Changes to the HIV Long Terminal Repeat and to HIV Integrase Differentially Impact HIV Integrase Assembly, Activity, and the Binding of Strand Transfer Inhibitors

Abstract

Human immunodeficiency virus (HIV) integrase enzyme is required for the integration of viral DNA into the host cell chromosome. Integrase complex assembly and subsequent strand transfer catalysis are mediated by specific interactions between integrase and bases at the end of the viral long terminal repeat (LTR). The strand transfer reaction can be blocked by the action of small molecule inhibitors, thought to bind in the vicinity of the viral LTR termini. This study examines the contributions of the terminal four bases of the nonprocessed strand (G 2 T 1 C ؊1 A ؊2 ) of the HIV LTR on complex assembly, specific strand transfer activity, and inhibitor binding. Base substitutions and abasic replacements at the LTR terminus provided a means to probe the importance of each nucleotide on the different functions. An approach is described wherein the specific strand transfer activity for each integrase/LTR variant is derived by normalizing strand transfer activity to the concentration of active sites. The key findings of this study are as follows. 1) The G 2 :C 2 base pair is necessary for efficient assembly of the complex and for maintenance of an active site architecture, which has high affinity for strand transfer inhibitors. 2) Inhibitor-resistant enzymes exhibit greatly increased sensitivity to LTR changes. 3) The strand transfer and inhibitor binding defects of a Q148R mutant are due to a decreased affinity of the complex for magnesium. 4) Gln 148 interacts with G 2 , T 1 , and C ؊1 at the 5 end of the viral LTR, with these four determinants playing important and overlapping roles in assembly, strand transfer catalysis and high affinity inhibitor binding.Integration, the insertion of a double-stranded DNA copy of the viral RNA genome into the host genome, is absolutely required for HIV 2 replication (1). As such, integration presents an attractive target for the development of small molecule inhibitors that could be used to treat HIV. Several integrase (IN) inhibitors that are progressing through clinical development (2, 3) have been shown to lower viral load in infected patients.The virus-encoded IN protein catalyzes two essential activities in the viral life cycle, 3Ј-processing and strand transfer. The 3Ј-processing reaction cleaves off the final two bases (5Ј-GT dinucleotide) from the 3Ј ends of the viral LTR. The strand transfer activity catalyzes the concerted insertion of the two viral 3Ј ends into the host genome with a 5-bp separation. To accomplish this, IN simultaneously positions the two 3Ј-hydroxyls of the LTRs for nucleophilic attack onto the phosphodiester bonds of the genomic DNA (4). In vitro, both reactions are catalyzed by divalent magnesium or manganese, although magnesium is thought to be the actual metal cofactor in cells (5).Suitable in vitro systems for studying these processes have been described, in which the full-length protein is minimally required to direct both 3Ј-processing and strand transfer (6, 7). In these systems, IN is typically allowed to form an in situ complex with a model D...

The Terminal (Catalytic) Adenosine of the HIV LTR Controls the Kinetics of Binding and Dissociation of HIV Integrase Strand Transfer Inhibitors

“…The apparent equilibrium dissociation constant, K d (apparent), was 21 nM, as previously reported (16 up to 643 nM). After equilibrium was reached, the kinetics of dissociation were monitored after adding a large molar excess of unlabeled 1.…”

“…PVT scintillation proximity assay (SPA) beads were from Amersham Biosciences. The d-spacer and aminadenosine monomers were from Glen Research, Sterling, VA. Recombinant IN enzymes were purified from an NL4-3 clone, as previously described (16 (16). LTR duplexes were prepared such that the 5′ end of the plus strand was synthesized with a biotin linker attached.…”

Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs

“…Viral DNA 3Ј end processing by IN is proposed to create a hydrophobic drug binding pocket in the space vacated by the removal of 3Ј-GT, bounded by the flexible active site loop (30) and the 5Ј-CA overhang of the viral DNA (32). Inhibitor binding is attenuated when either the flexibility of the active site loop is impaired (33) or the 5Ј-CA overhang of the viral DNA is deleted (32). Although the thiol group on the 5Ј-CA overhang cross-links efficiently to either K160C or Y143C, only cross-linked Y143C binds IN inhibitor.…”

Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors

“…Compared with the wild type (WT) enzyme, all variant enzymes were impaired with respect to assembly and multimerization into active complexes and their subsequent in vitro strand transfer abilities. Reduced susceptibility to one STI, STI 1, (23), was shown to be a result of decreased affinity of the inhibitor for the integrase enzymes containing the selected mutations. In addition, isogenic viruses harboring these resistance mutations showed defects in integration in cells.…”

“…The details of the assay have been reported previously (23). Briefly, viral LTR DNA is attached, via a biotin linker, to streptavidin-coated SPA PVT beads, after which the enzyme is added to form active complexes.…”