Unblocking of chain-terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism

Abstract

HIV-1 replication is inhibited by the incorporation of chain-terminating nucleotides at the 3 end of the growing DNA chain. Here we show a nucleotide-dependent reaction catalyzed by HIV-1 reverse transcriptase that can efficiently remove the chain-terminating residue, yielding an extendible primer terminus. Radioactively labeled 3-terminal residue from the primer can be transferred into a product that is resistant to calf intestinal alkaline phosphatase and sensitive to cleavage by snake venom phosphodiesterase. The products formed from different nucleotide substrates have unique electrophoretic migrations and have been identified as dinucleoside tri-or tetraphosphates. The reaction is inhibited by dNTPs that are complementary to the next position on the template (K i Ϸ 5 M), suggesting competition between dinucleoside polyphosphate synthesis and DNA polymerization. Dinucleoside polyphosphate synthesis was inhibited by an HIV-1 specific non-nucleoside inhibitor and was absent in mutant HIV-1 reverse transcriptase deficient in polymerase activity, indicating that this activity requires a functional polymerase active site. We suggest that dinucleoside polyphosphate synthesis occurs by transfer of the 3 nucleotide from the primer to the pyrophosphate moiety in the nucleoside dior triphosphate substrate through a mechanism analogous to pyrophosphorolysis. Unlike pyrophosphorolysis, however, the reaction is nucleotide-dependent, is resistant to pyrophosphatase, and produces dinucleoside polyphosphates. Because it occurs at physiological concentrations of ribonucleoside triphosphates, this reaction may determine the in vivo activity of many nucleoside antiretroviral drugs.

Molecular Mechanism by Which the K70E Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Resistance to Nucleoside Reverse Transcriptase Inhibitors

“…We hypothesize that this putative interaction between K65 and E70 may alter the location of K65, thereby affecting NRTI-TP incorporation. HIV-1 RT has the innate capacity to unblock NRTI-MPterminated primers in the presence of physiological concentrations of ATP or PPi (1,22). Biochemical studies show that TAMs confer resistance to AZT and other NRTI by accelerating the rate at which the terminal AZT-MP (or other NRTI-MP) is removed through phosphorolytic cleavage (3,21).…”

New substrates of DNA polymerases

“…As a result, compounds of the I and II types and DNA chains shortened by one nucleotide residue were generated [2,3]. We shall not analyze the biological signi¢cance of this reaction, but regard it from the viewpoint of molecular mechanisms of reactions catalyzed by DNA polymerases.…”

Dose–response curve slope is a missing dimension in the analysis of HIV-1 drug resistance

“…Additional mechanisms may also contribute. For example, resistance to zidovudine involves mutations that promote excision (24)(25)(26). Although the molecular mechanisms of resistance are well studied, the pharmacodynamics of resistance are less well understood.…”