Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31.

UNLABELLED: High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. IMPORTANCE: Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral replication, our findings suggest that HPV may utilize ATM activity to ensure localization of recombination factors to productively replicating viral genomes. The finding that E7 increases the levels of Rad51 and BRCA1 suggests that E7 contributes to productive replication by providing DNA repair factors required for viral DNA synthesis. Our studies not only imply a role for recombination in the regulation of productive HPV replication but provide further insight into how HPV manipulates the DDR to facilitate the productive phase of the viral life cycle.

Differential dependence on host cell glycosaminoglycans for infection of epithelial cells by high-risk HPV types.

Human papillomavirus (HPV) infection is the leading cause of cervical cancer world-wide. Here, we show that native HPV particles produced in a differentiated epithelium have developed different strategies to infect the host. Using biochemical inhibition assays and glycosaminoglycan (GAG)-negative cells, we show that of the four most common cancer-causing HPV types, HPV18, HPV31, and HPV45 are largely dependent on GAGs to initiate infection. In contrast, HPV16 can bind and enter through a GAG-independent mechanism. Infections of primary human keratinocytes, natural host cells for HPV infections, support our conclusions. Further, this renders the different virus types differentially susceptible to carrageenan, a microbicide targeting virus entry. Our data demonstrates that ordered maturation of papillomavirus particles in a differentiating epithelium may alter the virus entry mechanism. This study should facilitate a better understanding of the attachment and infection by the main oncogenic HPV types, and development of inhibitors of HPV infection.

Treatment of a human papillomavirus type 31b-positive cell line with benzo[a]pyrene increases viral titer through activation of the Erk1/2 signaling pathway.

Numerous epidemiological studies have implicated cigarette smoking as a cofactor in the progression to cervical cancer. Tobacco-associated hydrocarbons have been found in cervical mucus, suggesting a possible interaction with human papillomavirus (HPV)-infected cells. The polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) is a major component of cigarette smoke condensate that has received significant attention due to its ability to induce carcinogenesis. We have previously demonstrated by conventional methods for determining viral titer that high concentrations of BaP increase HPV31b titers within the context of organotypic raft cultures compared with the level for vehicle controls. However, a definitive mechanism for explaining this increase in viral titer was lacking. Here, we show that BaP treatment activates the Ras-Raf-Mek1/2-Erk1/2 signaling pathway. The importance of Erk1/2 pathway activation to the BaP-mediated increase in viral titer was determined by Erk pathway inhibition with multiple Erk1/2 pathway inhibitors. Finally, BaP treatment activated p90RSK and its downstream target CDK1. These data indicate that the Erk1/2 signaling pathway plays an important role in mediating the response to BaP treatment that ultimately leads to increased viral titers.