Hypersusceptibility to Substrate Analogs Conferred by Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Abstract

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contains four structural motifs (A, B, C, and D) that are conserved in polymerases from diverse organisms. Motif B interacts with the incoming nucleotide, the template strand, and key active-site residues from other motifs, suggesting that motif B is an important determinant of substrate specificity. To examine the functional role of this region, we performed "random scanning mutagenesis" of 11 motif B residues and screened replication-competent mutants for altered substrate analog sensitivity in culture. Single amino acid replacements throughout the targeted region conferred resistance to lamivudine and/or hypersusceptibility to zidovudine (AZT). Substitutions at residue Q151 increased the sensitivity of HIV-1 to multiple nucleoside analogs, and a subset of these Q151 variants was also hypersusceptible to the pyrophosphate analog phosphonoformic acid (PFA). Other AZT-hypersusceptible mutants were resistant to PFA and are therefore phenotypically similar to PFA-resistant variants selected in vitro and in infected patients. Collectively, these data show that specific amino acid replacements in motif B confer broad-spectrum hypersusceptibility to substrate analog inhibitors. Our results suggest that motif B influences RT-deoxynucleoside triphosphate interactions at multiple steps in the catalytic cycle of polymerization.Conversion of viral RNA to double-stranded DNA by reverse transcriptase (RT) is a defining step in the retroviral life cycle (8) and a key target of therapy for human immunodeficiency virus type 1 (HIV-1) infection (17). The 66-kilodalton subunit of HIV-1 RT contains four motifs (A, B, C, and D) that are similarly arranged in all known structures of replicative DNA and RNA polymerases (21). Three additional structural elements, motifs E and F and premotif A, are also conserved among RTs and viral RNA-dependent RNA polymerases (4,21,45,76). Together, motifs A, B, C, and F and premotif A form a closely packed protein framework that positions the templating nucleotide, the primer terminus, and incoming deoxynucleoside triphosphate (dNTP) at the RT active site (Fig. 1A). Amino acid substitutions within this conserved core can affect dNTP insertion fidelity, susceptibility to nucleoside analogs, and/or discrimination against ribonucleoside triphosphates (rNTPs) during DNA synthesis (40,49,64,72). Thus, these motifs influence the stringency and specificity of substrate incorporation by RT.Motif B is of particular interest because of its central position in the RT core structure (Fig. 1) (12, 24). Motif B contacts the template strand, the incoming dNTP, and each of the other motifs in the core structure (premotif A and motifs A, C, and F) (Fig. 1A), including residues within these other motifs that are known to affect dNTP substrate recognition (Fig. 1B) (12, 24). The importance of motif B in substrate selection is evident from studies of HIV-1 mutants resistant to nucleoside analogs (49, 72). Two amino acid substitutions in motif B a...

Extensive Mutagenesis of the Conserved Box E Motif in Duck Hepatitis B Virus P Protein Reveals Multiple Functions in Replication and a Common Structure with the Primer Grip in HIV-1 Reverse Transcriptase

“…In HIV-1 RT, the primer grip does not directly contact the incoming dNTP, but due to its scaffolding function, mutations can indirectly affect susceptibility to nucleoside analogs. For instance, the box E mutation F227A rendered the enzyme 8-fold hypersensitive to azidothymidine (AZT), and various HIV-1 RT mutants selected for resistance to PFA increased sensitivity to AZT (23,44). Both DHBV P protein mutations induced an about 3-fold, statistically significant resistance to PFA (mean IC 50 s of 189 M and 143 M, versus 55 M for wt DHBV), whereas sensitivity to lamivudine was about 2-fold enhanced (IC 50 s of 3.4 and 2.5 M, versus 6.8 M for wt DHBV) (see Fig.…”

Human Immunodeficiency Virus Types 1 and 2 Exhibit Comparable Sensitivities to Zidovudine and Other Nucleoside Analog Inhibitors In Vitro

“…The infectivity of wild-type HIV-2 ROD was 15 Ϯ 9 FFU/ng HIV-2 capsid p26. b EC 50 s were obtained with MAGIC-5A cells as previously described (22). The values shown in bold are significantly different from values for wild-type HIV-1 NL4-3 (P Ͻ 0.05; analysis of variance of log͓EC 50 ͔ values by use of Tukey's multiple-comparison test).…”

“…To obtain virus stocks from the infectious molecular clones, purified preparations of each wild-type or mutant plasmid were transfected into 293T-17 cells (293tsA1609 neo ) (17) using our previously published protocol (22). Culture supernatants were harvested 42 h after transfection, centrifuged at 1,500 ϫ g to remove residual cells, and stored in aliquots at Ϫ150°C.…”

“…(8). The MAGIC-5A cultures were infected at a multiplicity of infection of 300 focus-forming units (FFU) per 5 ϫ 10 4 cells and were fixed and stained 40 h after inoculation as previously described to ensure that the majority of the Lac ϩ foci arose from a single cycle of viral replication (22).…”

Human telomerase model shows the role of the TEN domain in advancing the double helix for the next polymerization step

“…Three conserved uncharged residues-Y717, Q833, and V867 (from motifs A, B′, and C, respectively)-form a hydrophobic pocket adjacent to the catalytic aspartates and take part in nucleotide binding (29). Residue V867 has been shown to alter human telomerase substrate specificity (37), and residue Q833 corresponds to Q151 in HIV-1 reverse transcriptase, where mutations cause hypersensitivity to substrate analogs (38). This pocket appears to hold the incoming deoxynucleotide in close proximity to the active site, for coordination with one of the Mg 2þ ions.…”