Project Summary/Abstract
Herpes Simplex Virus 1 (HSV-1) enters and infects cells by fusing its viral envelope with target cell
membranes. Membrane fusion is catalyzed by the HSV-1 fusogen, glycoprotein B (gB). Based on our
knowledge of other viral fusogens, gB is thought to irreversibly refold from its unstable prefusion form to its low
energy postfusion from through large conformational changes that merge the viral and target membranes.
However, unlike most enveloped viruses which catalyze fusion using a single protein, the fusion pathway in
HSV-1 is more complex and is unusual, requiring three additional glycoproteins, gD, gL, and gH. In the
hypothesized fusion pathway, gD binds a receptor on the target cell and activates the gH/gL complex, which
activates gB to refold. However, it is not known how gB is maintained in its metastable prefusion conformation
prior to fusion triggering, nor how gH activates gB to cause fusion. Our prior work suggests the cytoplasmic
regions of gB and gH regulate this process, with the gB cytoplasmic domain (CTD) stabilizing prefusion gB and
the gH cytotail (CT) activating gB to refold. Elucidating the mechanisms by which these regions regulate gB
fusogenic activity is essential for understanding how the viral glycoproteins accomplish membrane fusion
during viral infection. The objective of this proposal is to determine how the gB CTD stabilizes the prefusion
conformation prior to fusion triggering, and how the gH CT interacts with gB to cause fusion. In Aim 1, I will
identify regions in the gB CTD that are responsible for stabilizing gB by mutagenesis and an unfolding assay
and determine whether increased CTD stabilization decreases fusion. In Aim 2, I will identify residues involved
in the gH-gB interaction by mutagenesis and a protein-protein interaction assay, and their effect on fusion via a
cell-cell fusion assay. These experiments will illuminate how cytoplasmic regions of gB and gH interact and
regulate gB activity in the HSV-1 fusion pathway. The proposal is significant because it investigates the
unknown events between gH activation and gB refolding that currently limit our understanding of herpesvirus
fusion. The proposal is innovative because it uses original hypotheses to investigate a novel mechanism of
protein activity regulation in the unique herpesviral fusion pathway. Successful completion of the proposed
research would advance the HSV-1 field by increasing our understanding of the different steps in HSV-1 fusion
in molecular detail. In line with the mission of NIAID, this would be an important step towards better treating
and preventing HSV-1 infection. This fellowship will provide outstanding research training on my path to
becoming a physician-scientist.