Effect of DNA Modifications on DNA Processing by HIV-1 Integrase and Inhibitor Binding

Abstract

Integration of the viral cDNA into host chromosomes is required for viral replication. Human immunodeficiency virus integrase catalyzes two sequential reactions, 3-processing (3-P) and strand transfer (ST). The first integrase inhibitors are undergoing clinical trial, but interactions of inhibitors with integrase and DNA are not well understood in the absence of a co-crystal structure. To increase our understanding of integrase interactions with DNA, we examined integrase catalysis with oligonucleotides containing DNA backbone, base, and groove modifications placed at unique positions surrounding the 3-processing site. 3-Processing was blocked with substrates containing constrained sugars and ␣-anomeric residues, suggesting that integrase requires flexibility of the phosphodiester backbone at the 3-P site. Of several benzo[a]pyrene 7,8-diol 9,10-epoxide (BaP DE) adducts tested, only the adduct in the minor groove at the 3-P site inhibited 3-P, suggesting the importance of the minor groove contacts for 3-P. ST occurred in the presence of bulky BaP DE DNA adducts attached to the end of the viral DNA suggesting opening of the active site for ST. Positionspecific effects of these BaP DE DNA adducts were found for inhibition of integrase by diketo acids. Together, these results demonstrate the importance of DNA structure and specific contacts with the viral DNA processing site for inhibition by integrase inhibitors. HIV-12 integrase (integrase) catalyzes insertion of cDNA copies of the viral genome into human chromosomes. Integrase binds to the ends ("att" sites) of each viral long terminal repeat (LTR) through sequence-specific recognition of a conserved 5Ј-CA within the sequence 5Ј-GCAGT. In the first of two reactions, integrase cleaves the 3Ј-ends of the viral DNA, releasing the terminal 5Ј-GT dinucleotide (3Ј-processing, 3Ј-P). In the second reaction, the free 3Ј-hydroxyl of the conserved adenine provides the nucleophile for insertion of the viral cDNA into a chromosome (strand transfer, ST). Gap repair and ligation between the viral and cellular DNA are performed by cellular factors. (For recent reviews and insights on integration, see Refs. 1-5.)Determination of the molecular interactions between integrase and its DNA substrates (viral and chromosomal DNA) has proven challenging, and a co-crystal of these components remains elusive. Biochemical studies have revealed contact points between the viral DNA and integrase. Integrase has an absolute requirement for the conserved 5Ј-CA adjoining the 3Ј-P site (underlined in Fig. 1A). The efficiency of 3Ј-P is also dramatically decreased by changes to the G immediately 5Ј to the conserved CA dinucleotide (6 -8). The conserved adenine, substituted by 5-iododeoxyuracil as a photocross-linker, forms a photocross-link to Lys-159 of integrase (9). Residue Lys-159 also contacts the phosphate 5Ј to the conserved deoxyadenosine (10). Mutagenesis showed that Tyr-143 and probably Gln-148 interact with the 5Ј-overhang resulting from 3Ј-P (9, 11). Moreover, disulfide cross-linking ...

Selectivity for strand-transfer over 3′-processing and susceptibility to clinical resistance of HIV-1 integrase inhibitors are driven by key enzyme–DNA interactions in the active site

“…In the case of the WT IN, removing either of these two bases flanking the 3′-P site (positions +1 and −1) markedly reduced 3′-P while enabling RAL and DTG to inhibit 3′-P almost as efficiently as ST. An important difference between the SH and the WT enzymes, with respect to the viral DNA interactions (Figures 1 and 2) is our finding that the +1 guanine base (immediately 3′ from the conserved CA; see Figure 1A) is critical for optimum 3′-P activity in the case of the WT IN (34,45,46). By contrast removing this +1 guanine enables the SH mutant to regain 3′-P activity.…”

“…First, we compared the 3′-P activity of WT IN on a DNA substrate with the canonical CAGT sequence to the 3′-P activity on a substrate containing an abasic site at the −1 position relative to the cleavage site on the scissile strand (C_GT or 21Ab-1, Figure 1A). The conserved adenosine at the -1 position is known to be important for the activities of IN (34–37). Accordingly, 3′-P of this 21Ab-1 DNA was markedly reduced (Figure 1B).…”

“…Biochemical studies using recombinant IN and various DNA oligonucleotide substrates have established that IN recognizes the conserved CA dinucleotide (−2 and −1 positions, see Figure 1A) located near the 3′-end of the viral DNA for 3′-P (34,35,37,43–46). Here, we performed biochemical experiments and molecular modeling with the WT and the double-mutant (G140S-Q148H [SH]) that causes significant resistance to RAL and EVG and to a lesser extent to DTG (2,11,12,22,26).…”

HIV-1 Integrase-DNA Recognition Mechanisms

“…Probing effects of various DNA backbone, base, and groove modifications on IN catalytic activities suggested that IN requires flexibility of the phosphodiester backbone at the scissile bond [75]. The other study examined 2′-modified nucleosides and 1,3-propanediol insertions in various positions of the U5 sequence [76].…”

Probing HIV-1 Integrase Inhibitor Binding Sites with Position-Specific Integrase-DNA Cross-Linking Assays

“…All drug solutions were prepared from powder in 100% dimethyl sulfoxide. Drug concentrations producing 50% inhibition (IC 50 values, Table 1) are from previous reports Di Santo et al, 2006;Johnson et al, 2006b;Stagliano et al, 2006) or were determined as described below. Note that all the compounds shown display anti-HIV activity, except for bis-DKA and 5CITEP (summarized with references in Table 1).…”