MOLECULAR CHARACTERIZATION OF HIV VIRUSES GENERATED AFTER IN-VIVO LIGATION

During the course of infection, human immunodeficiency virus type 1 (H IV-I) displays wide genotypic and phenotypic differences. Construction of chimeric viruses is useful to determine the genotypic basis that u nderlies phenotypic variations, but the procedure is time-consuming. P reviously, it has been shown that co-transfection of truncated hemi-ge nomic HIV-1 proviral DNA can lead to generation of full-length infecti ous virus. In the study of HIV phenotypes, using this technique, it is important to determine whether recombination between the two hemigeno mes occurs without mutations. After co-transfection, progeny recombina nt viruses replicated at the same rate as the control. We purified pro geny viruses from culture supernatants and determined mutations at the recombination site. It appeared that correct in vivo ligation depende d on the purity of DNA and the restriction site used. It also appeared that some of the mutations observed affect replication, as progeny vi ruses bearing one of these mutations disappeared during in vitro cultu res, whereas other mutants did not. Although this technique is widely applied to generate chimeric viruses, the results should be evaluated with care, since mutations influencing the phenotype of the progeny vi ruses may have been introduced. (C) 1997 Elsevier Science B.V.