Mechanistic Basis of Zidovudine Hypersusceptibility and Lamivudine Resistance Conferred by the Deletion of Codon 69 in the HIV-1 Reverse Transcriptase Coding Region

Abstract

Deletions in the β3–β4 hairpin loop of human immunodeficiency virus type 1 reverse transcriptase (RT) are associated with the emergence of multidrug resistance. Common mutational patterns involve the deletion of Asp67 (Δ67) and mutations such as K70R and T215F or T215Y, or the deletion of Thr69 (Δ69) and mutations of the Q151M complex. Human immunodeficiency virus type 1 clones containing Δ69 in a multidrug-resistant sequence background, including the Q151M complex and substitutions K103N, Y181C, M184V, and G190A, showed high-level resistance to all tested nucleoside RT inhibitors. In a multidrug-resistant sequence context, the deletion increases viral replication capacity. By itself, Δ69 conferred increased susceptibility to β- d-(+)-3′-azido-3′-deoxythymidine (AZT) and β- l-(−)-2′,3′-dideoxy-3′-thiacytidine resistance. Here, we use transient kinetics to show that, in a wild-type sequence background, Δ69 does not affect the discrimination between AZT triphosphate and 2′-deoxythymidine 5′-triphosphate, but decreases the catalytic efficiency of the incorporation of β- l-(−)-2′,3′-dideoxy-3′-thiacytidine triphosphate relative to 2′-deoxycytidine 5′-triphosphate. In comparison with the wild-type RT, the Δ69 mutant showed decreased ability to excise primers terminated with AZT monophosphate in the presence of ATP or pyrophosphate (PPi). These data support the role of the excision mechanism in mediating AZT hypersusceptibility. In addition, we demonstrate that the deletion has no effect on resistance to foscarnet (a PPi analogue) on phenotypic and nucleotide incorporation assays carried out with viral clones and recombinant enzymes, respectively. The results of molecular modeling studies suggest that the side chains of Lys65, Asp67, and Lys219 could play an important role in AZT hypersusceptibility mediated by Δ69, whereas in the absence of Thr69, local structural rearrangements affecting the β3–β4 and β11a–β12 loops of the 66-kDa subunit of the RT could reduce the accessibility of the PPi donor to the terminating nucleotide at the 3′ end of the primer.