title: Reconstitution of wild type viral DNA in simian cells transfected with early and late SV40 defective genomes.
abstract: The DNAs of polyomaviruses ordinarily exist as a single circular molecule of approximately 5000 base pairs. Variants of SV40, BKV and JCV have been described which contain two complementing defective DNA molecules. These defectives, which form a bipartite genome structure, contain either the viral early region or the late region. The defectives have the unique property of being able to tolerate variable sized reiterations of regulatory and terminus region sequences, and portions of the coding region. They can also exchange coding region sequences with other polyomaviruses. It has been suggested that the bipartite genome structure might be a stage in the evolution of polyomaviruses which can uniquely sustain genome and sequence diversity. However, it is not known if the regulatory and terminus region sequences are highly mutable. Also, it is not known if the bipartite genome structure is reversible and what the conditions might be which would favor restoration of the monomolecular genome structure. We addressed the first question by sequencing the reiterated regulatory and terminus regions of E- and L-SV40 DNAs. This revealed a large number of mutations in the regulatory regions of the defective genomes, including deletions, insertions, rearrangements and base substitutions. We also detected insertions and base substitutions in the T-antigen gene. We addressed the second question by introducing into permissive simian cells, E- and L-SV40 genomes which had been engineered to contain only a single regulatory region. Analysis of viral DNA from transfected cells demonstrated recombined genomes containing a wild type monomolecular DNA structure. However, the complete defectives, containing reiterated regulatory regions, could often compete away the wild type genomes. The recombinant monomolecular genomes were isolated, cloned and found to be infectious. All of the DNA alterations identified in one of the regulatory regions of E-SV40 DNA were present in the recombinant monomolecular genomes. These and other findings indicate that the bipartite genome state can sustain many mutations which wtSV40 cannot directly sustain. However, the mutations can later be introduced into the wild type genomes when the E- and L-SV40 DNAs recombine to generate a new monomolecular genome structure.
authors.affiliation: University of Utah, Department of Cellular, Viral and Molecular Biology, Salt Lake City.
authors.forename: F J
authors.initials: FJ
authors.lastname: O'Neill
authors.forename: Y
authors.initials: Y
authors.lastname: Gao
authors.forename: X
authors.initials: X
authors.lastname: Xu
mesh_terms: D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001483:Base Sequence; D002213:Capsid / Q000235:genetics; D002460:Cell Line; D002522:Chlorocebus aethiops; D004274:DNA, Recombinant / Q000032:analysis; D004279:DNA, Viral / Q000737:chemistry*; D003673:Defective Viruses / Q000235:genetics; D016679:Genome, Viral; D005909:Glioblastoma; D006801:Humans; D008969:Molecular Sequence Data; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000952:Antigens, Polyomavirus Transforming; D004274:DNA, Recombinant; D004279:DNA, Viral