Viral-host interactions that influence the life cycle of DNA tumor viruses

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Project 2 Project Summary Abstract Oncogenic viruses cause 15-20% of human cancers. High-risk HPVs are associated with 5% of cancers, including cervical cancer (CC) as well as an increasing number of oropharyngeal squamous cell carcinomas (OSCC). RNA molecules, such as long non-coding RNAs (lncRNAs) and co-transcriptional R-loops (RNA:DNA hybrids) are associated with genomic instability and other hallmarks of cancer, but are understudied in the context of HPV infection. Our preliminary data indicate high-risk HPV31 positive cells have increased levels of R-loops compared to uninfected cells. Furthermore, we have identified several lncRNAs that are altered by HPV31 that influence epithelial differentiation, proliferation and survival. Whether these RNA molecules serve a pro-viral role and/or contribute to HPV pathogenesis is unclear. The DNA tumor viruses EBV and KSHV are also found in the oral cavity and cause oral cancers. Similarly to HPV, EBV and KSHV also use differentiation of the oral epithelium as a mechanism to activate lytic replication and virus production. Oral epithelial cells are the likely source of infectious virus in the saliva and a critical component of viral pathogenesis. However, the mechanisms that regulate the productive replication of HPV, as well as the latent/lytic phases of EBV and KSHV in the oral epithelium are not well characterized. These viruses likely employ similar strategies to uncouple proliferation from differentiation to promote both viral persistence and viral spread. KSHV and EBV alter the host lncRNA profile in other cell types, and R-loop forming sequences have been identified in KSHV and EBV viral genomes. Additionally, we have found that KSHV induces R-loop formation and DNA damage in oral epithelial cells. We hypothesize that HPV, EBV and KSHV reprogram the epithelial cell environment through R-loop formation/distribution and lncRNA expression in order to support the viral life cycle. Specific Aims to test this hypothesis are: (1) Determine the role of R-loops in HPV and KSHV pathogenesis by mapping R-loop distribution on cellular and viral DNA and determining if R-loops contribute to DNA damage in infected cells; (2) Determine how cellular lncRNAs contribute to the life cycle of HPV as well as KSHV and EBV in epithelial cells through a biased approach of overexpression/depletion studies of specific lncRNAs, and an unbiased approach through high throughput sequencing to examine global alterations in lncRNA expression. Understanding how R-loops and lncRNAs contribute to replication of these DNA tumor viruses may uncover a role for RNA molecules in facilitating viral persistence as well as identify cellular pathways that may be exploited for the treatment of viral- and potentially non-viral-associated cancers.