MOLECULAR MECHANISMS OF ALPHAVIRUS ENTRY AND EXIT

Project: Research project

Project Details

Description

DESCRIPTION (Adapted from Applicant's Summary): All enveloped viruses
use membrane fusion to infect a cell, and must bud through a cellular
membrane to produce progeny viruses. Molecular information on these
processes is critical to the understanding of viral disease, the
development of novel anti-viral agents, and as a model for cellular
membrane fusion reactions. The well-characterized alphavirus, Semliki
Forest virus (SFV), enters cells via low pH-triggered membrane fusion,
and exits by budding through the cell plasma membrane. Fusion is
mediated by the SFV spike protein, which undergoes a defined series of
conformational changes at acid pH. A critical feature of the SFV fusion
reaction is its striking dependence on the presence of cholesterol and
sphingolipid in the target bilayer. Dr. Kielian has also described a
novel requirement for cholesterol in the SFV exit pathway. The
overall goal of this grant is to define molecular features of the entry
and exit of alphaviruses from cells. Her major hypothesis is that both
SFV fusion and exit involve specific interactions of the viral spike
protein with cholesterol in the cell membrane.

She has isolated and characterized an SFV mutant (srf-3) that has
strikingly more efficient fusion and exit from cholesterol-depleted
cells than wt SFV. A single amino acid substitution in the E1 spike
subunit, a change of proline 226 to serine, is responsible for the
cholesterol-independence of both srf-3 fusion and exit. In vitro
mutagenesis of this domain and isolation of new srf mutants will be used
to define sequences that confer alphavirus cholesterol independence.
The hypothesis is that srf-3 is less cholesterol-dependent for key E1-
membrane interactions in fusion and exit. This will be tested by a
sensitive new fusion assay using purified lipid components, by
biochemical and immunological detection of E1 conformational changes and
membrane insertion, and by cryo-electron microscopy of control and
sterol-depleted virus.
Dr. Kielian has developed a biochemical assay for the final steps of
budding and release of cell surface spike proteins into SFV virions.
This assay is based on cell surface biotinylation of the spike proteins
and virus retrieval using streptavidin-conjugated magnetic particles.
This system will be used to determine the role of cholesterol in the
exit of wt SFV and srf-3, to characterize virus exit requirements in
intact cells, and as the basis of experiments to reconstitute the cell
surface budding of SFV in a semi-permeabilized cell system.
StatusFinished
Effective start/end date2/1/991/31/08

Funding

  • National Institute of General Medical Sciences: $350,700.00
  • National Institute of General Medical Sciences: $288,808.00
  • National Institute of General Medical Sciences: $35,000.00
  • National Institute of General Medical Sciences: $342,459.00
  • National Institute of General Medical Sciences: $285,151.00
  • National Institute of General Medical Sciences: $336,630.00
  • National Institute of General Medical Sciences: $293,196.00
  • National Institute of General Medical Sciences: $350,700.00
  • National Institute of General Medical Sciences: $365,200.00
  • National Institute of General Medical Sciences

ASJC

  • Biochemistry
  • Virology
  • Molecular Biology

Fingerprint

Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.