Transduction of cellular neo mRNA by retrovirus-mediated recombination.
Transduction of cellular oncogenes by retroviruses is thought to be a multistep process, involving transcriptional activation of a cellular gene by upstream proviral integration and joining of cellular DNA to retroviral transcriptional signals, followed by copackaging and recombination with a helper virus genome during reverse transcription. To examine the molecular mechanism of the reverse transcriptase-mediated recombination, we introduced into mouse fibroblast cells a variety of constructs in which the neo selectable marker was joined to flanking retroviruslike or cell-like sequences. After superinfection and copackaging with a replication-competent Mo-MuLVsupF virus, the formation of recombinant neo transducing viruses was assessed in a second round of virus infection by the ability to confer G418 resistance to infected cells. Our results showed that recombinant neo proviruses were generated from neo RNA containing either a 5' or 3' retroviral end, implying that one recombination event with helper virus RNA was sufficient to incorporate the neo gene into proviral DNA. Recombination occurred with an apparent frequency of 10(-4) to 10(-5) per replication cycle in the absence of homology between the two recombining partners. This frequency, however, increased at least 100-fold if homology was provided at the site of recombination. Our results support the hypothesis that neo-transducing viruses arise via reverse transcriptase-mediated recombination of RNA rather than by recombination proceeding through DNA intermediates. Unexpectedly, removal of the retroviral packaging site psi reduced the number of neo recombinants only slightly. Our data indicated that although RNAs lacking the psi site are poorly packaged into virions, those RNAs that are included in the virions undergo frequent recombination, even if there is no selection for recombination. Many of the neo recombinants formed with the psi- constructs had undergone additional recombinations and often incorporated the psi site from the helper RNA.
Department of Biochemistry, Beckman Center, Stanford University School of Medicine, California 94305-5307.
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Stuhlmann
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Dieckmann
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Berg
D004279:DNA, Viral / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics*; D005822:Genetic Vectors; D015151:Immunoblotting; D009355:Neomycin; D012333:RNA, Messenger / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012190:Retroviridae / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics; D014161:Transduction, Genetic; D014162:Transfection
D004279:DNA, Viral; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D009355:Neomycin