We have previously demonstrated that the human papillomavirus (HPV) genome replicates effectively in U2OS cells after transfection using electroporation. The transient extrachromosomal replication, stable maintenance, and late amplification of the viral genome could be studied for high-and low-risk mucosal and cutaneous papillomaviruses. Recent findings indicate that the cellular DNA damage response (DDR) is activated during the HPV life cycle and that the viral replication protein E1 might play a role in this process. We used a U2OS cell-based system to study E1-dependent DDR activation and the involvement of these pathways in viral transient replication. We demonstrated that the E1 protein could cause double-strand DNA breaks in the host genome by directly interacting with DNA. This activity leads to the induction of an ATM-dependent signaling cascade and cell cycle arrest in the S and G 2 phases. However, the transient replication of HPV genomes in U2OS cells induces the ATR-dependent pathway, as shown by the accumulation of ␥H2AX, ATR-interacting protein (ATRIP), and topoisomerase II␤-binding protein 1 (TopBP1) in viral replication centers. Viral oncogenes do not play a role in this activation, which is induced only through DNA replication or by replication proteins E1 and E2. The ATR pathway in viral replication centers is likely activated through DNA replication stress and might play an important role in engaging cellular DNA repair/recombination machinery for effective replication of the viral genome upon active amplification. P apillomaviruses are species-specific double-stranded DNA (dsDNA) viruses that infect the cutaneous and mucosal epithelia of many vertebrate species (1). Human papillomavirus (HPV) infections are widespread, and this virus is considered a common member of the human epithelial microflora (2). In many cases, infections with papillomaviruses are asymptomatic (3). Nearly 100 different HPV types have been identified (4); infections with low-risk viruses (e.g., HPV type 6b [HPV6b] and HPV11) might induce the formation of benign tumors, such as warts and condylomas, while other types (e.g., HPV16 and HPV18), which are referred to as high-risk types, have been shown to cause anogenital and head and neck cancers (reviewed in reference 5). The viral genomes are maintained in infected cells as extrachromosomal nuclear episomes. The proteins encoded by the E1 and E2 open reading frames (ORFs) load the cellular replication machinery at the origin of the HPV replication (reviewed in reference 6). The E1 protein is an origin recognition factor, which is loaded in a sequence-specific manner by the E2 protein at the replication origin, where it forms the E1 double-hexameric replicative helicase upon oligomerization (7-10). Before the initiation of replication, the oligomeric E1 protein unwinds dsDNA into two single strands, assembles into the double-hexameric, ATP-dependent replicative helicase, and loads the cellular replication complex at replication forks for the initiation of DNA replication (r...

We found that recircularized high-risk (type 16 and 18) and low-risk mucosal (type 6b and 11) and cutaneous (type 5 and 8) human papillomavirus (HPV) genomes replicate readily when delivered into U2OS cells by electroporation. The replication efficiency is dependent on the amount of input HPV DNA and can be followed for more than 3 weeks in proliferating cell culture without selection. Cotransfection of recircularized HPV genomes with a linear G418 resistance marker plasmid has allowed subcloning of cell lines, which, in a majority of cases, carry multicopy episomal HPV DNA. Analysis of the HPV DNA status in these established cell lines showed that HPV genomes exist in these cells as stable extrachromosomal oligomers. When the cell lines were cultivated as confluent cultures, a 3-to 10-fold amplification of the HPV genomes per cell was induced. Two-dimensional (2D) agarose gel electrophoresis confirmed amplification of mono-and oligomeric HPV genomes in these confluent cell cultures. Amplification occurred as a result of the initiation of semiconservative two-dimensional replication from one active origin in the HPV oligomer. Our data suggest that the system described here might be a valuable, cost-effective, and efficient tool for use in HPV DNA replication studies, as well as for the design of cell-based assays to identify potential inhibitors of all stages of HPV genome replication.Due to their association with distinctive human cancers, human papillomaviruses (HPVs) are widely studied. Papillomaviruses have been phylogenetically grouped into genera, species, types, subtypes, and variants (11), and more than 100 HPV types have been identified thus far. The best-characterized ␣-genus HPVs are associated with infections of the mucosal epithelia that lead to the induction of benign tumors. These viruses are divided into high-risk types (e.g., high-risk HPV type 16 [HR-HPV-16] and -18), which have the capability of inducing anogenital malignancies, and low-risk types (e.g., LR-HPV-6 and -11), which induce hyperproliferative mucosal lesions and are rarely associated with malignancy. Mucosal HPV infections tend to clear on their own, but in some cases, latent infection could be established and may persist for years. Cutaneous ␤-genus HPV infections are highly prevalent in the general population and tend to persist (14). Vaccines based on virus-like particles made up of the capsid protein L1 have been developed against and Gardasil/Silgard [Merck Research Laboratories]). Because these virus types are responsible for only a portion of all HPV-induced malignant and benign tumors, a clear need exists for vaccines or antivirals against a broader spectrum of pathogenic HPV types.Despite the differences in viral pathogenesis, progeny virion production invariably depends on cell differentiation and occurs only in terminally differentiated keratinocytes. HPVs require the host's replication machinery to reproduce their genomes, and these viruses have developed a unique replication strategy that is adapted to keratinocyte di...