An Exonuclease V-qPCR Assay to Analyze the State of the Human Papillomavirus 16 Genome in Cell Lines and Tissues

Curr Protoc Microbiol. 2020 Dec;59(1):e119. doi: 10.1002/cpmc.119.

Abstract

Integration of the human papillomavirus (HPV) genome into host cell chromosomes has been observed in a majority of HPV-positive cervical cancers and a subset of oral HPV-associated cancers. HPV integration also occurs in long-term cell culture. Screening for HPV integration can be labor intensive and yield results that are difficult to interpret. Here we describe an assay based on exonuclease V (ExoV/RecBCD) and quantitative polymerase chain reaction (qPCR) to determine if samples from cell lines and tissues contain episomal or integrated HPV. This assay can be applied to screen other small DNA viruses with episomal/linear genome configurations in their viral lifecycle and has the potential to be used in clinical settings to define viral genomic conformations associated with disease. © 2020 Wiley Periodicals LLC. Basic Protocol: Exonuclease V genomic DNA digestion and qPCR for detection of HPV16 genome configuration in cells Support Protocol: Exonuclease V analysis of HPV16 genome configuration in tissues Alternate Protocol: Determining HPV integration type or integrity of HPV episome.

Keywords: RecBCD; episomal; exonuclease V; human papillomavirus (HPV); integrated.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Line
  • DNA, Viral
  • Exodeoxyribonuclease V / analysis*
  • Exodeoxyribonuclease V / genetics*
  • Female
  • Genome, Viral*
  • Human papillomavirus 16 / enzymology*
  • Human papillomavirus 16 / genetics*
  • Humans
  • Papillomaviridae / genetics
  • Papillomavirus Infections / virology
  • Plasmids
  • Polymerase Chain Reaction / methods*
  • Uterine Cervical Neoplasms / virology
  • Virus Integration

Substances

  • DNA, Viral
  • Exodeoxyribonuclease V