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.
