Structural determinants of slippage-mediated mutations by human immunodeficiency virus type 1 reverse transcriptase

Single-base deletions at nucleotide runs or -1 frameshifting by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) result from template slippage during polymerization. In crystal structures of HIV-1 RT complexed with DNA-DNA template-primer, the palm subdomain in the template cleft contacts the template backbone near the proposed site of slippage via the Glu⁸⁹ side chain. We investigated the role of Glu⁸⁹ in frameshifting by perturbing this interaction. Substitutions with Asp, Gly, Ala, Val, Ser, Thr, Asn, or Lys were created in recombinant HIV RT, and frameshift frequencies of the resulting mutant RTs were measured. All substitutions led to reduced -1 frameshifting by HIV-1 RT (2-40-fold). Interestingly, the suppression of -1 frameshifting frequently coincided with an enhancement of +1 frameshifting (3-47-fold) suggesting that Glu⁸⁹ can influence the slippage of both strands. Glu⁸⁹ substitutions also led to reduced rates of dNTP misincorporation that paralleled reductions in -1 frameshifting, suggesting a common structural mechanism for both classes of RT error. Our results reveal a major influence of Glu⁸⁹ on slippage-mediated errors and dNTP incorporation fidelity. The crystal structure of HIV-1 RT reveals a salt bridge between Glu⁸⁹ and Lys¹⁵⁴, which may facilitate -1 frameshifting; this concept is supported by the observed reduction in -1 frameshifting for K154A and K154R mutants.