Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Veterinary Medical Sciences - Pathobiological Sciences

First Advisor

Konstantin G. Kousoulas


HSV-1(KOS)$\Delta$gK, a virus lacking the UL53 gene coding for glycoprotein K(gK) was constructed, characterized and compared to the partially deleted gK-null virus HSV-1(F)-gK$\beta$. $\Delta$gK and F-gK$\beta$ viruses produced microscopic plaques on Vero cells at 48 hours post infection. F-gK$\beta$ plaques at 72 hours post infection contained fused cells (syn), while $\Delta$gK plaques resembled wild-type KOS plaques ($syn\sp+$). $\Delta$gK replicated efficiently in exponentially dividing Vero cells whereas F-gK$\beta$ yields were 10 and 1000-fold lower than $\Delta$gK yields in HEp-2 or log-phase Vero cells, respectively. Electron micrographs of $\Delta$gK-infected Vero cells revealed large numbers of capsids in the nuclei of Vero, but not HEp-2 cells, while a small number of enveloped virions were observed in the cytoplasm of infected Vero and HEp-2 cells that failed to translocate to the extracellular spaces. KOS and $\Delta$gK replicated inefficiently in gK-transformed cells, with the exception of VK302 cells. Electron micrographs of the gK-transformed BL-1 cells infected with KOS revealed virions within perinuclear spaces, while there were no virions visualized in $\Delta$gK infected BL-1 cells. VK302 cells were the only gK-transformed cells that supported the replication and cellular egress of both KOS and $\Delta$gK. To fine-map the functional domains of gK involved in membrane fusion, a long-PCR site-directed mutagenesis system was developed that efficiently produced single base substitutions within larger than 4 kbp DNA fragments. Utilizing this system three different mutations were engineered and recombinant viruses containing one of the three mutations were isolated. The mutation at position 31 resulted in a $syn\sp+$ virus. This research revealed that gK is involved in nucleocapsid envelopment and cellular egress of infectious virions. Actively replicating cells can partially compensate for envelopment but not for the cellular egress deficiencies of $\Delta$gK. Interference of viral replication in gK-transformed cells suggests that the cellular gK can function in-trans to inhibit egress by preventing certain cellular factors required for viral egress to function efficiently. These results implicate that viral egress involves cellular receptors that are modified to act as "pilots" or "sorters" for virus transport and egress in a similar fashion to intracellular glycoprotein transport.