title: Methods to assess the nucleocytoplasmic shuttling of the HPV E1 helicase and its effects on cellular proliferation and induction of a DNA damage response.
abstract: Replication of the human papillomavirus (HPV) double-stranded DNA genome in the nucleus of infected cells relies on the viral proteins E1 and E2 in conjunction with the host DNA replication machinery. This process is tightly linked to the replication of cellular DNA, in part through the cyclin-dependent phosphorylation of E1, which inhibits its export out of the nucleus to promote its accumulation in this compartment during S-phase. It has been recently shown that accumulation of E1 in the nucleus, while a prerequisite for viral DNA replication, leads to the inhibition of cellular proliferation and the activation of a DNA damage response (DDR). Here we describe methods to monitor the subcellular localization of E1 and to assess the deleterious effects of its nuclear accumulation on cellular proliferation, cell cycle progression and the induction of a DDR, using a combination of colony formation assays, immunofluorescence microcopy, and flow cytometry approaches.
authors.affiliation: Laboratory of Molecular Virology, Institut de Recherches Cliniques de Montréal, 110 Pine Avenue West, Montreal, QC, Canada, H2W 1R7.
authors.forename: Michaël
authors.initials: M
authors.lastname: Lehoux
authors.forename: Amélie
authors.initials: A
authors.lastname: Fradet-Turcotte
authors.forename: Jacques
authors.initials: J
authors.lastname: Archambault
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001973:Bromodeoxyuridine / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D049109:Cell Proliferation; D004249:DNA Damage; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D027383:Papillomaviridae / Q000201:enzymology*; D010766:Phosphorylation; D021381:Protein Transport; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: C468783:H2AX protein, human; D006657:Histones; D004265:DNA Helicases; D001973:Bromodeoxyuridine