2002 ◽

Vol 76 (24) ◽

pp. 12712-12722 ◽

Cited By ~ 41

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.

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Spike Protein Fusion Peptide and Feline Coronavirus Virulence

Emerging Infectious Diseases ◽

10.3201/eid1807.120143 ◽

2012 ◽

Vol 18 (7) ◽

pp. 1089-1095 ◽

Cited By ~ 87

Author(s):

Hui-Wen Chang ◽

Herman F. Egberink ◽

Rebecca Halpin ◽

David J. Spiro ◽

Peter J.M. Rottier

Keyword(s):

Fusion Peptide ◽

Spike Protein ◽

Protein Fusion ◽

Feline Coronavirus

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Functions of the Stem Region of the Semliki Forest Virus Fusion Protein during Virus Fusion and Assembly

Journal of Virology ◽

10.1128/jvi.01679-06 ◽

2006 ◽

Vol 80 (22) ◽

pp. 11362-11369 ◽

Cited By ~ 22

Author(s):

Maofu Liao ◽

Margaret Kielian

Keyword(s):

Fusion Protein ◽

Membrane Fusion ◽

Semliki Forest Virus ◽

Virus Assembly ◽

Protein A ◽

Stem Length ◽

Membrane Fusion Protein ◽

Virus Fusion ◽

Target Membrane ◽

Domain Iii

ABSTRACT Membrane fusion of the alphaviruses is mediated by the E1 protein, a class II virus membrane fusion protein. During fusion, E1 dissociates from its heterodimer interaction with the E2 protein and forms a target membrane-inserted E1 homotrimer. The structure of the homotrimer is that of a trimeric hairpin in which E1 domain III and the stem region fold back toward the target membrane-inserted fusion peptide loop. The E1 stem region has a strictly conserved length and several highly conserved residues, suggesting the possibility of specific stem interactions along the trimer core and an important role in driving membrane fusion. Mutagenesis studies of the alphavirus Semliki Forest virus (SFV) here demonstrated that there was a strong requirement for the E1 stem in virus assembly and budding, probably reflecting its importance in lateral interactions of the envelope proteins. Surprisingly, however, neither the conserved length nor any specific residues of the stem were required for membrane fusion. Although the highest fusion activity was observed with wild-type E1, efficient fusion was mediated by stem mutants containing a variety of substitutions or deletions. A minimal stem length was required but could be conferred by a series of alanine residues. The lack of a specific stem sequence requirement during SFV fusion suggests that the interaction of domain III with the trimer core can provide sufficient driving force to mediate membrane merger.

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

The Journal of Cell Biology ◽

10.1083/jcb.140.1.91 ◽

1998 ◽

Vol 140 (1) ◽

pp. 91-99 ◽

Cited By ~ 108

Author(s):

Malini Vashishtha ◽

Thomas Phalen ◽

Marianne T. Marquardt ◽

Jae S. Ryu ◽

Alice C. Ng ◽

...

Keyword(s):

Membrane Fusion ◽

Semliki Forest Virus ◽

Single Point ◽

Cellular Membrane ◽

Spike Protein ◽

Single Amino Acid ◽

Progeny Virus ◽

Single Point Mutation ◽

Protein Subunit ◽

Single Amino Acid Change

Membrane fusion and budding are key steps in the life cycle of all enveloped viruses. Semliki Forest virus (SFV) is an enveloped alphavirus that requires cellular membrane cholesterol for both membrane fusion and efficient exit of progeny virus from infected cells. We selected an SFV mutant, srf-3, that was strikingly independent of cholesterol for growth. This phenotype was conferred by a single amino acid change in the E1 spike protein subunit, proline 226 to serine, that increased the cholesterol independence of both srf-3 fusion and exit. The srf-3 mutant emphasizes the relationship between the role of cholesterol in membrane fusion and virus exit, and most significantly, identifies a novel spike protein region involved in the virus cholesterol requirement.

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Membrane fusion process of Semliki Forest virus. I: Low pH-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells.

The Journal of Cell Biology ◽

10.1083/jcb.116.2.339 ◽

1992 ◽

Vol 116 (2) ◽

pp. 339-348 ◽

Cited By ~ 96

Author(s):

J M Wahlberg ◽

H Garoff

Keyword(s):

Membrane Protein ◽

Membrane Fusion ◽

Semliki Forest Virus ◽

Quaternary Structure ◽

Low Ph ◽

Host Cells ◽

Protein Oligomerization ◽

Protein Subunit ◽

New Host ◽

E1 Protein

The Semliki Forest virus (SFV) directs the synthesis of a heterodimeric membrane protein complex which is used for virus membrane assembly during budding at the surface of the infected cell, as well as for low pH-induced membrane fusion in the endosomes when particles enter new host cells. Existing evidence suggests that the E1 protein subunit carries the fusion potential of the heterodimer, whereas the E2 subunit, or its intracellular precursor p62, is required for binding to the nucleocapsid. We show here that during virus uptake into acidic endosomes the original E2E1 heterodimer is destabilized and the E1 proteins form new oligomers, presumably homooligomers, with altered E1 structure. This altered structure of E1 is specifically recognized by a monoclonal antibody which can also inhibit penetration of SFV into host cells as well as SFV-mediated cell-cell fusion, thus suggesting that the altered E1 structure is important for the membrane fusion. These results give further support for a membrane protein oligomerization-mediated control mechanism for the membrane fusion potential in alphaviruses.

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