HPV Life Cycle and R-loops

Project Summary 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 and establish genomes as low copy nuclear episomes. When HPV infected cells migrate from the basal layer, they become arrested in G1, and a subset are induced to re-enter G2/M phases in the most differentiated layers to allow for productive viral DNA replication in a process referred to as amplification. Our recent studies have shown that activation of both the ataxia telangiectasia (ATM) pathway as well as the ataxia telangiectasia and Rad3-related (ATR) DNA damage repair (DDR) pathways is critical for viral amplification in differentiated cells. Furthermore, activation of ATR but not ATM, is required for stable maintenance of viral episomes in undifferentiated cells. Our studies further showed that HPV proteins activate these repair pathways by inducing high levels of DNA breaks in both cellular and viral DNAs that are caused by aberrant R-loops along with the action of topoisomerases. R-loops are trimeric nucleic acid structures that consist of a hybrid between RNA and its complementary DNA strand along with the displaced single strand DNA. These are stable structures that form at promoter as well as transcription termination regions. R-loops play important roles in the normal regulation of transcription initiation and termination however failure to resolve aberrant R-loops leads to DNA break formation. Our studies indicate R-loop levels are increased by 5 to 10-fold in HPV positive cells and maintenance of these high levels is necessary for viral replication and transcription as either decreasing or increasing the amounts leads to impaired viral functions. Furthermore, our work indicates that these enhanced R-loop levels also regulate expression of cellular pathways that are important for viral pathogenesis. E6 is responsible for the high levels of R-loops in HPV positive cells through its inhibition of p53. This application will investigate the mechanisms by which R-loop levels are increased in HPV positive cells and why this is critical for the viral transcription as well as replication. Specifically, we will examine the role R-loops play in differentiation-dependent amplification and late viral gene expression. In addition, the role of E6 in regulating R-loop levels through stabilization will be examined along with investigating which downstream targets of p53 effects are responsible for regulating R-loop levels. Finally, a role for the cytosine deaminase, APOBCEC 3B, in regulating R-loop formation in HPV positive cells will be addressed. Overall, the proposed studies will provide important insights into why R-loops play a critical role in the HPV life cycle.