Complete Title of Thesis:
"Analysis of the role of Varicella open reading frame (ORF) 63 in the apoptotic pathway"
Prepared under the direction of: Donald H. Gilden, M.D.
Varicella zoster virus (VZV) infection produces chickenpox (varicella) upon primary infection, after which it becomes latent in multiple ganglia along the entire human neuraxis. Reactivation of latent VZV results in shingles (zoster) in infected individuals. The mechanisms of latency and reactivation of VZV are poorly understood.
VZV ORF 63 is present as duplicate copies in the varicella genome. VZV ORF 63 transcript and protein are detected during lytic infection in non-neuronal cells and during latency in infected human trigeminal ganglia. VZV ORF 63 is the most abundant transcript and protein detected in latently infected human ganglionic neurons. However, the requirement of this protein in the establishment of latent infection and reactivation is unclear.
I used simian varicella virus (SVV) a non-human counterpart of human VZV, to explore the requirement for varicella ORF 63 protein in viral replication. I generated a recombinant cosmid in which both copies of ORF 63 were deleted. SVV ORF 63 appeared to be required for replication in cell culture.
Previous studies indicate that VZV induces apoptosis (an energy dependent process of regulated cell death) in infected cells and that VZV ORF 63 may play a role in this process. The pathway of apoptosis utilized by VZV is unknown. I examined the role of VZV ORF 63 in virus-induced apoptosis. Here, I describe the pathway of VZV induced apoptosis by examining the markers of apoptosis, such as cleaved caspases, cytochrome c and Bcl-2 protein in uninfected and VZV-infected MeWo (human melanoma) cells over 72 hours. I determined that during lytic infection of non-neuronal cells, VZV induces apoptosis predominantly through the intrinsic pathway. This information lays a foundation for further examination of varicella-induced apoptosis.
To gain a better understanding of the role of varicella ORF 63 during apoptosis, I used a VZV 63 deleted virus (VZV del 63) to study the role of VZV ORF 63 protein on the apoptotic pathway in non-neuronal cells. VZV del 63 also uses the intrinsic pathway to induce apoptosis. However, VZV del 63 replicates with delayed kinetics and induced apoptosis in infected MeWo cells with slower kinetics compared to wild type virus. VZV ORF 63 does not appear to play a significant role in regulating apoptosis during lytic infection of non-neuronal cells.