Abstract
Primary HSV1 infection occurs in the keratinocytes that form the stratified epithelium of
human skin and mucosa. Despite HSV1 entry being a well studied aspect of the viral biology,
relatively few studies have been performed in the physiologically relevant keratinocyte cell.
In this thesis nTERT keratinocytes were used as a relevant, yet tractable, model to investigate
the role of the nectin1 in HSV1 entry.
A CRISPR-cas9 knockout (KO) of nectin1 was generated resulting in a cell line that was
refractory to efficient HSV1 infection. Re-introduction of the receptor restored susceptibility
to HSV1 infection. However, a small subpopulation of KO cells were still able to support
infection. At later times post-infection KO cells, which were originally resistant to infection,
also became infected. This was found to be via direct cell-to-cell spread of HSV1 in a process
that was dependent on both viral genome replication and nuclear egress. Transmission in the
KO cells was also reliant on viral glycoproteins gD and gE-gI, but siRNA depletion of the second
major entry receptor HVEM or cellular protein PTP1B had no effect on spread. These results
indicate that nectin1 is absolutely required for efficient HSV1 entry into keratinocytes, but is
dispensible for cell-to-cell spread.
Nectin1 was highly expressed on nTERT keratinocytes allowing observation of endogenous
nectin1 during HSV1 infection. Nectin1 was found to be specifically downregulated in HSV1
infected keratinocyte cells in a two step process. At early times post infection nectin1 formed
large macrodomains on the cell surface then, at later times post-infection, these domains
were removed or degraded in a proteasome dependent manner, that was also reliant on
gD synthesis