Retrovirus-mediated insertion of expressed and non-expressed genes at identical chromosomal locations.
During retrovirus replication, a cellularly derived tRNA is annealed to the viral RNA at the primer binding site (PBS) to prime reverse transcription, and both the tRNA and the PBS become copied and matched together on complementary proviral DNA strands prior to integration. Using a viral PBS single base pair mutant which affects provirus expression in undifferentiated cells, we show that reversion to wild type (wt) occurs at a frequency of approximately 50%. Daughter cell lines containing wt or mutant proviruses at identical chromosomal sites have been isolated, supporting a model where an integrated PBS-mismatched provirus was copied before mismatch correction could occur. Virus expression in daughter cells containing the mutant provirus was 100-fold higher than in cells bearing the wt counterpart. Additionally, proviral 5' DNA and cellular 5' flanking DNA became methylated in daughter cells containing wt but not mutant integrants. These results strongly support the current model of retrovirus reverse transcription, and indicate that the wt PBS region contains an element which suppresses virus expression and directs the methylation of viral and neighboring cellular DNA.
Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.
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B
Berwin
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Barklis
D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D005434:Flow Cytometry; D005814:Genes, Viral; D006651:Histocytochemistry; D008745:Methylation; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics*; D009154:Mutation; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D015183:Restriction Mapping; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase