title: Evidence supporting a role for TopBP1 and Brd4 in the initiation but not continuation of human papillomavirus 16 E1/E2-mediated DNA replication.
abstract: To replicate the double-stranded human papillomavirus 16 (HPV16) DNA genome, viral proteins E1 and E2 associate with the viral origin of replication, and E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated the Brd4 requirement for the transactivation properties of E2, while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More-recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here, we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2-containing nuclear foci, and the viral origin of replication is required for the efficient formation of these foci. Short hairpin RNA (shRNA) against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2-mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall, the results suggest that interactions between TopBP1 and E2 and between Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination.
  
  
  OBJECTIVE
  Human papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers, and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations, but antivirals targeting HPV16 are required for the alleviation of disease burden on the current, and future, generations. Targeting viral DNA replication that is mediated by two viral proteins, E1 and E2, in association with cellular proteins such as TopBP1 and Brd4 would have therapeutic benefits. This report suggests a role for these cellular proteins in the initiation of viral DNA replication by HPV16 E1-E2 but not for continuing replication. This is important if viral replication is to be effectively targeted; we need to understand the viral and cellular proteins required at each phase of viral DNA replication so that it can be effectively disrupted.
authors.affiliation: VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Department of Oral and Craniofacial Molecular Biology, Richmond, Virginia, USA University of Glasgow Institute of Infection, Immunology and Inflammation, Glasgow, United Kingdom.
authors.forename: Elaine J
authors.initials: EJ
authors.lastname: Gauson
authors.affiliation: University of Glasgow Institute of Infection, Immunology and Inflammation, Glasgow, United Kingdom.
authors.forename: Mary M
authors.initials: MM
authors.lastname: Donaldson
authors.affiliation: University of Glasgow Institute of Infection, Immunology and Inflammation, Glasgow, United Kingdom.
authors.forename: Edward S
authors.initials: ES
authors.lastname: Dornan
authors.affiliation: VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Department of Oral and Craniofacial Molecular Biology, Richmond, Virginia, USA.
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.affiliation: VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Department of Oral and Craniofacial Molecular Biology, Richmond, Virginia, USA.
authors.forename: Molly
authors.initials: M
authors.lastname: Bristol
authors.affiliation: Louisiana State University Health Sciences Center-Shreveport, Department of Microbiology and Immunology, Shreveport, Louisiana, USA.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Bodily
authors.affiliation: VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Department of Oral and Craniofacial Molecular Biology, Richmond, Virginia, USA Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia, USA immorgan@vcu.edu.
authors.forename: Iain M
authors.initials: IM
authors.lastname: Morgan
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D052162:Human papillomavirus 16 / Q000502:physiology*; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014779:Virus Replication; D000092468:Bromodomain Containing Proteins
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009856:Oncogene Proteins, Viral; D014157:Transcription Factors; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins; C093478:E1 protein, Human papillomavirus 16; C110513:TOPBP1 protein, human; C076036:oncogene protein E2, Human papillomavirus type 1