Impact of Primer-induced Conformational Dynamics of HIV-1 Reverse Transcriptase on Polymerase Translocation and Inhibition

Abstract

The rapid emergence and the prevalence of resistance mutations in HIV-1 reverse transcriptase (RT) underscore the need to identify RT inhibitors with novel binding modes and mechanisms of inhibition. Recently, two structurally distinct inhibitors, phosphonoformic acid (foscarnet) and INDOPY-1 were shown to disrupt the translocational equilibrium of RT during polymerization through trapping of the enzyme in the pre-and the post-translocation states, respectively. Here, we show that foscarnet and INDOPY-1 additionally display a shared novel inhibitory preference with respect to substrate primer identity. In RT-catalyzed reactions using RNA-primed substrates, translocation inhibitors were markedly less potent at blocking DNA polymerization than in equivalent DNA-primed assays; i.e. the inverse pattern observed with marketed non-nucleoside inhibitors that bind the allosteric pocket of RT. This potency profile was shown to correspond with reduced binding on RNA⅐DNA primer/template substrates versus DNA⅐DNA substrates. Furthermore, using site-specific footprinting with chimeric RNA⅐DNA primers, we demonstrate that the negative impact of the RNA primer on translocation inhibitor potency is overcome after 18 deoxyribonucleotide incorporations, where RT transitions primarily into polymerization-competent binding mode. In addition to providing a simple means to identify similarly acting translocation inhibitors, these findings suggest a broader role for the primer-influenced binding mode on RT translocation equilibrium and inhibitor sensitivity.HIV RT is a multifunctional enzyme that catalyzes the conversion of the single-stranded RNA HIV genome to doublestranded DNA that is then incorporated into the host genome by the HIV integrase. In the process, RT must support RNAdirected and DNA-directed DNA synthesis along with ribonuclease H (RNase H) 2 activities to create an integration-competent product. A key step during reverse transcription is the incomplete hydrolysis of the (ϩ)-stranded RNA that leaves behind a purine-rich RNA sequence referred to as the polypurine tract (PPT). The remnant RNA PPT sequence serves as a primer to initiate synthesis of the (ϩ)-strand DNA (second strand) (1-3). The unique structure of the RNA PPT sequence has been shown to be a key determinant of the RNase H cleavage specificity at the PPT/U3 junction (4). A recent report described the mechanism by which RT discriminates between polymerase and RNase H activities thereby enabling more efficient initiation of polymerization from the RNA PPT primer versus other remnant RNA primers (5). Using single-molecule spectroscopy experiments, it was shown that RT binds nucleic acid substrates in two distinct orientations in a manner that is governed by the sugar backbone composition of the four or five nucleotides at each end of the primer. Depending on the binding orientation, RT either initiates polymerization at the 3Ј-end of the primer (polymerase binding mode on a DNA primer), or alternatively, RNA hydrolysis through the RNase H domain (RNase H bindi...

Human Immunodeficiency Virus Reverse Transcriptase

“…Thus, assays can be developed to screen for novel translocation inhibitors. In this context it is important to note that both PFA and INDOPY-1 were shown to be ineffective in affecting the translocational equilibrium on RNA-primed reactions (Auger et al 2011 ). RT is able to fl ip between different conformations such as polymerase competent and RNase competent.…”

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

“…Like the natural dNTP substrate, INDOPY-1 can form a stable complex with HIV-1 RT and its primer⅐template substrate (6,8,45). These complexes are resistant to challenges with an enzyme trap, such as heparin.…”

HIV-1 Reverse Transcriptase Still Remains a New Drug Target: Structure, Function, Classical Inhibitors, and New Inhibitors with Innovative Mechanisms of Actions

“…In addition, the INDOPY-1 analog 1-(4-nitrophenyl)-2-oxo-2,5-dihydro-1H-pyrido[3,2-b]indole-3-carbonitrile (VRX329747) ( Figure 13 ) selected HIV-1 RT mutated at the amino acid residues M41L, A62V, S68N, G112S, V118I, and M184V, which are all located around the incoming nucleotide-binding site [ 112 ]. Further, binding and biochemical studies revealed that (i) the M184V mutation reduces the affinity to INDOPY-1, while the Y115F mutation facilitates the dNTP binding, and their combined effects enhance the ability of the enzyme to discriminate against the inhibitor [ 113 ]; (ii) RT complexed with INDOPY-1 is trapped in the posttranslocational state [ 113 ]; (iii) the INDOPY-1 has preference with respect to substrate primer identity since its binding to RT is higher on a DNA:DNA versus a RNA:DNA primer:template [ 114 ]; (iv) when assayed by steady-state kinetic analysis with homopolymeric template primers, INDOPY-1 inhibits RT-catalyzed DNA polymerization with a competitive [ 111 ] or mixed-type [ 112 ] mode with respect to dNTPs. Overall, these observations suggest that the binding site of the indolopyridones and nucleotide substrates can at least partially overlap and they are therefore proposed as Nucleotide competing RT inhibitors (NcRTIs).…”