HPV replication and its dependence on repair pathways

Human papillomaviruses (HPV) infect stratified squamous epithelia and link their productive life cycles to the differentiation of the host cell. HPVs infect cells in the basal layer of stratified epithelia and establish genomes as low copy nuclear episomes. When HPV infected cells migrate from the basal layer, they undergo differentiation and in suprabasal layers re-enter S/G2 phases to allow for vegetative viral DNA replication in a process referred to as amplification. HPV replication is regulated by viral and cellular factors that control cell cycle progression, differentiation, and DNA damage repair pathways. Our studies have shown that activation of two homologous recombination repair (HR) pathways, the ataxia telangiectasia (ATM) pathway as well as the ataxia telangiectasia and Rad3-related (ATR) pathway, is required for differentiation-dependent amplification of viral genomes. Two additional pathways that repair DNA breaks are referred to as non-homologous end joining and include the classical non-homologous end-joining pathway (c-NHEJ) and the alternate end joining pathway (alt-EJ). These pathways have been characterized as error-prone but recent work indicates c-NHEJ can be substantially error free. Importantly, we determined that levels of the activated c-NHEJ factors, such as pDNA- PK, are significantly increased in HPV positive cells relative to HFKs. Importantly, treatment of HPV positive cells with either of two drugs that inhibit pDNA-pK activation block viral replication with no effect on cell growth. Additional studies have shown that members of the alt-EJ pathway are also elevated in HPV positive cells but how they affect HPV replication is still unclear. In this application, we will investigate how c-NHEJ and alt-EJ function in HPV replication. We will determine if c-NHEJ factors associate with viral genomes and if knockdown of individual c-NHEJ factors affects replication. Additional studies will use reporters to measure total c-NHEJ activity and investigate if it is altered in comparison to normal keratinocytes. The viral factors responsible for increases will be determined along with the mechanisms mediating the choice between HR and c-NHEJ repair. These studies will provide insights into how activation of c-NHEJ affects HPV replication. A second set of studies will investigate how increases in the levels of alt-EJ factors contribute to regulation of HPV replication. Alt-EJ is an error prone activity that generates small deletions upon repair. Since HPV genomes are largely error free, how this pathway affects HPV replication is unclear. We will determine the factors involved and how they affect HPV replication. One member of this pathway is PARP1 which is regulated by cleavage by Caspase 3 proteases. PARP1 activates alt-EJ while the cleaved products inhibit. High levels of both are detected in HPV positive cells and how this regulates alt-EJ activity along with replication will be examined. Overall, this proposal describes studies aimed at understanding how HPV replication is controlled by non-homologous end joining repair pathways.