An amino acid in the central catalytic domain of three retroviral integrases that affects target site selection in nonviral DNA

Integrase can insert retroviral DNA into almost any site in cellular DNA; however, target site preferences are noted in vitro and in vivo. We recently demonstrated that amino acid 119, in the alpha2 helix of the central domain of the human immunodeficiency virus type 1 integrase, affected the choice of nonviral target DNA sites. We have now extended these findings to the integrases of a nonprimate lentivirus and a more distantly related alpharetrovirus. We found that substitutions at the analogous positions in visna virus integrase and Rous sarcoma virus integrase changed the target site preferences in five assays that monitor insertion into nonviral DNA. Thus, the importance of this protein residue in the selection of nonviral target DNA sites is likely to be a general property of retroviral integrases. Moreover, this amino acid might be part of the cellular DNA binding site on integrase proteins.     

Weak palindromic consensus sequences are a common feature found at the integration target sites of many retroviruses

Integration into the host genome is one of the hallmarks of the retroviral life cycle and is catalyzed by virus-encoded integrases. While integrase has strict sequence requirements for the viral DNA ends, target site sequences have been shown to be very diverse. We carefully examined a large number of integration target site sequences from several retroviruses, including human immunodeficiency virus type 1, simian immunodeficiency virus, murine leukemia virus, and avian sarcoma-leukosis virus, and found that a statistical palindromic consensus, centered on the virus-specific duplicated target site sequence, was a common feature at integration target sites for these retroviruses.     

Role of the nonspecific DNA-binding region and alpha helices within the core domain of retroviral integrase in selecting target DNA sites for integration

Retroviral integrase plays an important role in choosing host chromosomal sites for integration of the cDNA copy of the viral genome. The domain responsible for target site selection has been previously mapped to the central core of the protein (amino acid residues 49-238). Chimeric integrases between human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) were prepared to examine the involvement of a nonspecific DNA-binding region (residues 213-266) and certain alpha helices within the core domain in target site selection. Determination of the distribution and frequency of integration events of the chimeric integrases narrowed the target site-specifying motif to within residues 49-187 and showed that alpha 3 and alpha 4 helices (residues 123-166) were not involved in target site selection. Furthermore, the chimera with the alpha 2 helix (residues 118-121) of FIV identity displayed characteristic integration events from both HIV-1 and FIV integrases. The results indicate that the alpha 2 helix plays a role in target site preference as either part of a larger or multiple target site-specifying motif.     

Use of Patient-Derived Human Immunodeficiency Virus Type 1 Integrases To Identify a Protein Residue That Affects Target Site Selection

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.     

Irreversible inhibition of human immunodeficiency virus type 1 integrase by dicaffeoylquinic acids

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses require integration of a double-stranded DNA copy of the RNA genome into the host cell chromosome for productive infection. The viral enzyme, integrase, catalyzes the integration of retroviral DNA and represents an attractive target for developing antiretroviral agents. We identified several derivatives of dicaffeoylquinic acids (DCQAs) that inhibit HIV-1 replication in tissue culture and catalytic activities of HIV-1 integrase in vitro. The specific step at which DCQAs inhibit the integration in vitro and the mechanism of inhibition were examined in the present study. Titration experiments with different concentrations of HIV-1 integrase or DNA substrate found that the effect of DCQAs was exerted on the enzyme and not the DNA. In addition to HIV-1, DCQAs also inhibited the in vitro activities of MLV integrase and truncated variants of feline immunodeficiency virus integrase, suggesting that these compounds interacted with the central core domain of integrase. The inhibition on retroviral integrases was relatively specific, and DCQAs had no effect on several other DNA-modifying enzymes and phosphoryltransferases. Kinetic analysis and dialysis experiments showed that the inhibition of integrase by DCQAs was irreversible. The inhibition did not require the presence of a divalent cation and was unaffected by preassembling integrase onto viral DNA. The results suggest that the irreversible inhibition by DCQAs on integrase is directed toward conserved amino acid residues in the central core domain during catalysis.     

Juxtaposition of two viral DNA ends in a bimolecular disintegration reaction mediated by multimers of human immunodeficiency virus type 1 or murine leukemia virus integrase.

Integration of retroviral DNA involves a coordinated joining of the two ends of a viral DNA molecule into precisely spaced sites on target DNA. In this study, we designed an assay that requires two separate oligonucleotides to be brought together via interactions between integrase promoters to form a "crossbones" substrate that mimics the integration intermediate. The crossbones substrate contains two viral DNA ends, each joined to one strand of target DNA and separated by a defined length of target DNA. We showed that purified integrases of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV) could mediate a concerted strand cleavage-ligation between the two half-substrates at one or both viral DNA joining sites (trans disintegration). Another major product, termed fold-back, resulted from an intramolecular attack on the phosphodiester bond at the viral-target DNA junction by the 3'-OH group of the same DNA molecule (cis disintegration). The activity of integrase on the crossbones substrate depended on the presence of viral DNA sequences. For trans disintegration, the optimal length of target DNA between the viral DNA joining sites of the crossbones substrate corresponded to the spacing between the staggered joints formed on two opposite strands of target DNA during retroviral DNA integration in vivo. The activity of integrases on crossbones did not require complementary base pairing between the two half-substrates, indicating that the half-substrates were juxtaposed solely through protein-DNA interactions. The crossbones assay, therefore, measures the ability of integrase to juxtapose two viral DNA ends, an activity which heretofore has been difficult to detect by using purified integrase in conventional assays. Certain mutant integrases that were otherwise inactive with the crossbones substrate could complement one another, indicating that no single protomer in the integrase multimer requires a complete set of functional domains either for catalytic activity or for juxtaposition of the two viral DNA ends by the active multimer.     

Biochemical Characterization of Novel Retroviral Integrase Proteins

Integrase is an essential retroviral enzyme, catalyzing the stable integration of reverse transcribed DNA into cellular DNA. Several aspects of the integration mechanism, including the length of host DNA sequence duplication flanking the integrated provirus, which can be from 4 to 6 bp, and the nucleotide preferences at the site of integration, are thought to cluster among the different retroviral genera. To date only the spumavirus prototype foamy virus integrase has provided diffractable crystals of integrase-DNA complexes, revealing unprecedented details on the molecular mechanisms of DNA integration. Here, we characterize five previously unstudied integrase proteins, including those derived from the alpharetrovirus lymphoproliferative disease virus (LPDV), betaretroviruses Jaagsiekte sheep retrovirus (JSRV), and mouse mammary tumor virus (MMTV), epsilonretrovirus walleye dermal sarcoma virus (WDSV), and gammaretrovirus reticuloendotheliosis virus strain A (Rev-A) to identify potential novel structural biology candidates. Integrase expressed in bacterial cells was analyzed for solubility, stability during purification, and, once purified, 3′ processing and DNA strand transfer activities in vitro. We show that while we were unable to extract or purify accountable amounts of WDSV, JRSV, or LPDV integrase, purified MMTV and Rev-A integrase each preferentially support the concerted integration of two viral DNA ends into target DNA. The sequencing of concerted Rev-A integration products indicates high fidelity cleavage of target DNA strands separated by 5 bp during integration, which contrasts with the 4 bp duplication generated by a separate gammaretrovirus, the Moloney murine leukemia virus (MLV). By comparing Rev-A in vitro integration sites to those generated by MLV in cells, we concordantly conclude that the spacing of target DNA cleavage is more evolutionarily flexible than are the target DNA base contacts made by integrase during integration. Given their desirable concerted DNA integration profiles, Rev-A and MMTV integrase proteins have been earmarked for structural biology studies.