Structures of type-1 human immunodeficiency virus (HIV-1) reverse transcriptase (RT) have been determined in several forms, but only one contains an RNA/DNA hybrid. Here we report three structures of HIV-1 RT complexed with a non-nucleotide RT inhibitor (NNRTI) and an RNA/DNA hybrid. In the presence of an NNRTI, the RNA/DNA structure differs from all prior nucleic acid bound to RT including the RNA/DNA hybrid. The enzyme structure also differs from all previous RT–DNA complexes. As a result, the hybrid has ready access to the RNase H active site. These observations indicate that an RT–nucleic acid complex may adopt two structural states, one competent for DNA polymerization and the other for RNA degradation. RT mutations that confer drug resistance but are distant from the inhibitor-binding sites often map to the unique RT–hybrid interface that undergoes conformational changes between two catalytic states.

Recent crystallographic data suggest that conserved residues in the connection subdomain and C-terminal ribonuclease H (RNase H) domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) contact the nascent DNA primer and modulate the trajectory of the template relative to the RNase H catalytic center. Within the RNase H domain, these residues include Thr473, Glu475, Lys476, Tyr501, and Ile505, while His539 and Asn474 interact with the scissile phosphate of the RNA template. Amino acid substitutions at several of these positions were evaluated in the context of hydrolysis of nonspecific RNA-DNA hybrids and substrates mimicking specific RNase H-mediated events. With the exception of mutant I505G, which exhibited a dimerization defect, substituting alanine at positions 473-476 and 501 had minimal consequences for DNA synthesis on duplex and hybrid DNA and RNA substrates. In contrast, the efficiency with which most mutants catalyzed polymerization-independent RNase H cleavage was sharply reduced. This deficiency was more pronounced when mutant enzymes were challenged to process the (+) strand polypurine tract (PPT) primer from either (+) RNA or a PPT/(+) DNA RNA/DNA chimera. Reduced polymerization-independent RNase H activity also significantly influenced the rate of DNA strand transfer, suggesting the donor template must be reduced in size below 13 nt before this event proceeds.

High-throughput screening of National Cancer Institute libraries of synthetic and natural compounds identified the vinylogous ureas 2-amino-5, 6,7,8-tetrahydro-4H-cyclohepta[b] thiophene-3-carboxamide (NSC727447) and N-[3-(aminocarbonyl)-4,5-dimethyl-2-thienyl]-2-furancarboxamide (NSC727448) as inhibitors of the ribonuclease H (RNase H) activity of HIV-1 and HIV-2 reverse transcriptase (RT).A Yonetani-Theorell analysis demonstrated that NSC727447, and the active-site hydroxytropolone RNase H inhibitor β-thujaplicinol were mutually exclusive in their interaction with the RNase H domain. Mass spectrometric protein footprinting of the NSC727447 binding site indicated that residues Cys280 and Lys281 in helix I of the thumb subdomain of p51 were affected by ligand binding. Although DNA polymerase and pyrophosphorolysis activities of HIV-1 RT were less sensitive to inhibition by NSC727447, protein footprinting indicated that NSC727447 occupied the equivalent region of the p66 thumb. Sitedirected mutagenesis using reconstituted p66/p51 heterodimers substituted with natural or nonnatural amino acids indicates that altering the p66 RNase H primer grip significantly affects inhibitor sensitivity. NSC727447 thus represents a novel class of RNase H antagonists with a mechanism of action differing from active site, diva-lent metal-chelating inhibitors that have been reported.Although an absolute requirement for reverse transcriptase (RT)-associated ribonuclease H (RNase H) activity for human immunodeficiency virus (HIV) replication was documented almost two decades ago (1,2), development of potent and selective RNase H inhibitors has been surprisingly slow compared with the nucleoside and non-nucleoside DNA polymerase inhibitors currently in clinical use. Recently, however,4), diketo acids (5,6), and dihydroxytropolones (7) have shown promise by specifically inhibiting RNase H activity of HIV-1 and HIV-2 RT, and in some instances acting synergistically with clinically approved RT inhibitors. The preliminary crystal structure of an N-hydroxyimide bound to the RNase H domain of HIV-1 RT (4) suggests that it sequesters the divalent metal cofactor, laying the foundation for rational design of improved inhibitors. Increasing the diversity of RNase H © 2008 American Chemical Society * Corresponding author, slegrice@ncifcrf.gov.. Supporting Information Available:This material is free of charge via the Internet. In order to examine the NSC727447 binding site, we performed mass spectrometric protein footprinting based on biotin modification of exposed lysine residues in the free protein and the protein-inhibitor complex (8-10). Cys280 and Lys281, located in helix I of the thumb subdomain, were protected from modification by inhibitor binding. Proximity between the p51 thumb subdomain and the p66 RNase H domain implies that inhibitor binding adjacent to the catalytic center affects either divalent metal coordination or positioning of the nucleic acid substrate in the active site. Although DNA polymerase activity was less sensi...