Formation of a Quaternary Complex of HIV-1 Reverse Transcriptase with a Nucleotide-competing Inhibitor and Its

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 mecha- nism. Binding studies and site-specific footprinting experi- ments 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 usu- ally accommodates the incoming nucleotide substrate. Struc- ture-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 hypersus- ceptible phenotype with respect to INDOPY-1. The same muta- tional 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 pyrophos- phate 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