title: Formation of a quaternary complex of HIV-1 reverse transcriptase with a nucleotide-competing inhibitor and its ATP enhancer.
abstract: Nucleotide-competing reverse transcriptase inhibitors were shown to bind reversibly to the nucleotide-binding site of the reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1). Here, we show that the presence of ATP can enhance the inhibitory effects of the prototype compound INDOPY-1. We employed a combination of cell-free and cell-based assays to shed light on the underlying molecular mechanism. Binding studies and site-specific footprinting experiments demonstrate the existence of a stable quaternary complex with HIV-1 RT, its nucleic acid substrate, INDOPY-1, and ATP. The complex is frozen in the post-translocational state that usually accommodates the incoming nucleotide substrate. Structure-activity relationship studies show that both the base and the phosphate moieties of ATP are elements that play important roles in enhancing the inhibitory effects of INDOPY-1. In vitro susceptibility measurements with mutant viruses containing amino acid substitutions K70G, V75T, L228R, and K219R in the putative ATP binding pocket revealed unexpectedly a hypersusceptible phenotype with respect to INDOPY-1. The same mutational cluster was previously shown to reduce susceptibility to the pyrophosphate analog phosphonoformic acid. However, in the absence of INDOPY-1, ATP can bind and act as a pyrophosphate donor under conditions that favor formation of the pre-translocated RT complex. We therefore conclude that the mutant enzyme facilitates simultaneous binding of INDOPY-1 and ATP to the post-translocated complex. Based on these data, we propose a model in which the bound ATP traps the inhibitor, which, in turn, compromises its dissociation.
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada.
authors.forename: Maryam
authors.initials: M
authors.lastname: Ehteshami
authors.forename: Monique
authors.initials: M
authors.lastname: Nijhuis
authors.forename: Jean A
authors.initials: JA
authors.lastname: Bernatchez
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Ablenas
authors.forename: Suzanne
authors.initials: S
authors.lastname: McCormick
authors.forename: Dorien
authors.initials: D
authors.lastname: de Jong
authors.forename: Dirk
authors.initials: D
authors.lastname: Jochmans
authors.forename: Matthias
authors.initials: M
authors.lastname: Götte
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D019380:Anti-HIV Agents / Q000737:chemistry*; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry; D004795:Enzyme Stability; D017245:Foscarnet / Q000737:chemistry; D057809:HEK293 Cells; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D007211:Indoles / Q000737:chemistry*; D009570:Nitriles / Q000737:chemistry*; D011485:Protein Binding; D011728:Pyridones / Q000737:chemistry*; D013329:Structure-Activity Relationship
chemical_list: C517167:5-methyl-1-(4-nitrophenyl)-2-oxo-2,5-dihydro-1H-pyrido(3,2-b)indole-3-carbonitrile; D019380:Anti-HIV Agents; D004279:DNA, Viral; D007211:Indoles; D009570:Nitriles; D011728:Pyridones; D017245:Foscarnet; D000255:Adenosine Triphosphate; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase
keywords: DNA Polymerase; Drug Action; Drug Resistance; HIV-1; Reverse Transcription