Defining the DNA substrate binding sites on HIV-1 integrase.
ABSTRACT A tetramer model for human immunodeficiency virus type 1 (HIV-1) integrase (IN) with DNA representing long terminal repeat (LTR) termini was previously assembled to predict the IN residues that interact with the LTR termini; these predictions were experimentally verified for nine amino acid residues [Chen, A., Weber, I. T., Harrison, R. W. & Leis, J. (2006). Identification of amino acids in HIV-1 and avian sarcoma virus integrase subsites required for specific recognition of the long terminal repeat ends. J. Biol. Chem., 281, 4173-4182]. In a similar strategy, the unique amino acids found in avian sarcoma virus IN, rather than HIV-1 or Mason-Pfizer monkey virus IN, were substituted into the structurally related positions of HIV-1 IN. Substitutions of six additional residues (Q44, L68, E69, D229, S230, and D253) showed changes in the 3' processing specificity of the enzyme, verifying their predicted interaction with the LTR DNA. The newly identified residues extend interactions along a 16-bp length of the LTR termini and are consistent with known LTR DNA/HIV-1 IN cross-links. The tetramer model for HIV-1 IN with LTR termini was modified to include two IN binding domains for lens-epithelium-derived growth factor/p75. The target DNA was predicted to bind in a surface trench perpendicular to the plane of the LTR DNA binding sites of HIV-1 IN and extending alongside lens-epithelium-derived growth factor. This hypothesis is supported by the in vitro activity phenotype of HIV-1 IN mutant, with a K219S substitution showing loss in strand transfer activity while maintaining 3' processing on an HIV-1 substrate. Mutations at seven other residues reported in the literature have the same phenotype, and all eight residues align along the length of the putative target DNA binding trench.
Article: The (52-96) C-terminal domain of Vpr stimulates HIV-1 IN-mediated homologous strand transfer of mini-viral DNA.[show abstract] [hide abstract]
ABSTRACT: Viral integrase (IN) and Vpr are both components of the human immunodeficiency virus type 1 (HIV-1) pre-integration complex. To investigate whether these proteins interact within this complex, we investigated the effects of Vpr and its subdomains on IN activity in vitro. When a 21mer oligonucleotide was used as a donor and acceptor, both Vpr and its C-terminal DNA-binding domain [(52-96)Vpr] inhibited the integration reaction, whereas the (1-51)Vpr domain did not affect IN activity. Steady-state fluorescence anisotropy showed that both full-length and (52-96)Vpr bind to the short oligonucleotide, thereby extending previous observations with long DNA. The concentrations of the two proteins required to inhibit IN activity were consistent with their affinities for the oligonucleotide. The use of a 492 bp mini-viral substrate confirmed that Vpr can inhibit the IN-mediated reaction. However, the activity of (52-96)Vpr differed notably since it stimulated specifically integration events involving two homologous mini-viral DNAs. Order of addition experiments indicated that the stimulation was maximal when IN, (50-96)Vpr and the mini-viral DNA were allowed to form a complex. Furthermore, in the presence of (50-96)Vpr, the binding of IN to the mini-viral DNA was dramatically enhanced. Taken together, these data suggest that (52-96)Vpr stimulates the formation of a specific complex between IN and the mini-viral DNA.Nucleic Acids Research 06/2003; 31(10):2694-702. · 8.03 Impact Factor
Article: Use of patient-derived human immunodeficiency virus type 1 integrases to identify a protein residue that affects target site selection.[show abstract] [hide abstract]
ABSTRACT: To identify parts of retroviral integrase that interact with cellular DNA, we tested patient-derived human immunodeficiency virus type 1 (HIV-1) integrases for alterations in the choice of nonviral target DNA sites. This strategy took advantage of the genetic diversity of HIV-1, which provided 75 integrase variants that differed by a small number of amino acids. Moreover, our hypothesis that biological pressures on the choice of nonviral sites would be minimal was validated when most of the proteins that catalyzed DNA joining exhibited altered target site preferences. Comparison of the sequences of proteins with the same preferences then guided mutagenesis of a laboratory integrase. The results showed that single amino acid substitutions at one particular residue yielded the same target site patterns as naturally occurring integrases that included these substitutions. Similar results were found with DNA joining reactions conducted with Mn(2+) or with Mg(2+) and were confirmed with a nonspecific alcoholysis assay. Other amino acid changes at this position also affected target site preferences. Thus, this novel approach has identified a residue in the central domain of HIV-1 integrase that interacts with or influences interactions with cellular DNA. The data also support a model in which integrase has distinct sites for viral and cellular DNA.Journal of Virology 09/2001; 75(16):7756-62. · 5.40 Impact Factor