HPV induced R-loops and expression of innate immune genes

About This Grant

Project Summary/Abstract Human papillomaviruses (HPV) infect stratified epithelia and link their productive life cycles to differentiation. The HPV life cycle is regulated by viral proteins acting together with cellular factors that control cell cycle progression, differentiation, and DNA damage repair. This application will investigate the pathways that regulate the productive replication of high-risk papillomaviruses. We have previously shown that activation of both the ataxia telangiectasia (ATM) pathway as well as the ataxia telangiectasia and Rad3-related (ATR) DNA damage repair (DDR) pathway is necessary for differentiation-dependent amplification of viral genomes. In addition, ATR activation was found to play a critical role in the stable maintenance of episomes in undifferentiated cells. Our studies further indicate these pathways are activated by the high numbers of DNA breaks caused by aberrant R-loops. R-loops are trimeric nucleic acid structures consisting 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 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. We have shown that R-loop levels are increased by over 10-fold in HPV positive cells, and maintenance of these high levels is necessary for viral replication and transcription. Furthermore, our work indicates that these enhanced R-loop levels help to regulate the expression of important cellular pathways, including the repression of innate immune regulatory genes. Two major enzymes act to regulate R-loops; RNAse H1 is an R-loop specific RNase that degrades the RNA moiety while senataxin (SETX) is a helicase that unwinds R-loops as well as acts at termination sites that contain R-loops to recruit the exonuclease XRN2 for mRNA cleavage. Our recent work shows that both SETX and RNAse H1 contribute to repression of innate immune gene expression in HPV positive cells while at the same time both are required for high level viral transcription. SETX acts to regulate resolution of R-loops particularly those at 3’ termination sequences along with activating the m6A RNA methylation pathway. Recruitment of the m6A catalytic enzyme Mettl3 is mediated by SETX to methylate RNAs in R-loops and is important for their resolution and proper termination of transcription. RNAse H1 acts primarily to remove RNA moieties in R-loops. These two enzymes appear to work through complementary but different mechanisms and this application proposes to investigate how this leads to repression of innate immune gene expression along with activation of viral expression and replication.