Molecular mechanisms of HSV-1 entry and membrane fusion.
Herpesviruses are a large family of enveloped viruses that establish lifelong infections
in their hosts. Human herpesviruses (HHV) include the causative agents of chicken pox,
mononucleosis, and Kaposi's sarcomas. Herpes Simplex Virus (HSV) commonly causes painful
mucocutaneous sores, occasionally keratitis, and encephalitis in severe cases.
Herpesvirus entry into cells is mediated ... read moreby the viral fusogen glycoprotein B (gB), which
is thought to refold from the prefusion to the postfusion form in a series of large
conformational changes that energetically couple refolding to membrane fusion. HSV entry
and cell-to-cell fusion requires gB, gH/gL, and gD, of which all but gD are conserved in
all herpesviruses known to date. In this work, we investigated the roles of the
cytoplasmic domain of gB and gH in regulating cell-cell fusion, and the application of
the vesicular stomatitis virus (VSV) pseudotype system to the HSV-1 fusion
glycoproteins. Previous work proposed a model of the gB cytoplasmic domain based on
biochemical and functional data. Several gB cytodomain mutations may relieve fusion
repression by altering membrane interaction, while the mechanism of other hyperfusogenic
mutants is not known. We tested several predictions made by this model, and several
possible mechanisms for the regulation of gB by the cytoplasmic domain. In addition, we
characterized the entry of VSV pseudotyped with HSV-1 gB, gH/gL, and gD into permissive
cells that are models of the three routes of entry used by HSV-1. Here we show that all
hyperfusogenic mutants increase fusion levels by the same general mechanism, where
fusion overall is faster. The hyperfusogenic mutants still require gH/gL for fusion,
including the cytoplasmic domain of gH. Based on gH cytoplasmic domain truncations, we
propose that the gH cytoplasmic domain activates gB by destabilization of the gB
cytoplasmic domain. The VSV pseudotyped with HSV-1 gB, gH/gL, and gD entered cells by
endocytosis and required low pH because entry was sensitive to inhibitors of endosomal
low pH. Entry of the pseudotyped VSV into Vero cells did not occur at the plasma
membrane as reported for HSV. Thus, while gB, gH/gL, and gD are sufficient for viral
entry by endocytosis, the virions do not recapitulate the cell-specific entry routes
used by HSV. The pseudotyped VSV has potential as a tool to investigate the fundamental
requirements for HSV fusion and cell entry.
Thesis (Ph.D.)--Tufts University, 2016.
Submitted to the Dept. of Biochemistry.
Advisor: Katya Heldwein.
Committee: Andrew Bohm, Michael Forgac, and Marta Gaglia.
Keywords: Biochemistry, and Virology.read less
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