Mechanisms and consequences of human papillomavirus 16 E2 regulation of host gene transcription - ABSTRACT Human papillomaviruses (HPV) are causative agents in ano-genital and head and neck cancers and are responsible for around 5% of cancers worldwide. In order to identify and develop novel approaches for targeting these viral diseases, we must enhance our understanding of the viral life cycle. HPV16 is the most common type that causes cancer (high-risk, HR-HPV). During its life cycle, the virus exists as an 8kbp DNA plasmid. A viral protein that mediates viral replication is E2, a DNA binding protein that forms homodimers that bind to 12bp palindromic DNA target sequences. E2 has three functions during the viral life cycle: first, it binds to target DNA sites around the viral origin of replication and recruits the viral helicase E1 to the origin. E1 then forms a di-hexameric complex that initiates viral replication in association with host cellular factors. Second, E2 mediates segregation of the episomal viral genome into daughter nuclei during cell division by simultaneously interacting with viral and host chromatin acting as a bridge to locate the viral genomes to daughter nuclei. Third, E2 can regulate viral and host transcription. The focus of this proposal is on how E2 regulates transcription from the host genome to facilitate the viral life cycle. Using RNA-seq data from isogenic N/Tert-1-Vec (Vector control), N/Tert-1+E2 (expressing the E2 protein only) and N/Tert-1+HPV16 (containing the episomal viral genome, this model demonstrates several aspects of the HPV16 life cycle in organotypic raft cultures) we demonstrated that E2 regulates host transcription that is important during the viral life cycle. This proposal will focus on E2 repression of innate immune gene transcription (IIG) and suppression of epithelial to mesenchymal transition (EMT) via repression of TWIST1. The repression of IIGs is via DNA methylation and here we demonstrate that E2 is in a cellular complex with DNMT1, providing a potential mechanism (E2 recruitment of DNMT1 to IIG promoters) for E2 repression. E2 represses TWIST1 via a direct interaction with the TWIST1 promoter; DNA methylation is not involved. The TWIST1 promoter is activated by STAT3 and here we demonstrate that E2 interacts with STAT3, providing a potential mechanism of how E2 is repressing TWIST1. In this proposal, we will investigate the repression of IIG IFIT1. The IFIT1 protein can bind to and disrupt HPV18 E1-E2 replication, and we have demonstrated that it binds to HPV16 E1, and preliminary studies demonstrate it can attenuate E1-E2 replication. Using novel CRISPR technology we have increased the levels of IFIT1 in a host of HPV16 and E2 positive cells. We will investigate whether elevated TWIST1 interferes with the HPV16 life cycle. We will carry out similar experiments with TWIST1. Recently we demonstrated that E2 sensitizes cells to cisplatin, an important process that could contribute to the better outcomes for E2 expressing HPV+ cancer patients. We will investigate the roles of IIGs and TWIST1 in this process. Overall, our results will advance our understanding of how E2 regulates host transcription, and provide potential novel therapeutic approaches for combatting HPV16 infections and cancers.
