Abstract 1637 Molecular mechanism of HPV DNA replication initiation, mediated by E1 and E2 proteins

Human papillomaviruses (HPVs) cause virtually all cervical cancers, the second leading cause of death by cancer among women, as well as a substantial portion of oropharyngeal cancers. Despite the availability of a vaccine that targets 9 of the over 100 variants, HPV-induced cancer and infections continue to be an important clinical and societal burden, as these vaccines do not offer any therapeutic effect against pre-existing infections or HPV-associated cancers. HPV is a double stranded DNA virus that encodes two replicative proteins, E1 (DNA Helicase) and E2 (DNA replication initiator protein). The E1 and E2 proteins play important roles in the regulation of viral DNA replication and transcription of the oncoproteins of HPV. In this study, we focused on elucidating the interaction between E1 and E2 proteins, which are the essential players in HPV DNA replication, using purified full-length recombinant proteins obtained from E. coli. Through in vitro binding assays, such as Electrophoretic Mobility Shift Assay (EMSA), we identified a crucial interaction between the E1 helicase and E2 protein. Notably, E2 exhibited specific binding to its binding sites at the origin, thereby attracting the E1 helicase to the origin site. This interaction facilitates the formation of the E1-E2-DNA preinitiation complex, a crucial step in initiating HPV DNA replication. Further investigations utilizing ATPase and helicase assays with E1 and E2, we found that E2 regulates E1 activity, leading to reduced ATPase and helicase functions, suggesting a potential mechanism for E2 dissociation from the replication fork when E1 helicase binds to DNA. This dissociation may enable the E1 helicase to efficiently carry out the unwinding of the DNA, a critical step in the initiation of HPV DNA replication. This study illuminates DNA replication mechanisms and E1-E2 protein regulatory dynamics, advancing our understanding of HPV biology and paving the way for targeted therapeutic interventions against HPV-associated diseases.