       Document 0409
 DOCN  M9620409
 TI    Genetic analysis of human immunodeficiency virus type 1 integrase and
       the U3 att site: unusual phenotype of mutants in the zinc finger-like
       domain.
 DT    9602
 AU    Masuda T; Planelles V; Krogstad P; Chen IS; Department of Microbiology &
       Immunology, UCLA School of Medicine; 90095, USA.
 SO    J Virol. 1995 Nov;69(11):6687-96. Unique Identifier : AIDSLINE
       MED/96013761
 AB    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.
 DE    Amino Acid Sequence  Animal  Base Sequence  Binding Sites  Cell Line
       Cercopithecus aethiops  Conserved Sequence  DNA
       Nucleotidyltransferases/CHEMISTRY/*GENETICS/*METABOLISM  DNA,
       Viral/GENETICS/METABOLISM  Gene Expression  Genome, Viral  Human
       HIV-1/*ENZYMOLOGY/*GENETICS  Kinetics  Molecular Sequence Data
       Mutagenesis, Site-Directed  Oligodeoxyribonucleotides  Phenotype
       Recombinant Proteins/BIOSYNTHESIS/CHEMISTRY/METABOLISM  RNA-Directed DNA
       Polymerase/METABOLISM  Support, Non-U.S. Gov't  Support, U.S. Gov't,
       P.H.S.  Transfection  Virus Integration  *Zinc Fingers  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

