title: Analysis of JC virus DNA replication using a quantitative and high-throughput assay.
abstract: Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication.
authors.affiliation: Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: Jong
authors.initials: J
authors.lastname: Shin
authors.affiliation: Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Phelan
authors.affiliation: Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: Panharith
authors.initials: P
authors.lastname: Chhum
authors.affiliation: Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: Nazym
authors.initials: N
authors.lastname: Bashkenova
authors.affiliation: Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: Sung
authors.initials: S
authors.lastname: Yim
authors.affiliation: Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Parker
authors.affiliation: Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7; Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec, Canada.
authors.forename: David
authors.initials: D
authors.lastname: Gagnon
authors.affiliation: Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA 02111, USA.
authors.forename: Ole
authors.initials: O
authors.lastname: Gjoerup
authors.affiliation: Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7; Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec, Canada.
authors.forename: Jacques
authors.initials: J
authors.lastname: Archambault
authors.affiliation: Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111, USA. Electronic address: Peter.Bullock@tufts.edu.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Bullock
mesh_terms: D045744:Cell Line, Tumor; D004261:DNA Replication / Q000502:physiology*; D004279:DNA, Viral / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D006801:Humans; D007577:JC Virus / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; D014764:Viral Proteins
keywords: C33A cells; NF-1; Nuclear localization of T-ag; Replication of JCV DNA; Screening for inhibitors of JCV replication; Stimulation of replication by the non-coding control region