Molecular Determinants of Multi-nucleoside Analogue Resistance in HIV-1 Reverse Transcriptases Containing a Dipeptide Insertion in the Fingers Subdomain

Abstract

Human immunodeficiency virus type 1 isolates having dipeptide insertions in the fingers subdomain of the reverse transcriptase (RT) show high level resistance to 3-azido-3-deoxythymidine (AZT) and other nucleoside analogues. Insertions are usually associated with thymidine analogue resistance mutations, such as T215Y. The resistance phenotype correlates with increased ATP-dependent phosphorolytic activity, which facilitates removal of thymidine analogues from inhibitor-terminated primers. In this report, we show that substituting Thr, Ser, or Asn for Tyr-215 in a multidrug-resistant RT, bearing a Ser-Ser insertion between codons 69 and 70, leads to AZT and stavudine resensitization through the loss of the ATP-mediated removal activity. The mutation D67N, which is rarely found in insertion-containing strains, had no effect on excision and a minor influence on resistance. Substituting Tyr-215 had a larger effect than deleting the dipeptide insertion. The presence of both the insertion and mutation T215Y in the wild-type BH10 RT conferred significant ATP-mediated removal activity and moderate resistance to AZT. However, resistance levels and unblocking activities were lower than those observed with the multidrug-resistant enzyme. Removal reactions can be inhibited by the next complementary dNTP. Both Tyr-215 and the dipeptide insertion affect RT-DNA⅐DNA-dNTP ternary complex formation, an effect that was not detected in the presence of foscarnet. Based on crystal structures of binary and ternary complexes of HIV-1 RT, we propose that Tyr-215 exerts its action by facilitating a proper orientation of the pyrophosphate donor molecule, whereas the effects on dNTP binding are indirect and could be related to significant conformational changes occurring during polymerization.Human immunodeficiency virus type 1 (HIV-1) 1 reverse transcriptase (RT) replicates the viral genomic RNA to synthesize a double-stranded DNA that integrates into the host genome. The viral enzyme is multifunctional, possessing RNAand DNA-dependent DNA polymerase, RNase H, strand transfer, and strand displacement activities (1). HIV-1 RT is a heterodimeric enzyme composed of two polypeptide chains of 66 and 51 kDa, with subdomains termed fingers, thumb, palm, and connection in both subunits and an RNase H domain in the larger subunit only.HIV-1 RT is an important target for chemotherapeutic intervention in the control of AIDS. Antiretroviral drugs targeting the viral polymerase include nucleoside analogue inhibitors (NRTIs), acyclic nucleoside phosphonates, and non-nucleoside RT inhibitors (NNRTIs) (reviewed in Ref. 2). Inside the cell, nucleoside derivatives are converted to their active triphosphate forms by host cell kinases and are then incorporated into the HIV-1 genome by the viral RT. Because nucleoside analogues lack a 3Ј-OH group, their incorporation blocks elongation of the growing DNA chain. On the other hand, NNRTIs bind to an allosteric site located 10 -15 Å away from the polymerase active site, distorting the geometry and/or the mobility...

Insertions in the Human Immunodeficiency Virus Type 1 Protease and Reverse Transcriptase Genes: Clinical Impact and Molecular Mechanisms

“…Strains with these inserts almost always have a T-to-S point mutation at codon 69, leading to the accepted nomenclature of T69SϩXX to describe the presence of these inserts. The most commonly inserted amino acids (XX) found in patient-derived strains are SG, SS, and SA, although at least 22 other combinations of codon 69 mutations and insert amino acids have been reported (40,67). A small number of single amino acid inserts at this position have been reported, as have inserts of 5, 8, 11, and 15 amino acids (34,39,40,62).…”

“…Thymidine analog mutations (TAMs) are significantly associated with T69SϩXX inserts, as approximately 95% of insertcontaining strains also possess a T215Y/F mutation, 65% have an M41L mutation, and 52% have an L210W mutation (40). Uncommon variants at codon 67 are also frequently present (31,39,40,59,67).…”

“…The most commonly inserted amino acids (XX) found in patient-derived strains are SG, SS, and SA, although at least 22 other combinations of codon 69 mutations and insert amino acids have been reported (40,67). A small number of single amino acid inserts at this position have been reported, as have inserts of 5, 8, 11, and 15 amino acids (34,39,40,62). At the nucleotide level, inserts appear to be duplications of neighboring genetic sequences (67), likely generated during reverse transcription by RT stalling and slippage (3,20), although one report suggested that a 15-amino-acid insert was generated through intramolecular recombination (34).…”

Thymidine Analogue Resistance Suppression by V75I of HIV-1 Reverse Transcriptase

“…Chain Terminator Excision Assays-RT-catalyzed DNA rescue reactions were performed with D38-25PGA DNA duplexes, as previously described (42,43). Briefly, the phosphorylated template-primer (30 nM) was preincubated at 37°C for 10 min in the presence of the corresponding RT at 12-24 nM active enzyme concentration in 50 mM Hepes buffer (pH 7.0), containing 15 mM NaCl, 15 mM magnesium acetate, 130 mM potassium acetate, 1 mM dithiothreitol, and 5% (w/v) polyethylene glycol 6000.…”

Amino acid residues in HIV-2 reverse transcriptase that restrict the development of nucleoside analogue resistance through the excision pathway

“…Chain terminator excision assays -RT-catalyzed DNA rescue reactions were carried out as previously described with template/primers D38/25PGA, D38T/25PGA, and 31Trna/21P (54,55). Reactions were carried out in 70 mM Hepes pH 7.0 buffer containing 20 mM magnesium acetate, 20 mM NaCl, 130 mM potassium acetate, 1 mM DTT and 5% (w/v) polyethylene glycol 6000.…”