Journal of Molecular Biology
Defining the DNA Substrate Binding Sites on HIV-1 Integrase
Introduction
The integrase (IN) of human immunodeficiency virus type 1 (HIV-1) is an attractive target for therapeutic development, as it is essential for early steps in viral replication and there are no homologues in the eukaryotic system for which inhibitors would negatively affect host viability. This enzyme is both necessary and sufficient to catalyze the insertion of viruses into host DNA.1, 2, 3 In the first step or 3′ processing reaction, two deoxyribonucleotides are removed from the 3′ end of long terminal repeat (LTR) strands containing the highly conserved CA dinucleotides. In the second step or strand transfer reaction, the newly created 3′ ends undergo a staggered nucleophilic attack on two strands of the target DNA. These structures are resolved and repaired by host cell enzymes, resulting in an integrated copy of the viral DNA, with the gene-encoding sequence colinear to the viral RNA and flanked by a 4- to 6-bp duplication of the target DNA, depending upon the viral IN.
Design of inhibitors has been hampered by the lack of crystal structures available for full-length IN monomers or higher-order IN oligomers, let alone in complex with DNA. Partial structures with two of the three domains have been reported, and these were used to assemble a model of a tetramer HIV-1 IN with bound LTR DNAs.4 The model was then used to predict residues that were in close proximity to the viral DNA. To verify these predictions, a structural alignment of the primary sequences of HIV-1, simian immunodeficiency virus (SIV), and avian sarcoma virus (ASV) INs5 was used to identify residues that were unique to each virus enzyme, since viral IN specifically recognizes its cognate LTR end substrates. The unique amino acids from ASV IN were substituted into the equivalent structural position of HIV-1 IN. Substitution of the ASV IN residues conferred on the HIV-1 IN mutants the partial ability to cleave an ASV substrate. Multiple residues of HIV-1 IN were demonstrated to alter specificity (V72, S153, K160, I161, G163, Q164, V165, H171, and L172,4 shown in red in Fig. 1) for only one of the two LTR ends. In this report, we have identified six additional HIV-1 IN residues that influence the selection of the LTR end substrates for 3′ processing. These include Q44, L68, E69, D229, S230, and D253, which align, along the two LTR binding grooves, with previous residues that alter recognition.
In addition, the structural model was modified to include the host protein lens-epithelium-derived growth factor (LEDGF)/p75 IN binding domains and to predict a trench on the HIV-1 IN surface that may accommodate the target DNA. The putative binding site for target DNA is positioned roughly perpendicular to the LTR binding sites. Consistent with this interpretation, we have identified in the literature a series of amino acids along the length of one side of this trench, where point amino acid substitutions result in enzymes that lose the ability to strand transfer with little or no effect on 3′ processing. In addition, we have identified a residue on the opposite wall of the trench, K219, where serine substitution displays the same phenotype. This residue was found in a peptide that was previously demonstrated to cross-link to the target DNA.7
Section snippets
Prediction of additional HIV-1 IN residues interacting with LTR DNA ends
In the original selection of amino acids that could affect the recognition of LTR ends, we used the structural alignment of SIV, HIV-1, and ASV INs to identify those residues that were unique. We subsequently observed that HIV-1 IN was capable of 3′ processing a U5 SIV, but not a Mason–Pfizer monkey virus, LTR DNA substrate (data not shown). Therefore, we examined the structural alignment of HIV-1 and ASV INs with the Mason–Pfizer monkey virus IN sequence.5 This analysis identified additional
Discussion
The homotetramer form of IN catalyzes all of its known enzymatic activities. While dimers of IN are capable of catalyzing 3′ processing and strand transfer reactions, they do not support a concerted DNA integration reaction.27 For this reason, a homotetramer model was assembled. In the model, two of the four subunits are depicted with major contacts with the LTR and target DNAs. The remaining two subunits are available for contacts with proteins that interact with IN,28, 29 including the host
Reagents
[γ-33P]ATP (2500 Ci/mmol) was purchased from Perkin Elmer Life Sciences. HiTrap™ Chelating HP resin and HiTrap™ Heparin HP resin were purchased from GE Healthcare Life Sciences (Piscataway, NJ). T4 polynucleotide kinase was obtained from USB (Cleveland, OH). IPTG was obtained from Roche (Indianapolis, IN). The Slide-A-Lyzer Dialysis cassette (molecular weight cutoff, 10,000) was obtained from Pierce (Rockford, IL). CentriPrep centrifugal filter devices with YM-10 MW membranes were obtained from
Acknowledgements
We thank Yuan-Fang Wang for assistance with modeling and preparation of structural figures. This work was supported, in part, by United States Public Health Service grants AI054143 (to J.L.), GM6290, and GM065762 (to I.T.W. and R.W.H.). J.D. was supported, in part, by the Training Program in Viral Replication T32 AI060523.
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2010, Journal of Biological ChemistryCitation Excerpt :There are currently no crystal structures available containing full-length HIV IN. Thus, the intermolecular contacts observed in two-domain crystal structures have been used to construct plausible models of higher oligomeric forms of the IN protein (18, 25, 26, 34, 35, 37–39, 62). Indeed, the models we consider here are based on similar logic.