The Core Domain of HIV-1 Integrase Recognizes Key Features of Its DNA Substrates

Abstract

We investigated which features of the substrate specificity of human immunodeficiency virus type 1 (HIV-1) integrase could be assigned to the central domain of the 288-residue HIV-1 integrase protein, composed of amino acids 50 -212. This domain contains the active site and shares structural homology with a large family of polynucleotidyl transferases. Using model substrates with defined alterations in critical features we found that this domain alone is sufficient for recognition of: 1) the phylogenetically conserved CA/TG base pairs near the viral DNA end; 2) the 5-terminal dinucleotide that is left unpaired after end processing; and 3) target DNA flanking the site of joining. Future efforts aimed at identifying specific amino acids involved in recognition of these key substrate features can now be targeted at this domain.Integration of a double-stranded DNA copy of the retroviral genome into a host cell chromosome is essential for viral replication. Genetic and biochemical studies have shown that retroviral integration requires two viral components, integrase and the U3 and U5 att sites, which are phylogenetically conserved sequences at the ends of viral DNA. Analysis in vivo and in vitro has revealed that integrase processively catalyzes two chemical steps. In 3Ј-end processing, integrase cleaves two nucleotides from the 3Ј-ends of the double-stranded viral DNA, leaving a phylogenetically invariant CA dinucleotide sequence at the 3Ј-termini of the recessed viral DNA ends. In the second chemical step, integrase catalyzes the joining of the two 3Ј-recessed viral DNA ends to sites on opposite strands of a target DNA, separated by 5 base pairs (bps) 1 in the case of HIV-1. The gaps that flank this product of the joining reaction are repaired by a pathway that remains uncharacterized. The chemical reactions catalyzed by integrase can be studied in vitro using model oligonucleotide substrates and purified recombinant protein.We evaluated the specificity of the isolated core domain of HIV-1 integrase. A minimal catalytically active peptide has been identified by deletion analysis, comprised of amino acids 50 -186 (1). To ensure reasonable dynamic range in our assays, we used a slightly larger, more active polypeptide, consisting of amino acids 50 -212 (1). The three-dimensional structure of this domain has been solved, and it reveals striking structural homology to a larger family of polynucleotidyl transferases (2). This domain contains the phylogenetically conserved DD35E motif that defines the active site (3-8). Using model substrates with defined alterations in features known to be critical for the catalytic specificity of full-length integrase, we assessed the ability of the isolated core domain to distinguish between altered and wild-type substrates.Integrase can carry out a concerted cleavage-ligation reaction, termed disintegration, on a model substrate that mimics the product of integration of a single viral DNA end, yielding a free cleaved viral DNA end and a ligated target DNA strand (9). A divalent meta...

Role of the Nonspecific DNA-binding Region and α Helices within the Core Domain of Retroviral Integrase in Selecting Target DNA Sites for Integration

“…The reaction condition was identical to that described earlier in tion event (44,52). This DNA-binding region recognizes key features of viral DNA substrates and may be responsible for viral DNA specificity (19,41,52). In addition to viral DNA, UV cross-linking studies with HIV-1 integrase and a dumbbell disintegration substrate have identified residues 49 -69 and 139 -152 to be in close proximity to target DNA.…”

“…C, PCR-based assay. The reaction condition was identical to that described earlier in tion event (44,52). This DNA-binding region recognizes key features of viral DNA substrates and may be responsible for viral DNA specificity (19,41,52).…”

“…Whether this particular DNA-binding region is involved in dictating integration site preference is not known. The core domain is also able to bind target DNA as observed in in vitro assays with the dumbbell disintegration substrate that mimics a single-ended integration event (52). In addition, specific peptides within the core have been identified to be in close proximity to target DNA in UV cross-linking studies (53).…”

HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase

“…Disintegration substrates, such as the dumbbell used in Figs. 2 and 3, mimic integration intermediates and thus should occupy both the donor and target DNA binding sites in the active complex (27)(28)(29). L-731,988 was not bound in the presence of disintegration substrates, suggesting that the target DNA may occupy a site required for DKA binding.…”

“…High-affinity binding (i) requires assembly on specific viral DNA donor substrates, with the U5 end preferred over U3, (ii) is competed with by DNAs that are competent to serve as target substrates for strand transfer or occupy the target site in the context of the disintegration reaction (7,(27)(28)(29), and (iii) requires an intact integrase catalytic center in the context of an active multimer. The ability to function as a donor substrate in strand transfer and 3Ј processing correlates with the ability to promote inhibitor binding because the U5 end is more efficient than U3 in both contexts (20).…”