| Yhteenveto: | Herpes simplex virus type 1 is an enveloped human pathogen that can be
engineered for oncolytic virus therapy. The viral genome encodes the US3
protein kinase which regulates many cellular and viral processes during infection.
For example, US3 protein kinase is a known inhibitor of caspase-3 activation.
Caspase-3 activates caspase-activated DNAse, which fragments DNA during
apoptosis. The connection between US3 and DNA fragmentation has yet to be
demonstrated. This thesis investigates the effect of viral US3 protein kinase on
cellular DNA damage. In these studies, we compared the amount of DNA
damage in cells infected with herpes simplex type 1 US3 deletion virus and the
repair virus with reinserted US3 gene. Cell viability and the amount of DNA
damage and nuclear caspase-activated DNAse during infections were analyzed
with the comet assay, trypan blue staining, and confocal microscopy. We found
that infected cells with US3 deletion virus had increased DNA damage 12 hours
post infection and had higher fluorescence intensity in DNA damage loci at 16
hours post infection compared to US3 repair virus infected cells. Based on the
comet assay, it was found that herpes infection caused DNA damage in the host
genome regardless of whether it contained the US3 protein. The intensity of
caspase-activated DNAse in the nucleus was decreased in the US3 deletion virus
infection compared to repair virus infection at eight and twelve hours post
infection. The viability of US3 deletion virus infected cells decreased significantly
between twelve and sixteen hours post infection. As a conclusion, DNA damage
was more extensive in US3 deletion virus infected cells than in US3 repair virus
infected cells. In conclusion, our results suggest that US3 protects the host cell
genome from DNA damage during herpes infection. However, the higher
amount of the DNA damage in US3 deletion virus infection could not be
attributed to an increased presence of caspase-activated DNAse in the nucleus. It
is highly likely that the US3 protein may phosphorylate yet unknown cellular
and viral proteins, and these phosphorylation events potentially affect the
amount of DNA damage in the infected cell.
|
|---|