MOLECULAR ANALYSIS OF ALPHAVIRUS MEMBRANE FUSION PROTEIN

Project: Research project

Project Details

Description

The entry of viruses into their host cells is a key step in the virus
infection pathway, and a potential point for therapeutic intervention.
The enveloped alphavirus, Semliki Forest virus (SFV), infects cells via
endocytosis followed by a membrane fusion reaction triggered by the acid
pH present in intracellular vacuoles. The alphavirus family is comprised
of 26 related viruses, some of which are significant pathogens of humans
or domestic animals. Major elements of the endocytic infection pathway
first described for SFV are also used by a number of other virus families
that include important human and veterinary pathogens. A crucial issue
in studies of all of these viruses is the molecular mechanism of membrane
fusion, a critical function for both viruses and cells. Our goal is to
define the molecular features of a membrane fusion reaction, using the
well defined SFV system and a combination of biochemical, genetic, and
immunological approaches.

SFV fusion is mediated by the heterotrimeric viral spike protein, which
undergoes an ordered series of conformational changes following exposure
to acid pH that culminate in membrane fusion. These conformational
changes will be localized by determining the binding sites for a series
of monoclonal antibodies specific for the acid form of the spike protein.
Binding sites will be defined by competition assays, by identifying the
amino acids that comprise the antibody epitopes, and by functional assays
of the effects of antibodies in virus fusion.

The role of specific spike protein domains in fusion will be evaluated
by analysis of our previously obtained virus and spike protein fusion
mutants. The mechanisms by which these mutations affect fusion will be
determined by analysis of the series of known molecular events that lead
to fusion, including acid-dependent conformational changes and
interactions with the target membrane. An infectious SFV clone will be
used to analyze the effects of the spike protein mutations on virus
assembly, infectivity, and fusion. Revertants of a mutation that blocks
virus fusion will be selected and characterized for their genotype and
fusion mechanism.

The SFV E1 spike protein subunit interacts with the target membrane to
trigger fusion. A proteolytically truncated form of E1 will be used to
analyze the biochemical nature of E1's interaction with the membrane and
its requirement for specific lipids, and to identify and characterize the
E1 domain involved in membrane binding. A similar but genetically
truncated form of E1 will be prepared and assayed for functional activity
and suitability for structural studies.
StatusFinished
Effective start/end date8/15/957/31/96

Funding

  • National Institute of General Medical Sciences

ASJC

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Virology
  • Microbiology

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