Abstract
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 Glu(89) side chain. We investigated the role of Glu(89) 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 Glu(89) can influence the slippage of both strands. Glu(89) 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 Glu(89) on slippage-mediated errors and dNTP incorporation fidelity. The crystal structure of HIV-1 RT reveals a salt bridge between Glu(89) and Lys(154), which may facilitate -1 frameshifting; this concept is supported by the observed reduction in -1 frameshifting for K154A and K154R mutants.
Highlights
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
The first crystal structure of HIV-1 RT complexed with double-stranded DNA template-primer showed specific structural elements termed primer grip and template grip, which correctly position the templating base and the primer 3Ј terminus for the insertion of incoming Deoxynucleotide triphosphates (dNTPs) (Fig. 1) [7]
The structure of HIV-1 RT complexed with template-primer and dNTP showed that the 3–4 hairpin of the fingers subdomain provides key contacts for the templating nucleotide and the incoming dNTP [8]
Summary
Recognized as one of the factors responsible for high genetic variation in HIV, which leads to the rapid emergence of drug-resistant HIV variants. It was shown that the (Ϫ)-2Ј-deoxy-3Ј-thiacytidine-resistance mutation M184V, affecting a residue located near the incoming dNTP, conferred an increased fidelity of dNTP incorporation on HIV-1 RT (4 – 6). The structure of HIV-1 RT complexed with template-primer and dNTP showed that the 3–4 hairpin of the fingers subdomain provides key contacts for the templating nucleotide and the incoming dNTP [8] The importance of this region for dNTP selection and fidelity is further supported by reports of several mutations in the 3–4 hairpin that affect RT fidelity (9 –12). Substitution at Glu may affect polymerase fidelity by changing the conformation of the template-primer duplex, thereby altering the geometry of the dNTP-binding site [25]. Substitutions at Glu increased the fidelity of dNTP incorporation showing the influence of Glu on multiple types of replication errors
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