title: A cell-free replication system for human polyomavirus JC DNA.
abstract: The human polyomavirus JC virus (JCV) establishes persistent infections in most individuals and is the etiologic agent of progressive multifocal leukoencephalopathy. In this report, we describe the establishment of a soluble cell-free system that is capable of replicating exogenous plasmid DNA containing the JCV origin of replication. Replication in this system is completely dependent on the addition of JCV large T antigen (TAg). To prepare JCV TAg for replication analysis, a recombinant baculovirus containing the JCV TAg-coding sequence was generated. TAg expressed in insect cells was purified by metal chelate chromatography. JCV TAg supported initiation of JCV DNA replication in the presence of DNA polymerase alpha-primase, replication protein A, and topoisomerase I in a dose-dependent manner and was also capable of supporting DNA replication in crude human cell extracts. Point mutation of TAg-binding site I strongly diminished TAg binding and concomitantly reduced JCV DNA replication in vivo and in vitro by approximately 50%. Point mutation of TAg-binding site II or deletion of the early palindrome completely abolished replication of JCV origin-containing plasmid DNA in vivo and in vitro, marking these sequences as essential components of the JCV core origin. A comparison of several TAgs showed that simian virus 40 TAg, but not mouse polyomavirus (PyV) TAg, supported replication of a plasmid containing a JCV origin. These findings provide evidence that replication in the cell-free system faithfully mimics JCV DNA replication in vivo. Therefore, it may be a useful tool for future analysis of interactions between JCV and its host cell.
authors.affiliation: Abteilung Biochemie, Institut für Molekulare Biotechnologie, Jena, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Nesper
authors.forename: R W
authors.initials: RW
authors.lastname: Smith
authors.forename: A R
authors.initials: AR
authors.lastname: Kautz
authors.forename: E
authors.initials: E
authors.lastname: Sock
authors.forename: M
authors.initials: M
authors.lastname: Wegner
authors.forename: F
authors.initials: F
authors.lastname: Grummt
authors.forename: H P
authors.initials: HP
authors.lastname: Nasheuer
mesh_terms: D000818:Animals; D000957:Antigens, Viral, Tumor / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016367:Baculoviridae; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002474:Cell-Free System; D002846:Chromatography, Affinity; D019915:DNA Primase; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005909:Glioblastoma; D006801:Humans; D007577:JC Virus / Q000235:genetics / Q000502:physiology*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011120:Polyomavirus / Q000502:physiology; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D051716:Replication Protein A; D017384:Sequence Deletion; D018411:Spodoptera; D014162:Transfection; D014407:Tumor Cells, Cultured; D014779:Virus Replication
chemical_list: D000957:Antigens, Viral, Tumor; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; C497167:RPA1 protein, human; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A; C497168:Rpa1 protein, mouse; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004264:DNA Topoisomerases, Type I