Katzman M, Sudol M; NIH Conference on Retroviral Integrase.
NIH Conf Retroviral Integr NIH Conf Retroviral Integr 1995 Bethesda Md. 1995 Jan 19-20; (Participants' abstracts and posters, abstract no. 4).
Department of Medicine, Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA
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.
Publication Types:
Keywords:
- Animals
- Binding Sites
- Chimera
- DNA
- DNA Nucleotidyltransferases
- DNA Primers
- DNA, Viral
- HIV-1
- Integrases
- Recombinant Fusion Proteins
- Sheep
- Visna-maedi virus
- enzymology
Other ID:
UI: 102212968
From Meeting Abstracts