title: Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31.
abstract: High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination.
  
  
  OBJECTIVE
  Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral replication, our findings suggest that HPV may utilize ATM activity to ensure localization of recombination factors to productively replicating viral genomes. The finding that E7 increases the levels of Rad51 and BRCA1 suggests that E7 contributes to productive replication by providing DNA repair factors required for viral DNA synthesis. Our studies not only imply a role for recombination in the regulation of productive HPV replication but provide further insight into how HPV manipulates the DDR to facilitate the productive phase of the viral life cycle.
authors.affiliation: Lineberger Comprehensive Cancer Center and Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: William H
authors.initials: WH
authors.lastname: Chappell
authors.affiliation: Lineberger Comprehensive Cancer Center and Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Dipendra
authors.initials: D
authors.lastname: Gautam
authors.affiliation: Lineberger Comprehensive Cancer Center and Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Suzan T
authors.initials: ST
authors.lastname: Ok
authors.affiliation: Lineberger Comprehensive Cancer Center and Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Johnson
authors.affiliation: Lineberger Comprehensive Cancer Center and Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Anacker
authors.affiliation: Lineberger Comprehensive Cancer Center and Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA camoody@med.unc.edu.
authors.forename: Cary A
authors.initials: CA
authors.lastname: Moody
mesh_terms: D019313:BRCA1 Protein / Q000378:metabolism*; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000821:virology; D005347:Fibroblasts / Q000821:virology; D005786:Gene Expression Regulation; D054884:Host-Pathogen Interactions; D058952:Human papillomavirus 31 / Q000254:growth & development / Q000502:physiology*; D006801:Humans; D050725:Papillomavirus E7 Proteins / Q000378:metabolism; D051135:Rad51 Recombinase / Q000378:metabolism*; D059767:Recombinational DNA Repair; D014158:Transcription, Genetic; D015854:Up-Regulation; D014779:Virus Replication
chemical_list: D019313:BRCA1 Protein; C492913:BRCA1 protein, human; C502189:E7 protein, Human papillomavirus type 31; D050725:Papillomavirus E7 Proteins; C495153:RAD51 protein, human; D051135:Rad51 Recombinase