Molecular characterization of HIV viruses generated after in vivo ligation

During the course of infection, human immunodeficiency virus type 1 (HIV-1) displays wide genotypic and phenotypic differences. Construction of chimeric viruses is useful to determine the genotypic basis that underlies phenotypic variations, but the procedure is time-consuming. Previously, it has been shown that co-transfection of truncated hemi-genomic HIV-1 proviral DNA can lead to generation of full-length infectious virus. In the study of HIV phenotypes, using this technique, it is important to determine whether recombination between the two hemigenomes occurs without mutations. After co- transfection, progeny recombinant viruses replicated at the same rate as the control. We purified progeny viruses from culture supernatants and determined mutations at the recombination site. It appeared that correct in vivo ligation depended on the purity of DNA and the restriction site used. It also appeared that some of the mutations observed affect replication, as progeny viruses bearing one of these mutations disappeared during in vitro cultures, 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 viruses may have been introduced.