title: HIV-1 integrase strand transfer inhibitors stabilize an integrase-single blunt-ended DNA complex.
abstract: Integration of human immunodeficiency virus cDNA ends by integrase (IN) into host chromosomes involves a concerted integration mechanism. IN juxtaposes two DNA blunt ends to form the synaptic complex, which is the intermediate in the concerted integration pathway. The synaptic complex is inactivated by strand transfer inhibitors (STI) with IC(50) values of ∼20 nM for inhibition of concerted integration. We detected a new nucleoprotein complex on a native agarose gel that was produced in the presence of >200 nM STI, termed the IN-single DNA (ISD) complex. Two IN dimers appear to bind in a parallel fashion at the DNA terminus, producing an ∼32-bp DNase I protective footprint. In the presence of raltegravir (RAL), MK-2048, and L-841,411, IN incorporated ∼20-25% of the input blunt-ended DNA substrate into the stabilized ISD complex. Seven other STI also produced the ISD complex (≤5% of input DNA). The formation of the ISD complex was not dependent on 3'OH processing, and the DNA was predominantly blunt ended in the complex. The RAL-resistant IN mutant N155H weakly forms the ISD complex in the presence of RAL at ∼25% level of wild-type IN. In contrast, MK-2048 and L-841,411 produced ∼3-fold to 5-fold more ISD than RAL with N155H IN, which is susceptible to these two inhibitors. The results suggest that STI are slow-binding inhibitors and that the potency to form and stabilize the ISD complex is not always related to inhibition of concerted integration. Rather, the apparent binding and dissociation properties of each STI influenced the production of the ISD complex.
authors.affiliation: Saint Louis University Health Sciences Center, Institute for Molecular Virology, Doisy Research Center, 1100 South Grand Boulevard, St. Louis, MO 63104, USA.
authors.forename: Sibes
authors.initials: S
authors.lastname: Bera
authors.forename: Krishan K
authors.initials: KK
authors.lastname: Pandey
authors.forename: Ajaykumar C
authors.initials: AC
authors.lastname: Vora
authors.forename: Duane P
authors.initials: DP
authors.lastname: Grandgenett
mesh_terms: D020029:Base Pairing / Q000235:genetics; D055162:Biocatalysis / Q000187:drug effects; D002232:Carbocyanines / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D024882:Drug Resistance, Viral / Q000187:drug effects; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes / Q000378:metabolism; D019427:HIV Integrase / Q000378:metabolism*; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D020128:Inhibitory Concentration 50; D019429:Integrase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D007651:Keto Acids / Q000737:chemistry / Q000494:pharmacology; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D011760:Pyrrolidinones / Q000737:chemistry / Q000494:pharmacology; D000068898:Raltegravir Potassium; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D019429:Integrase Inhibitors; D007651:Keto Acids; C524755:L-841,411; D050505:Mutant Proteins; D011760:Pyrrolidinones; C089187:cyanine dye 3; D000068898:Raltegravir Potassium; D004247:DNA; D019427:HIV Integrase; D003850:Deoxyribonuclease I; C514801:p31 integrase protein, Human immunodeficiency virus 1