Human papillomaviruses (HPVs) are the etiologic agents of cervical and other epithelial cancers. Persistence of infections by high-risk HPV types is the single greatest risk factor for malignant progression. Although prophylactic vaccines have been developed targeting high risk HPV types, there is a continuing need to better understand the cell-host interactions that underlie persistent benign infection and progression to cancer. In this review, we summarize the molecular events that facilitate the differentiation-dependent HPV life cycle, how the life cycle is organized to facilitate viral persistence, and how the activities of HPV regulatory proteins result in malignancy. Human papillomavirus infection leading to cancerViruses cause approximately 15% of human cancers, and of these nearly half are attributable to human papillomaviruses (HPVs) 1 . Infections by HPVs in the genital tract are the most common sexually transmitted viral infection 2 . Although progression to cancer is a rare event, the high prevalence of HPV infection makes HPV-related cancers, including cervical and other anogenital cancers, among the most common malignancies. It is estimated that over 450,000 new cases of cervical cancer are diagnosed each year making this the second most prevalent cancer in women worldwide 1 . The effective utilization of the Papanicolaou (Pap) smear to recognize cellular abnormalities associated with HPV infection has resulted in a reduction in the number of cases of cervical cancer in the US by 80% in the last 50 years. Despite this decrease, approximately 11,000 women are diagnosed with cervical cancer annually in the US and approximately 3700 die of this disease 2 . In addition, highrisk HPV infection is responsible for approximately 3,000 cases of anal cancer and over 3500 cases of oropharyngeal cancer annually in the United States 2 .Papillomaviruses are small, non-enveloped DNA viruses that infect stratified squamous and cutaneous epithelia 3 . Over 120 different types of HPVs have been identified and approximately one-third of these types specifically target epithelial cells in the genital tract. The remaining types infect epithelial cells in other tissues including cutaneous epithelia. The HPV types that infect the genital mucosa can be divided into two groups. The high-risk (HR) types, including HPV16, 18, 31 and others, are frequently found in cervical cancers. By contrast, the low-risk (LR) types, including HPV6 and 11, also infect the genital epithelia, but are rarely detected in malignancies 4 . Two prophylactic vaccines (Gardasil (Merck) and Ceravrix (GSK)) against the HR types HPV16 and 18 were recently approved

Infection by human papillomaviruses (HPV) leads to the formation of benign lesions, warts, and in some cases, cervical cancer. The formation of these lesions is dependent upon increased expression of proangiogenic factors. Angiogenesis is linked to tissue hypoxia through the activity of the oxygen-sensitive hypoxia-inducible factor 1a (HIF-1a). Our studies indicate that the HPV E7 protein enhances HIF-1 transcriptional activity whereas E6 functions to counteract the repressive effects of p53. Both high-and low-risk HPV E7 proteins were found to bind to HIF-1a through a domain located in the N-terminus. Importantly, the ability of E7 to enhance HIF-1 activity mapped to the C-terminus and correlated with the displacement of the histone deacetylases HDAC1, HDAC4, and HDAC7 from HIF-1a by E7. Our findings describe a novel role of the E7 oncoprotein in activating the function of a key transcription factor mediating hypoxic responses by blocking the binding of HDACs.

To replicate the double-stranded human papillomavirus 16 (HPV16) DNA genome, viral proteins E1 and E2 associate with the viral origin of replication, and E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated the Brd4 requirement for the transactivation properties of E2, while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More-recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here, we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2-containing nuclear foci, and the viral origin of replication is required for the efficient formation of these foci. Short hairpin RNA (shRNA) against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2-mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall, the results suggest that interactions between TopBP1 and E2 and between Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination. IMPORTANCEHuman papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers, and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations, but antivirals targeting HPV16 are required for the alleviation of disease burden on the current, and future, generations. Targeting viral DNA replication that is mediated by two viral proteins, E1 and E2, in association with cellular proteins such as TopBP1 and Brd4 would have therapeutic benefits. This report suggests a role for these cellular proteins in the initiation of viral DNA replication by HPV16 E1-E2 but not for continuing replication. This is important if viral replication is to be effectively targeted; we need to understand the viral and cellular proteins required at each phase of viral DNA replication so that it can be effectively disrupted. Human papillomaviruses (HPVs) are double-stranded DNA viruses that infect the epithelium and cause a variety of human diseases. Human papillomavirus 16 (HPV16) is the most commonly found HPV in cervical cancer (found in around 50% of cases) and also in head and neck cancer (around 90% of the HPVpositive cases) (see reference 1 for a recent review). Two viral proteins, E1 and E2, are required for viral replication. E2 has a carboxyl terminus DNA binding and d...

Human papillomaviruses (HPV) are the causative agents of cervical cancer and have been shown to increase expression of pro-angiogenic factors from infected cells. Many angiogenic factors are regulated by hypoxia inducible factor 1α (HIF-1α). We investigated whether HPV31 affects the levels of HIF-1α under normal and hypoxic conditions. Our studies indicate that cells containing complete HPV31 genomes showed enhanced levels of HIF-1α upon treatment with the hypoxia mimic DFO, which resulted from protein stabilization and lead to increased expression of some but not all HIF-1α target genes. Both HPV E6 and E7 were able independently to enhance induction of HIF-1α upon DFO treatment. Enhancement of HIF-1α stability was not restricted to high risk HPV types, as HPV11, a low risk HPV type, mediated a similar effect. These findings shed light on mechanisms by which HPV contributes to angiogenesis both in benign cervical lesions and in cervical cancers.

Human papillomaviruses (HPV) regulate their differentiation-dependent life cycles by activating a number of cellular pathways, such as the DNA damage response, through control of post-translational protein modification. Sirtuin 1 (SIRT1) is a protein deacetylase that modulates the acetylation of a number of cellular substrates, resulting in activation of pathways controlling gene expression and DNA damage repair. Our studies indicate that SIRT1 levels are increased in cells containing episomes of high-risk HPV types through the combined action of the E6 and E7 oncoproteins. Knockdown of SIRT1 in these cells with shRNAs impairs viral activities including genome maintenance, amplification and late gene transcription, with minimal effects on cellular proliferation ability. Abrogation of amplification was also seen following treatment with the SIRT1 deacetylase inhibitor, EX-527. Importantly, SIRT1 binds multiple regions of the HPV genome in undifferentiated cells, but this association is lost upon of differentiation. SIRT1 regulates the acetylation of Histone H1 (Lys26) and H4 (Lys16) bound to HPV genomes and this may contribute to regulation of viral replication and gene expression. The differentiation-dependent replication of high-risk HPVs requires activation of factors in the Ataxia Telangiectasia Mutated (ATM) pathway and SIRT1 regulates the recruitment of both NBS1 and Rad51 to the viral genomes. These observations demonstrate that SIRT1 is a critical regulator of multiple aspects of the high-risk HPV life cycle.