Characterizing Critical Residues for the Interaction of Human Immunodeficiency Virus Type 1 Integrase with DNA SubstratesPublic Deposited
A tetramer model for HIV-1 Integrase (IN) with DNA representing the LTR termini was previously assembled which predicted amino acid residues on the enzyme surface that interact with the LTR termini (Chen et al, 2006). A separate structural alignment of HIV-1, SIV, and ASV INs predicted which of these residues were unique. By substituting the unique amino acids found in ASV IN into the structurally related positions of HIV-1, nine amino acid residues were shown to partially change the specificity for 3' processing from HIV-1 to ASV duplex oligo substrates. Using a similar strategy, but with a structural alignment that substituted MPMV for the SIV sequence, six additional residues (Q44, L68, E69, D229, S230 and D253) were identified that interact with the LTR DNA and change the 3' processing specificity of the enzyme. All fifteen residues align along a sixteen base pair length of the LTR termini asymmetrically positioned relative to each strand of the DNA. The tetramer model for HIV-1 IN with LTR termini was modified to include two IN binding domains for LEDGF/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 LEDGF. Consistent with this hypothesis is the finding that a HIV-1 IN mutant with a K219S substitution displays an activity phenotype where there is a loss in strand transfer with little change in 3' processing activity towards HIV-1 substrates. Mutations at seven other residues reported in the literature have the same activity phenotype and align along the opposite face of the putative target DNA binding trench. Integrase is now an attractive target for antiretroviral therapy. A small-molecule inhibitor of IN was recently approved by the FDA for human therapy, providing a proof-of-principle for development of additional compounds. When infected cells are put under selection of these inhibitors, mutations arise in IN that result in resistance to the drugs. Examining the in vitro enzymatic activities of INs with changes at these residues provides insight into both the mechanism of action of the compound and the function of secondary and tertiary mutations. In this work, I show that mutation at a second position that arises during drug selection complements the enzymatic defect of the primary drug-resistance mutation in the 3' end processing reaction.