No abstract

The human immunodeficiency virus type 1 (HIV-1) life cycle involves the reverse transcription of the viral RNA genome into cDNA and subsequent integration of the viral DNA by integrase (IN) into the host chromosomes. Although the amino acid sequences of INs differ significantly between viruses, INs share three conserved structural domains and their associated functions (13). The N-terminal domain (ϳ50 residues) contains a zinc-binding region (7), promotes multimerization (46), and is necessary for 3Ј-OH processing and strand transfer. The catalytic core domain (CCD) (ϳ162 residues) contains the highly conserved acidic D, D-35-E motif (27) that is involved in coordinating Mg 2ϩ for 3Ј-OH processing and strand transfer activities (4, 13). The catalytic core is also involved in target binding for strand transfer (3,20,26,40). The C-terminal domain (ϳ35 residues) binds to the viral DNA ϳ6 to 9 bp from the long terminal repeat (LTR) end (15); the C-terminus and CCD are also involved in IN multimerization (1, 24, 29a).IN, along with the reverse transcriptase and protease, is an antiretroviral target (2,35,38). Highly active antiretroviral therapy, consisting of various combinations of reverse transcriptase and protease inhibitors, has significantly decreased HIV-1 replication in humans. The emergence of multidrugresistant HIV-1 mutants and undesirable side effects associated with certain drug combinations necessitates continuing efforts to develop novel and effective combinational therapies. The addition of inhibitors of HIV-1 IN function would enhance highly active antiretroviral therapy. Raltegravir (MK-0518), an analog of the strand transfer inhibitor L-870,810 used in this report, is in phase III human clinical trials (18,33).Oligonucleotide-based assays in vitro have identified a large number of compounds that inhibit HIV-1 IN activities in vitro (25), the majority of which are ineffective at preventing HIV-1 replication in cell culture. The "strand transfer inhibitors" were identified as being effective against recombinant IN, suppressed HIV-1 replication in cell culture and in vivo, and were so named because of their selectivity in both cases towards inhibiting strand transfer over 16,21,22,38). The first generation of strand transfer inhibitors possessed a 1,3-diketo acid (DKA) pharmacophore, which served as a template in the development of the naphthyridine carboxamide inhibitors. They are structurally analogous to and function identically to DKA inhibitors but exhibit improved metabolic and pharmacokinetic properties and are represented here by compounds L-870,810 and L-870,812 (21, 23). DKA-mediated inhibition is accomplished by the contact of the DKA moiety with the divalent metal ion in the CCD of IN (19,38), and efficient inhibitor binding occurs only with IN bound to the viral DNA substrate (14,22,38

We report the efficient concerted integration of a linear virus-like DNA donor into a 2.8 kbp circular DNA target by integrase (IN) purified from avian myeloblastosis virus. The donor was 528 bp, contained recessed 3' OH ends, was 5' end labeled, and had a unique restriction site not found in the target. Analysis of concerted (full-site) and half-site integration events was accomplished by restriction enzyme analysis and agarose gel electrophoresis. The donor also contained the SupF gene that was used for genetic selection of individual full-site recombinants to determine the host duplication size. Two different pathways, involving either one donor or two donor molecules, were used to produce full-site recombinants. About 90% of the full-site recombinants were the result of using two donor molecules per target. These results imply that juxtapositioning an end from each of two donors by IN was more efficient than the juxtapositioning of two ends of a single donor for the full-site reaction. The formation of preintegration complexes containing integrase and donor on ice prior to the addition of target enhanced the full-site reaction. After a 30 min reaction at 37 degrees C, approximately 20-25% of all donor/target recombinants were the result of concerted integration events. The efficient production of full-site recombinants required Mg2+; Mn2+ was only efficient for the production of half-site recombinants. We suggest that these preintegration complexes can be used to investigate the relationships between the 3' OH trimming and strand transfer reactions.