GENETIC-ANALYSIS OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTEGRASE ANDTHE U3 ATT SITE - UNUSUAL PHENOTYPE OF MUTANTS IN THE ZINC FINGER-LIKE DOMAIN

Retroviral integration is the step which leads to establishment of the provirus. cis- and trans-acting regions of the human immunodeficiency type 1 (HIV-1) retrovirus genome, including the attachment site (att) at the ends of the unintegrated viral DNA and the conserved domains w ithin the integrase (IN) protein, have been identified as being import ant for integration. We investigated the role of each of these regions in the context of an infectious HIV-1 molecular clone through point m utagenesis of the att site and the zinc finger-like and catalytic doma ins of IN. The effect of each mutation on integration activity was exa mined by using a single-step infection system with envelope-pseudotype virus. The relative integration efficiency was estimated by monitorin g the levels of viral DNA over time in the infected cells. The integra tion activities of catalytic domain point mutants and att site deletio n mutants were estimated to be 0.5 and 5% of wild-type activity, respe ctively. However, in contrast with previous in vitro cell-free integra tion studies, alteration of the highly conserved CA dinucleotide resul ted in a mutant which still retained 40% of wild type integration acti vity. The relative levels of expression of each mutant, as measured by a luciferase reporter gene, correlated with levels of integration. Th is observation is consistent with those of previous studies indicating that integration is an obligatory step for retroviral gene expression . Interestingly, we found that three different HIV-1 constructs bearin g point mutations in the zinc finger-like domain synthesized much lowe r levels of viral DNA after infection, suggesting impairment of these mutants before or at the initiation of reverse transcription. Western blot (immunoblot) analysis demonstrated wild-type levels of reverse tr anscriptase within the mutant virions. In vitro endogenous reverse tra nscription assays indicated that all three mutants in the zinc finger- like domain had wild-type levels of reverse transcriptase activity. Th ese data indicate that in addition to integration, IN may have an effe ct on the proper course of events in the viral life cycle that precede integration.