Expression of the viral polypeptides E1 and E2 is necessary and sufficient for replication of BPV in mouse C127 cells. By providing these factors from heterologous expression vectors we have identified a minimal origin fragment from BPV that contains all the sequences required in cis for replication of BPV in short term replication assays. This same sequence is also required for stable replication in the context of the entire viral genome. The identified region is highly conserved between different papillomaviruses, and is unrelated to the previously identified plasmid maintenance sequences. The minimal ori sequence contains a binding site for the viral polypeptide E1, which we identify as a sequence specific DNA binding protein, but surprisingly, an intact binding site for the viral transactivator E2 at the ori is not required. The isolated origin shows an extended host region for replication and replicates efficiently in both rodent and primate cell lines.

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...

In HPV–related cancers, the “high-risk” human papillomaviruses (HPVs) are frequently found integrated into the cellular genome. The integrated subgenomic HPV fragments express viral oncoproteins and carry an origin of DNA replication that is capable of initiating bidirectional DNA re-replication in the presence of HPV replication proteins E1 and E2, which ultimately leads to rearrangements within the locus of the integrated viral DNA. The current study indicates that the E1- and E2-dependent DNA replication from the integrated HPV origin follows the “onion skin”–type replication mode and generates a heterogeneous population of replication intermediates. These include linear, branched, open circular, and supercoiled plasmids, as identified by two-dimensional neutral-neutral gel-electrophoresis. We used immunofluorescence analysis to show that the DNA repair/recombination centers are assembled at the sites of the integrated HPV replication. These centers recruit viral and cellular replication proteins, the MRE complex, Ku70/80, ATM, Chk2, and, to some extent, ATRIP and Chk1 (S317). In addition, the synthesis of histone γH2AX, which is a hallmark of DNA double strand breaks, is induced, and Chk2 is activated by phosphorylation in the HPV–replicating cells. These changes suggest that the integrated HPV replication intermediates are processed by the activated cellular DNA repair/recombination machinery, which results in cross-chromosomal translocations as detected by metaphase FISH. We also confirmed that the replicating HPV episomes that expressed the physiological levels of viral replication proteins could induce genomic instability in the cells with integrated HPV. We conclude that the HPV replication origin within the host chromosome is one of the key factors that triggers the development of HPV–associated cancers. It could be used as a starting point for the “onion skin”–type of DNA replication whenever the HPV plasmid exists in the same cell, which endangers the host genomic integrity during the initial integration and after the de novo infection.

Papillomavirus genomes are maintained as multicopy nuclear plasmids in transformed cells. To address the mechanisms by which the viral DNA is stably propagated in the transformed cells, we have constructed a cell line CH04.15 expressing constitutively the viral proteins E1 and E2, that are required for initiation of viral DNA replication. We show that these viral proteins are necessary and sufficient for stable extrachromosomal replication. Using the cell line CH04.15, we have shown that the bovine papillomavirus‐1 (BPV‐1) minimal origin of replication (MO) is absolutely necessary, but is not sufficient for stable extrachromosomal replication of viral plasmids. By deletion and insertion analysis, we identified an additional element (minichromosome maintenance element, MME) in the upstream regulatory region of BPV‐1 which assures stable replication of the MO‐containing plasmids. This element is composed of multiple binding sites for the transcription activator E2. MME appears to function in the absence of replication but requires E1 and E2 proteins for activity. In contrast to, for example, Epstein‐Barr virus oriP, stably maintained BPV‐1 plasmids are not subject to once‐per‐cell cycle replication as determined by density labelling experiments. These results indicate that papillomavirus episomal replicators replicate independently of the chromosomal DNA of their hosts.

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...

The bovine papillomavirus type I transcriptional activator E2 is essential for replication of bovine papillomavirus DNA, yet most of the high-affinity binding sites for E2 are dispensable. Here we demonstrate an absolute requiremuent for a binding site for the E2 polypeptide as a cis-acting replication element, establishing that site-specific binding ofE2 to the origin is a prerequisite for bovine papillomavirus replication in vivo. The position and distance of the E2 binding site relative to the other origin of replication components are flexible, but function at a dance requires hig-affnity E2 binding sites. Thus, low-afnit binding sites function only when located close to the origin of replication, while activity at greater distances requires multimerized high-affinity E2 binding sites. The requirement for E2, although different in some respects, shows distinct similarities to what has been termed replication enhancers and may provide insight into the function of this class of DNA replication element.

Development of invasive cervical cancer upon infection by 'high-risk' human papillomavirus (HPV) in humans is a stepwise process in which some of the initially episomal 'high-risk' type of HPVs (HR-HPVs) integrate randomly into the host cell genome. We show that HPV replication proteins E1 and E2 are capable of inducing overamplification of the genomic locus where HPV origin has been integrated. Clonal analysis of the cells in which the replication from integrated HPV origin was induced showed excision, rearrangement and de novo integration of the HPV containing and flanking cellular sequences. These data suggest that papillomavirus replication machinery is capable of inducing genomic changes of the host cell that may facilitate the formation of the HPV-dependent cancer cell.

Papillomaviruses establish their productive life cycle in stratified epithelium or mucosa, where the undifferentiated proliferating keratinocytes are the initial targets for the productive viral infection. Papillomaviruses have evolved mechanisms to adapt to the normal cellular growth control pathways and to adjust their DNA replication and maintenance cycle to contend with the cellular differentiation. We provide overview of the papillomavirus DNA replication in the differentiating epithelium and describe the molecular interactions important for viral DNA replication on all steps of the viral life cycle.