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...

No abstract

Determining the mechanism of HPV18 replication is paramount for identifying possible drug targets against HPV infection. We used two-dimensional and three-dimensional gel electrophoresis techniques to identify replication intermediates arising during the initial amplification of HPV18 episomal genomes. We determined that the first rounds of HPV18 replication proceed via bidirectional theta structures; however, a notable accumulation of almost fully replicated HPV18 genomes indicates difficulties with the completion of theta replication. We also observed intermediates that were created by a second replication mechanism during the initial amplification of HPV18 genomes. The second replication mechanism does not utilize specific initiation or termination sequences and proceeds via a unidirectional replication fork. We suggest a significant role for the second replication mechanism during the initial replication of the HPV18 genome and propose that the second replication mechanism is recombination-dependent replication.

We describe the extensive and progressive oligomerization of human papillomavirus (HPV) genomes after transfection into the U2OS cell line. The HPV genomic oligomers are extrachromosomal concatemeric molecules containing the viral genome in a head-to-tail orientation. The process of oligomerization does not depend on the topology of the input DNA, and it does not require any other viral factors besides replication proteins E1 and E2. We provide evidence that oligomerization of the HPV18 and HPV11 genomes involves homologous recombination. We also demonstrate oligomerization of the HPV18 and HPV11 genomes in SiHa, HeLa, and C-33 A cell lines and provide examples of oligomeric HPV genomes in clinical samples obtained from HPVinfected patients. Human papillomaviruses (HPV) are important pathogens that cause different epithelial hyperplastic lesions, most commonly manifesting as benign warts or papillomas. Over 100 HPV types have been identified to date (1). These epitheliotropic viruses can be categorized based on their ability to infect mucosal or cutaneous keratinocytes. The mucosal viruses can be further subdivided into low-and high-risk HPVs. A potential for malignant progression is characteristic of high-risk HPV types, such as HPV18, HPV16, HPV31, and HPV45, whereas types such as HPV6 and HPV11 do not show similar associations and are considered low risk (2). Essentially all cervical carcinomas (3) and a quarter of reported head and neck cancers (4) are associated with HPV infections.HPV is a small DNA virus with an approximately 8-kbp genome. During infection of stratified cutaneous or mucosal epithelia, the viral genomes replicate as multicopy extrachromosomal genetic elements in the nuclei of host cells. HPV genomes undergo a three-phase replication cycle linked to the host cell differentiation program (5). The first stage of HPV DNA replication occurs in undifferentiated basal keratinocytes after infection and is referred to as transient amplificational replication. During the first phase, the viral replication factors are produced and the HPV genome is amplified up to 100 of copies per cell during the S phase of the cell cycle. After initial amplification, the expression of viral replication proteins is downregulated to a level sufficient for the stable maintenance phase of episomal genomes in HPV-infected basal cells. Upon differentiation of the infected cells, the regulated expression of viral proteins initiates a second amplification of the viral genome, the production of capsid proteins, and the assembly of viral particles in the uppermost layers of terminally differentiated epithelium. The mechanisms regulating the switch from the initial HPV genome replication to HPV genome maintenance and, subsequently, to vegetative amplification are not entirely understood.Replication of the HPV genome is carried out by the cellular replication machinery, which is directed to the viral origin by the viral replication proteins E1 and E2. The mechanism underlying the initiation of DNA replication is well described...

This unit includes the necessary information to conduct neutral/neutral and neutral/alkaline two-dimensional and neutral/neutral/alkaline three-dimensional agarose gel electrophoresis. The methodology has been optimized over the years to gain a better outcome from the hard-to-interpret signals of human papilloma virus replication intermediates obtained from two- and three-dimensional agarose gels. Examples of typical results and interpretation of replication intermediate patterns are included, and the outcomes of multiple-dimension assays are assessed using previously published experimental data. © 2017 by John Wiley & Sons, Inc.