A cell-free replication system for human polyomavirus JC DNA
| Autor: | J. Nesper, Elisabeth Sock, Armin R. Kautz, Richard W.P. Smith, F. Grummt, Heinz-Peter Nasheuer, Michael Wegner |
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| Rok vydání: | 1997 |
| Předmět: |
DNA Replication
DNA polymerase Recombinant Fusion Proteins viruses Immunology DNA Primase Spodoptera Transfection Virus Replication Origin of replication Microbiology Chromatography Affinity Cell Line Mice Plasmid Replication factor C Replication Protein A Virology Tumor Cells Cultured Animals Humans Point Mutation Antigens Viral Tumor Replication protein A Sequence Deletion Binding Sites Base Sequence Cell-Free System biology DNA replication virus diseases RNA Nucleotidyltransferases JC Virus Molecular biology nervous system diseases DNA-Binding Proteins DNA Topoisomerases Type I Oligodeoxyribonucleotides nervous system Viral replication Insect Science Mutagenesis Site-Directed biology.protein Primase Glioblastoma Polyomavirus Baculoviridae Research Article |
| Zdroj: | Europe PubMed Central |
| ISSN: | 1098-5514 0022-538X |
| DOI: | 10.1128/jvi.71.10.7421-7428.1997 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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