Date of this Version
Rogers, Adam. "Telomere-Related Factors and Human Papillomavirus Genome Maintenance." PhD Dissertation, University of Nebraska. May 2013.
Human papillomaviruses (HPVs) are small DNA tumor viruses identified by their characteristic ability to replicate as a nuclear plasmid in mitotically active basal keratinocytes. A key characteristic of the HPV life cycle is the establishment of a stable maintenance phase wherein the virus replicates at low copy number, which likely occurs in cells expressing little to no E1 and E2. It is thought that HPV16 replicates in a once-per-cell-cycle manner during this portion of its life cycle and presumably interacts with host chromosome replication and maintenance factors to facilitate this replication. The adaptive radiation of papillomaviruses in response to changing host factors was well demonstrated in this work with an examination of the evolution of the Papillomaviruses’ E2 proteins and cognate binding sites as the virus has adapted to infect new body tissues. Additionally, the yeast model of HPV replication we utilize in our laboratory showed a varying ability to replicate in S. cerevisiae, again demonstrating that the replication environment plays a significant role in the long-term success of papillomaviruses.
To further investigate these cellular factors, we investigated the role telomeric maintenance factors may play in these processes. We have performed ChIP assays that have shown that components of the telomere maintenance complex (shelterin) can bind to at least four sites in the HPV genome, each of which contain nine-base telomere-repeat sequences (TTAGGGTTA). We have shown that mutating these sites has a detrimental effect on the virus’s ability to replicate under certain conditions. The shelterin complex interacts with a number of important chromosome replication and maintenance proteins with such diverse functions as DNA replication, chromosome segregation, and DNA repair, making it an ideal target for coercion by a DNA virus utilizing a stable low-copy replication strategy. Interaction between Telomer Repeat Binding Factor 2 (TRF2) and Epstein Barr Virus Nuclear Antigen 1 (EBNA1) protein (a structural and functional homologue of E2) is required for replication of plasmids containing the Epstein Barr Virus latent origin of replication. Kaposi’s Sarcoma Herpesvirus (KSHV) Latency Associated Nuclear Antigen (LANA) protein (another homologue of E2) also interacts with TRF2. Results from our Far-Western and co-immunoprecipitation assays show that E2 interacts with TRF2 and other shelterin components. In summary, these results suggest that TRF2, TRF1, Rap1, Pot1, and Tin2, plus certain DNA repair proteins, may regulate the maintenance phase of the HPV lifecycle. E2 appears to be capable of mediating these interactions.
Advisor: Peter C. Angeletti