Mechanisms of mutations inhibiting fusion and infection by Semliki Forest virus

Margaret Kielian, Matthew R. Klimjack, Swati Ghosh, Wayne A. Duffus

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Abstract

Semliki Forest virus (SFV) infects cells by an acid-dependent membrane fusion reaction catalyzed by the virus spike protein, a complex containing E1 and E2 transmembrane subunits. E1 carries the putative virus fusion peptide, and mutations in this domain of the spike protein were previously shown to shift the pH threshold of cell-cell fusion (G91A), or block cell-cell fusion (G91D). We have used an SFV infectious clone to characterize virus particles containing these mutations. In keeping with the previous spike protein results, G91A virus showed limited secondary infection and an acid-shifted fusion threshold, while G91D virus was noninfectious and inactive in both cell-cell and virus-liposome fusion assays. During the low pH-induced SFV fusion reaction, the E1 subunit exposes new epitopes for monoclonal antibody (mAb) binding and forms an SDS-resistant homotrimer, the virus associates hydrophobically with the target membrane, and fusion of the virus and target membranes occurs. After low pH treatment, G91A spike proteins were shown to bind conformation-specific mAbs, associate with target liposome membranes, and form the E1 homotrimer. However, both G91A membrane association and homotrimer formation had an acid-shifted pH threshold and reduced efficiency compared to wt virus. In contrast, studies of the fusion-defective G91D mutant showed that the virus efficiently reacted with low pH as assayed by mAb binding and liposome association, but was essentially inactive in homotrimer formation. These results suggest that the G91D mutant is noninfectious due to a block in a late step in membrane fusion, separate from the initial reaction to low pH and interaction with the target membrane, and involving the lack of efficient formation of the E1 homotrimer.

Original languageEnglish (US)
Pages (from-to)863-872
Number of pages10
JournalJournal of Cell Biology
Volume134
Issue number4
StatePublished - Aug 1996

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Semliki forest virus
Viruses
Mutation
Infection
Liposomes
Membrane Fusion
Membranes
Cell Fusion
Acids
Monoclonal Antibodies
Virus Internalization
Proteins
Coinfection
Virion
Epitopes
Clone Cells
Peptides

ASJC Scopus subject areas

  • Cell Biology

Cite this

Mechanisms of mutations inhibiting fusion and infection by Semliki Forest virus. / Kielian, Margaret; Klimjack, Matthew R.; Ghosh, Swati; Duffus, Wayne A.

In: Journal of Cell Biology, Vol. 134, No. 4, 08.1996, p. 863-872.

Research output: Contribution to journalArticle

Kielian, M, Klimjack, MR, Ghosh, S & Duffus, WA 1996, 'Mechanisms of mutations inhibiting fusion and infection by Semliki Forest virus', Journal of Cell Biology, vol. 134, no. 4, pp. 863-872.
Kielian, Margaret ; Klimjack, Matthew R. ; Ghosh, Swati ; Duffus, Wayne A. / Mechanisms of mutations inhibiting fusion and infection by Semliki Forest virus. In: Journal of Cell Biology. 1996 ; Vol. 134, No. 4. pp. 863-872.
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