Show simple item record

dc.contributor.authorDuffus, Wayne Andrew
dc.date.accessioned2018-07-12T18:42:42Z
dc.date.available2018-07-12T18:42:42Z
dc.date.issued1994
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 55-06, Section: B, page: 2057.;Advisors: Margaret Kielian.
dc.identifier.urihttps://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:9428018
dc.identifier.urihttps://hdl.handle.net/20.500.12202/3551
dc.description.abstractThe E1 and E2 spike protein subunits of Semliki Forest virus (SFV) form a heterodimeric complex. The dimer interaction is very important during membrane fusion and budding. Using an SFV infectious clone, we have characterized the effects of mutations within the putative fusion peptide of the E1 subunit on entry, dimerization and virus assembly. These mutations were previously demonstrated to block spike protein membrane fusion activity (G91D) or cause an acid shift in the pH threshold of fusion (G91A). During infection of BHK cells at 37{dollar}\sp\circ{dollar}C the assembly of mutant spike proteins into virus was blocked, and a soluble fragment of E1 was released into the medium. Incubation of infected cells at reduced temperature (28{dollar}\sp\circ{dollar}C) dramatically decreased E1 cleavage and permitted assembly of morphologically normal virus particles. Pulse-labeling studies showed that the assembly defect was rapidly reversible by temperature. Using either infected cells or virus particles assembled at 28{dollar}\sp\circ{dollar}C and conditions that preserved the wild type dimer, mutant E1 and E2 did not co-immunoprecipitate or sediment as a dimer on sucrose gradients. Chemical cross-linking studies revealed that both G91D and G91A mutant viruses contained E1-E2 dimers, as did wt virus. The mutant E1-E2 dimers, in contrast to wt dimers, were easily disrupted by solubilization in non-ionic detergent. Our studies thus demonstrate that mutations in the putative fusion peptide of SFV confer a strong and reversible budding defect. The altered assembly phenotype of the G91D and G91A mutants correlates with decreased stability of the E1-E2 dimer. Both wt and G91A mutant virus particles were infectious at 28{dollar}\sp\circ{dollar}C. In contrast, the G91D mutant was not infectious even during 28{dollar}\sp\circ{dollar}C conditions in which virus particles formed. The ability to produce the E1 homotrimer, the putative fusion-active structure, was investigated for each mutant by low pH treatment in the presence of a target membrane. The G91A mutant formed the E1 homotrimer, although rather less efficiently than wt virus while the G91D mutant was unable to form E1 homotrimers. The G91D mutant thus appears to be blocked in membrane fusion activity, presumably due to a block in the formation of the fusion-active E1 homotrimer.
dc.publisherProQuest Dissertations & Theses
dc.subjectCellular biology.
dc.subjectMolecular biology.
dc.titleOligomerization, assembly and entry of Semliki Forest virus spike protein mutants
dc.typeDissertation


Files in this item

FilesSizeFormatView

There are no files associated with this item.

This item appears in the following Collection(s)

Show simple item record