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

被引:133
作者
MASUDA, T
PLANELLES, V
KROGSTAD, P
CHEN, ISY
机构
[1] UNIV CALIF LOS ANGELES,SCH MED,DEPT MED,DIV HEMATOL,LOS ANGELES,CA 90095
[2] UNIV CALIF LOS ANGELES,SCH MED,DEPT MICROBIOL & IMMUNOL,LOS ANGELES,CA 90095
[3] UNIV CALIF LOS ANGELES,SCH MED,DEPT PEDIAT,DIV INFECT DIS,LOS ANGELES,CA 90095
关键词
D O I
10.1128/JVI.69.11.6687-6696.1995
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 within the integrase (IN) protein, have been identified as being important for integration. We investigated the role of each of these regions in the context of an infectious HIV-1 molecular clone through point mutagenesis of the att site and the zinc finger-like and catalytic domains of IN. The effect of each mutation on integration activity was examined by using a single-step infection system with envelope-pseudotype virus. The relative integration efficiency was estimated by monitoring the levels of viral DNA over time in the infected cells. The integration activities of catalytic domain point mutants and att site deletion mutants were estimated to be 0.5 and 5% of wild-type activity, respectively. However, in contrast with previous in vitro cell-free integration studies, alteration of the highly conserved CA dinucleotide resulted in a mutant which still retained 40% of wild type integration activity. The relative levels of expression of each mutant, as measured by a luciferase reporter gene, correlated with levels of integration. This 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 bearing point mutations in the zinc finger-like domain synthesized much lower 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 transcriptase within the mutant virions. In vitro endogenous reverse transcription assays indicated that all three mutants in the zinc finger-like domain had wild-type levels of reverse transcriptase activity. These data indicate that in addition to integration, IN may have an effect on the proper course of events in the viral life cycle that precede integration.
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页码:6687 / 6696
页数:10
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