Document 0696
 DOCN  M9590696
 TI    Functional analysis of chimeras between hiv-1 and visna virus integrase
       enzymes.
 DT    9509
 AU    Katzman M; Sudol M; Department of Medicine, Department of Microbiology
       and; Immunology, The Pennsylvania State University College of; Medicine,
       Hershey, PA
 SO    NIH Conf Retroviral Integrase. 1995 Jan 19-20;:(Participants' abstracts
       and posters, abstract no. 4). Unique Identifier : AIDSLINE AIDS/95920023
 AB    Three regions of HIV IN have been defined: the N-terminus (amino acid
       residues 1-49) contains a conserved zinc finger motif and is required
       for optimal enzymatic activity, the central region (residues 50-186) has
       a highly conserved D,D(35)E motif, and the non-conserved C-terminus
       (residues 187-288), which also is required for optimal activity, has
       non-specific DNA binding activity. Although the active site that
       mediates the three phosphoryl transfer reactions exhibited by retroviral
       integrases (i.e., 3'end processing, strand transfer and disintegration)
       has been mapped to the central region, the locations of the viral and
       host DNA binding sites have not been determined. We have found that the
       purified integrases of HIV-1 and visna virus exhibit varying degrees of
       specific processing activity on oligonucleotide substrates derived from
       either end of HIV-1 or visna virus linear DNA, and demonstrate
       distinctly different patterns of strand transfer or integration site
       preferences on these same substrates. In an attempt to map the regions
       of IN that are responsible for viral DNA specificity (the putative viral
       DNA binding site) and target site selection (the putative target DNA
       binding site), we constructed and purified chimeric proteins in which
       all three regions of the two integrases were reciprocally exchanged. By
       convention, we refer to the wild type proteins as HHH and VVV, and to
       the six chimeras as VHH, VVV, HVH, VHV, HHV, and VVH, where the three
       letters represent the N-terminus, central region, and C-terminus,
       respectively, and H (HIV-1) or V (visna) indicates the source of that
       region. We found that all six chimeric proteins, like the wild type
       integrases, were active for disintegration, demonstrating that the
       active site of each chimera was maintained in a functional conformation.
       In contrast, although the chimeric HVV IN exhibited specific processing
       activity, the other chimeras had varying degrees of non-specific
       nuclease activity, indicating disregulation of this activity within the
       context of these chimeras. The HVV IN also demonstrated significant
       strand transfer activity, with a pattern of preferred integration sites
       reminiscent of, but not identical with, that of the wild type VVV IN.
       The only other chimera with significant integration activity was HHV,
       which produced a novel pattern of longer products. These preliminary
       results indicate that whereas the active site of the central region
       readily maintains a functional conformation, viral DNA specificity and
       target DNA selectivity may involve complex interactions between regions
       of IN.
 DE    DNA Nucleotidyltransferases/ANTAGONISTS & INHIB/*METABOLISM
       HIV-1/*ENZYMOLOGY  Kinetics  Magnesium/*METABOLISM  Spectrometry,
       Fluorescence/*METHODS  MEETING ABSTRACT

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