Replication of the papillomavirus genome is initiated by the assembly of a complex between the viral E1 and E2 proteins at the origin. The E1 helicase is comprised of a C-terminal ATPase/helicase domain, a central domain that binds to the origin, and an N-terminal regulatory region that contains nuclear import and export signals mediating its nucleocytoplasmic shuttling. We previously reported that nuclear accumulation of E1 has a deleterious effect on cellular proliferation which can be prevented by its nuclear export. Here we have shown that nuclear accumulation of E1 from different papillomavirus types blocks cell cycle progression in early S phase and triggers the activation of a DNA damage response (DDR) and of the ATM pathway in a manner that requires both the origin-binding and ATPase activities of E1. Complex formation with E2 reduces the ability of E1 to induce a DDR but does not prevent cell cycle arrest. Transient viral DNA replication still occurs in S-phase-arrested cells but surprisingly is neither affected by nor dependent on induction of a DDR and of the ATM kinase. Finally, we provide evidence that a DDR is also induced in human papillomavirus type 31 (HPV31)-immortalized keratinocytes expressing a mutant E1 protein defective for nuclear export. We propose that nuclear export of E1 prevents cell cycle arrest and the induction of a DDR during the episomal maintenance phase of the viral life cycle and that complex formation with E2 further safeguards undifferentiated cells from undergoing a DDR when E1 is in the nucleus.Human papillomaviruses (HPVs) are small double-stranded DNA viruses that infect the differentiating epithelium of the skin or mucosa (reviewed in references 10 and 91). About 25 types infect the anogenital tract (6, 19), characterized either as low-risk or high-risk types according to their association with benign or malignant hyperproliferative lesions. Clinically, low-risk HPV types cause benign warts while high-risk types are associated with lesions that can progress to cancer (28,56,63,86).The HPV life cycle is dependent on the differentiation program that keratinocytes undergo within a stratified epithelium. Viral DNA replication is required during the three distinct phases of the viral life cycle (reviewed in references 30 and 36). Upon infection of cells from the basal layer of the epithelium, the viral genome is established as a nuclear episome and is replicated by up to 50 to 100 copies (reviewed in reference 30). These episomes are then maintained at a constant copy number by low levels of replication in the lower layers of the epithelium. During this maintenance phase, viral DNA replication is thought to occur only once per cell cycle, during S phase, and in synchrony with replication of the host DNA (32). Finally, as the infected keratinocytes reach the uppermost differentiated layers of the epithelium, the copy number of the viral episome is amplified to very high levels (reviewed in reference 30), presumably through multiple rounds of replication in S-phase-arrested c...

Polyoma- and papillomaviruses genome replication is initiated by the binding of large T antigen (LT) and of E1 and E2, respectively, at the viral origin (ori). Replication of an ori-containing plasmid occurs in cells transiently expressing these viral proteins and is typically quantified by Southern blotting or PCR. To facilitate the study of SV40 and HPV31 DNA replication, we developed cellular assays in which transient replication of the ori-plasmid is quantified using a firefly luciferase gene located in cis to the ori. Under optimized conditions, replication of the SV40 and HPV31 ori-plasmids resulted in a 50- and 150-fold increase in firefly luciferase levels, respectively. These results were validated using replication-defective mutants of LT, E1 and E2 and with inhibitors of DNA replication and cell-cycle progression. These quantitative and high-throughput assays should greatly facilitate the study of SV40 and HPV31 DNA replication and the identification of small-molecule inhibitors of this process.

Approximately 20% of all cancers are associated with infectious agents. Among them, human papillomaviruses (HPVs) are very common and are now recognized as the etiological agent of cervical cancer, the second most common cancer in women worldwide, and they are increasingly linked with other forms of dysplasia. Carcinogenesis is a complex and multistep process requiring the acquisition of several genetic and/or epigenetic alterations. HPV-induced neoplasia, however, is in part mediated by the intrinsic functions of the viral proteins. In order to replicate its genome, HPV modulates the cell cycle, while deploying mechanisms to escape the host immune response, cellular senescence and apoptosis. As such, HPV infection leads directly and indirectly to genomic instability, further favouring transforming genetic events and progression to malignancy. This review aims to summarize our current understanding of the molecular mechanisms exploited by HPV to induce neoplasia, with an emphasis on the role of the 2 viral oncoproteins E6 and E7. Greater understanding of the role of HPV proteins in these processes will ultimately aid in the development of antiviral therapies, as well as unravel general mechanisms of oncogenesis.