Recombination-dependent oligomerization of human papillomavirus genomes upon transient DNA 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 HPV-infected patients.

Development of a cellular assay system to study the genome replication of high- and low-risk mucosal and cutaneous human papillomaviruses.

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.

Interaction of the Human Papillomavirus E1 Helicase with UAF1-USP1 Promotes Unidirectional Theta Replication of Viral Genomes.

Human papillomaviruses (HPVs) are important pathogens with a significant medical burden. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism. In this report, we provide evidence that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 deubiquitinating enzyme complex, a member of the Fanconi anemia DNA damage response pathway, is required for the completion of the bidirectional theta replication of the HPV11 genome and the subsequent initiation of the unidirectional replication. We show that unidirectional replication proceeds via theta structures and is supported by the cellular Bloom helicase, which interacts directly with E1 and whose engagement in HPV11 replication requires UAF1-USP1 activity. We propose that the unidirectional replication of the HPV11 genome initiates from replication fork restart events. These findings suggest a new role for the Fanconi anemia pathway in HPV replication.IMPORTANCE Human papillomaviruses (HPVs) are important pathogens that replicate their double-stranded circular DNA genome in the nucleus of infected cells. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism, and the onset of viral replication requires the engagement of cellular DNA damage response pathways. In this study, we showed that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 complex is necessary for the completion of bidirectional replication and the subsequent initiation of the unidirectional replication mechanism. Our results suggest HPVs may use the cellular Fanconi anemia DNA damage pathway to achieve the separation of daughter molecules generated by bidirectional theta replication. Additionally, our results indicate that the unidirectional replication of the HPV genome is initiated from restarted bidirectional theta replication forks.

Analysis of Human Papillomavirus Genome Replication Using Two- and Three-Dimensional Agarose Gel Electrophoresis.

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.

Initial amplification of the HPV18 genome proceeds via two distinct replication mechanisms.

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.