Homologous recombination of copackaged retrovirus RNAs during reverse transcription.
According to prevailing models, the high frequency of recombination in retroviruses occurs during reverse transcription of two genetically different genomes copackaged into virion particles. This view has been tested in our studies of the mechanism of recombination within homologous sequences of two retroviral genomes during a single round of virus replication and in the absence of helper virus. The recombination substrates were Moloney murine leukemia virus-based vectors, each of which contains an altered defective neomycin gene (neo) under the transcriptional control of the 5' long terminal repeat; the 3' sequences of each construct contain either the Moloney murine leukemia virus or simian virus 40 large-T polyadenylation sequence. One neo gene contained a linker insertion mutation at the 5' end (neo minus), and the other contained a deletion and linker insertion at the 3' end (neo delta 3). Each of the mutant neo constructs was introduced into the packaging helper cell line psi 2 by sequential cotransfection, and individual psi 2 double transformants were selected. Supernatant fluids from the cloned psi 2 double transformants were used to infect NIH 3T3 cells, and recombinant neo+ proviruses were detected by their ability to confer G418 resistance during infection of NIH 3T3 cells. Our results show that (i) recombination between a homologous sequence of about 560 bp occurred with a frequency of about 10(-4) per virus replication cycle; (ii) recombination occurred only after the viral RNAs had been packaged into particles, i.e., recombination between the two vector DNAs or between viral RNAs prior to packaging was not detected; and (iii) copackaging of two different genomic RNAs as a heterodimer is a prerequisite for recombination. Furthermore, our results indicate that recombination can occur during the DNA negative-strand synthesis of reverse transcription.
Department of Biochemistry, Beckman Center, Stanford University School of Medicine, California 94305-5307.
H
H
Stuhlmann
P
P
Berg
D016475:3T3 Cells; D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D004352:Drug Resistance, Microbial / Q000235:genetics; D005814:Genes, Viral; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics* / Q000502:physiology; D009355:Neomycin / Q000494:pharmacology; D011533:Proviruses / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011995:Recombination, Genetic; D014158:Transcription, Genetic / Q000235:genetics*; D014161:Transduction, Genetic; D014779:Virus Replication
D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase; D009355:Neomycin