A cell-free replication system for human polyomavirus JC DNA.
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.
Nesper
J
J
Abteilung Biochemie, Institut für Molekulare Biotechnologie, Jena, Germany.
Smith
R W
RW
Kautz
A R
AR
Sock
E
E
Wegner
M
M
Grummt
F
F
Nasheuer
H P
HP
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
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