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VIRUS ENTRY AND EXIT
The research in our lab focuses on the molecular mechanisms of
virus-membrane fusion and budding, using primarily the alphavirus
Semliki Forest virus (SFV) and the flavivirus dengue
virus (DV).
Why are these viruses important? Flaviviruses and alphaviruses cause
severe human and animal illnesses such as encephalitis and hemorrhagic
fever. Many of these viruses are pathogens identified by the Centers for
Disease Control as being especially important agents of emerging
infectious diseases and/or potential bioterrorist agents (category A-C
pathogens). These include the flaviviruses dengue, West Nile, Japanese
encephalitis and yellow fever viruses, and the alphaviruses Venezuelan,
eastern, and western equine encephalitis viruses. Dengue virus (DV),
a category A pathogen, is of particular concern as it has dramatically
reemerged to become endemic in >100 countries, with an estimated 100
million cases of dengue infection per year.
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Antiviral strategies for the flaviviruses and alphaviruses are
urgently needed. SFV has been a critical model system to study the
alphaviruses and flaviviruses since it is highly defined biochemically
and structurally, has a very robust infection cycle and an easily
manipulated infectious clone, and has low pathogenicity. |
How do alphaviruses and flaviviruses enter cells?
The entry of both alphaviruses and flaviviruses takes advantage of cellular
endocytosis,
the pathway used by cells to take up many extracellular molecules such
as hormones and nutrients. Virus binds to receptors on the plasma
membrane and is then internalized by endocytosis and delivered to the
endosome compartment.
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Endosomes
have a mildly acidic pH that acts to trigger the fusion of the virus
membrane with that of the endosome. |
We know a lot about what happens during low pH-triggering.
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(A) The alphavirus membrane contains a
dimer of 2 transmembrane glycoproteins, E2 (shown in grey) and
E1 (shown in color). (B) Low pH causes the dissociation of the
E2/E1 heterodimer, releasing E1 from its regulation by E2. (C)
E1 is the membrane fusion protein and inserts into the target
membrane in a cholesterol-dependent reaction. (D, E) E1 then
rearranges to form a highly stable E1 homotrimer that has a
“hairpin”, folded-back conformation. The conformational
transition to the E1 trimeric hairpin brings the virus and
target membranes together and drives membrane fusion. In
collaboration with Dr. Félix Rey and his colleagues, we
determined the structure of the homotrimer conformation of SFV
E1. Based on this structural and functional information, we can
now address how the membrane fusion process works at the
molecular level. |
Inhibition of alphavirus and flavivirus fusion. The flavivirus and
alphavirus membrane fusion proteins are structurally and functionally
similar and are therefore grouped together as members of the class II
virus fusion proteins. Using the structures as a guide, our lab has
recently developed fragments of the SFV and DV fusion proteins that act
as dominant-negative inhibitors of SFV and DV fusion and infection.
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Based on this information, we are working to establish general screens
for inhibitors of class II fusion reactions. Such inhibitors will serve
as lead compounds to develop new antiviral therapies.
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How does virus budding work? Our understanding of the pathways by which
enveloped viruses assemble and bud is very limited, even though these
are key steps in the production of new infectious virus particles. Virus
budding can be targeted to the membranes of various cellular
compartments including the endoplasmic reticulum, Golgi, nucleus and
plasma membrane. Budding of different viruses also differs in
requirements for membrane proteins and/or the virus core.
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Budding of the alphaviruses (see lifecycle picture) occurs at the plasma membrane, is
dependent on the specific interaction of the E2 cytoplasmic tail with
the viral nucleocapsid, and produces highly organized virus particles
containing 240 copies each of E2, E1 and capsid. How does this happen
and what controls budding? Is alphavirus budding a self-driven process
or is it dependent on cellular energy, chaperones, and other components?
We have developed assays for the budding of cell surface E2/E1 into
alphavirus particles, enabling us to address the mechanism of budding.
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What are the important questions to address next? This is an exciting
time for studies of virus entry and exit. There are many important areas
of research, including the molecular mechanism of the homotrimer during
membrane fusion, the identification and use of fusion inhibitors,
mutagenesis of virus infectious clones to characterize fusion protein
function, the mechanism of E1-membrane insertion and the role of
cholesterol, the structure and function of the E1 transmembrane and stem
regions, the regulation of virus budding and the role of cellular
factors in budding.
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