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, 96 (18), 10027-32

Lamivudine (3TC) Resistance in HIV-1 Reverse Transcriptase Involves Steric Hindrance With Beta-Branched Amino Acids

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Lamivudine (3TC) Resistance in HIV-1 Reverse Transcriptase Involves Steric Hindrance With Beta-Branched Amino Acids

S G Sarafianos et al. Proc Natl Acad Sci U S A.

Abstract

An important component of triple-drug anti-AIDS therapy is 2', 3'-dideoxy-3'-thiacytidine (3TC, lamivudine). Single mutations at residue 184 of the reverse transcriptase (RT) in HIV cause high-level resistance to 3TC and contribute to the failure of anti-AIDS combination therapy. We have determined crystal structures of the 3TC-resistant mutant HIV-1 RT (M184I) in both the presence and absence of a DNA/DNA template-primer. In the absence of a DNA substrate, the wild-type and mutant structures are very similar. However, comparison of crystal structures of M184I mutant and wild-type HIV-1 RT with and without DNA reveals repositioning of the template-primer in the M184I/DNA binary complex and other smaller changes in residues in the dNTP-binding site. On the basis of these structural results, we developed a model that explains the ability of the 3TC-resistant mutant M184I to incorporate dNTPs but not the nucleotide analog 3TCTP. In this model, steric hindrance is expected for NRTIs with beta- or L- ring configurations, as with the enantiomer of 3TC that is used in therapy. Steric conflict between the oxathiolane ring of 3TCTP and the side chain of beta-branched amino acids (Val, Ile, Thr) at position 184 perturbs inhibitor binding, leading to a reduction in incorporation of the analog. The model can also explain the 3TC resistance of analogous hepatitis B polymerase mutants. Repositioning of the template-primer as observed in the binary complex (M184I/DNA) may also occur in the catalytic ternary complex (M184I/DNA/3TCTP) and contribute to 3TC resistance by interfering with the formation of a catalytically competent closed complex.

Figures

Figure 1
Figure 1
Chemical structures of deoxythymidine and various NRTIs, with aligned C1′ and C4′ atoms of the nucleoside rings. Such alignment highlights the unique direction (arrows pointing down) of the β-l-nucleosides ring, projecting in the opposite direction from the other nucleoside and nucleoside analog inhibitors (arrows pointing up).
Figure 2
Figure 2
Superposition of the polymerase active sites of wild-type HIV-1 RT/DNA/Fab and M184I HIV-1 RT/DNA/Fab. The wild-type RT complex is shown in white, the mutant RT complex in cyan. The wild-type and M184I RT structures were superimposed on the basis of the core of the p66 palm subdomains (residues 107 to 112 and 151 to 215 of their corresponding p66 subunits).
Figure 3
Figure 3
Ribbon diagram of the superimposed polymerase active sites of wild-type HIV-1 RT/DNA/Fab and M184I HIV-1 RT/DNA/Fab. The wild-type protein and DNA are shown in gray, the mutant protein in red, and DNA in the mutant RT/DNA complex in yellow.
Figure 4
Figure 4
Schematic illustration of proposed steric conflict between 3TCTP and 184I. The van der Waals volume of the side chain of I184 (green) is shown to overlap (red) with the sulfur of the β-l-oxathiolane ring of the incoming 3TCTP (yellow). A dCTP molecule (green) is shown superimposed on 3TCTP (white) in a way that maintains the same base pairing with a modeled template strand and proximity to the 3′-OH, but without steric conflict with I184. The model predicts similar steric hindrance with the two other β-branched amino acids (Val and Thr). The side chains of these amino acids are shown in an orientation similar to the I184 observed in our structure. This model is supported by the reported resistance of M184V and M184T RT to 3TCTP. An analogous model can explain resistance of analogous mutants of HBV RT to 3TCTP.

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