Clinical Significance of HIV Reverse-transcriptase Inhibitor-resistance Mutations

Shiro Ibe; Wataru Sugiura

Disclosures

Future Microbiol. 2011;6(3):295-315. 

In This Article

NRTI-resistance Mutations & their Mechanism of Drug Resistance

In 1989, drug-resistant HIV-1 was isolated from patients treated with zidovudine,[33,34] leading to identification of the first cluster of drug-resistance amino acid mutations (D67N, K70R, T215F/Y and K219Q) in RT.[35] Thereafter, no antiretroviral has been found to successfully avoid the emergence of drug-resistant variants. According to the latest list of drug-resistance mutations for HIV clinical practitioners,[36] 16 amino acid positions associated with NRTI resistance are mapped in the 3D structure of HIV-1 RT (Figure 3). Interestingly, all 16 amino acid positions are located in the fingers and palm subdomains of RT, which surround the incoming nucleotide.[22]

Figure 3.

Nucleos(t)ide analog reverse-transcriptase inhibitor-resistance amino acid positions in the 3D structure of HIV-1 reverse transcriptase. A total of 16 amino acid positions associated with nucleos(t)ide reverse-transcriptase inhibitor resistance36 are shown in red. Two magnesium ions necessary for DNA polymerization are shown as tiny brown balls near the deoxythymidine-5'-triphosphate (dTTP; magenta). The fingers and palm subdomains of the p66 subunit are shown in blue and green wire-frame, respectively. A triad of aspartic acid residues at positions 110, 185 and 186 within the DNA polymerase catalytic site are shown in yellow. The DNA template-primer is shown in dark gray. (A) Amino acid positions of thymidine analog-associated mutations (TAMs): M41L, D67N, K70R, L210W, T215Y/F and K219Q/E. (B) Amino acid positions for the multinucleoside reverse-transcriptase inhibitor-resistance complex of Q151M with A62V, V75I, F77L and F116Y mutations. (C) Amino acid positions for K65R, 69-insertion, L74V, Y115F and M184V/I mutations.

The most prevalent NRTI-resistance mutation recently identified in large-scale analyses of drug-resistant viral genotypes was the M184V/I mutation (>38%, M184V predominant).[37–40] This high prevalence of the M184V/I mutation in therapy-failure cases was explained by the frequent first-line therapy use of lamivudine, emtricitabine and abacavir (drugs corresponding to the M184V/I mutation). In addition, acquisition of the M184V mutation alone is sufficient for the virus to exhibit high-level resistance to both lamivudine and emtricitabine.[41–43] The M184V/I mutation was first identified in the course of viral passages with lamivudine or emtricitabine, and simultaneously characterized as the mutation that restored susceptibility of zidovudine-resistant variants to zidovudine.[43,44] While the M184V-mediated zidovudine resensitization was confirmed in vivo during 6 months of zidovudine plus lamivudine therapy,[41] the resensitization was transiently observed in most zidovudine-experienced patients (69%, 20/29) during 1 year of combination therapy.[45] Furthermore, M184V-containing RT was shown in most biochemical studies to have drastically reduced binding activity to lamivudine- or emtricitabine-5'-triphosphate.[46–49] Concerning the molecular mechanism of resistance, structural studies show that the M184V/I mutation creates steric hindrance that interferes with lamivudine-5'-triphosphate binding to the active site of RT.[22,50]

The second most prevalent NRTI-resistance mutation in drug-resistant viral genotypes was identified as a series of thymidine analog-associated mutations (TAMs): M41L, D67N, K70R, L210W, T215Y/F and K219Q/E.[37–40] The prevalence of TAMs ranges from 19 to 42%,[37] 7–20%[38] and 13–25%,[40] and T215Y/F commonly shows the highest prevalence. Focusing on the T215 mutation patterns, several studies proposed two different TAM pathways: TAM-1 (M41L+L210W+T215Y) and TAM-2 (K70R+T215F+K219Q).[51–55] However, the significance of these two pathways is still controversial, and indeed the definition of TAM-1 and -2 differs among the studies. Regarding the clinical impact of the pathways, two studies reported a relationship between the TAM-1 profile and poor virological responses to tenofovir-containing salvage therapy.[53,54] In another study, patients with the TAM-2 profile demonstrated a better virological response to stavudine-containing therapy than to zidovudine-containing therapy.[55] Importantly, the accumulation of TAMs leads to a progressive decrease in drug susceptibility for all the approved NRTIs, referred to as multi-NRTI resistance.[56,57] The mechanism of drug resistance by TAMs has been well studied, with zidovudine resistance as a model. TAM-containing RT demonstrated enhanced activity for dinucleoside polyphosphate synthesis, a pyrophosphorolysis-related reaction in which an incorporated zidovudine-5'-monophosphate is removed from the end of terminated DNA[58,59] (Figure 4). TAM-containing RT can continue elongating viral DNA, even in the presence of NRTIs, through a mechanism termed 'excision'. Excision has only been shown in structural studies when the chain-terminated end is positioned at the nucleotide-binding site (N site) of RT (Figure 4).[60,61] Interestingly, the most prevalent TAM, T215Y/F, appeared to be the key mutation to make a direct contact with ATP, the most likely pyrophosphate donor for the excision reaction.[58] Of note, adding the M184V mutation to the complex of TAMs increased viral susceptibility to zidovudine, stavudine and tenofovir.[56,62,63] This favorable interference may explain in part why the combination of two NRTIs, a TAM-inducible drug (zidovudine, stavudine and tenofovir), and an M184V-inducible drug (lamivudine and emtricitabine) has functioned well as the backbone of HAART. RT containing both M184V and TAMs demonstrated a relatively reduced activity for excision of the chain terminator, but the number and/or combination of TAMs seemed to impact the M184V-mediated increase in drug susceptibility.[64–66]

Figure 4.

ATP-mediated chain-terminator excision. Thymidine analog-associated mutation-containing reverse transcriptase has enhanced activity for dinucleoside polyphosphate synthesis, in which an incorporated nucleos(t)ide analog reverse-transcriptase inhibitor monophosphate (red) is removed from the end of terminated DNA (bottom). The most likely pyrophosphate donor for the excision reaction is adenosine-5'-triphosphate (ATP; blue). After removing the nucleos(t)ide analog reverse-transcriptase inhibitor monophosphate, the mode of DNA polymerization becomes active again (bottom). The P site and N site of HIV-1 reverse transcriptase are shown as purple and green boxes, respectively.
dNTP: Deoxynucleotide triphosphate; N site: Nucleotide-binding site; P site: Priming site.

Most of the other NRTI-resistance mutations were identified as low prevalence (<5%).[37–40] Among these, the K65R mutation (prevalence: 2.5–4.9%) should be noted owing to its interesting characteristics. K65R was first identified as a mutation conferring resistance to didanosine, zalcitabine, lamivudine,[67,68] and its wide spectrum of cross-resistance to all approved NRTIs except for zidovudine has been well characterized.[69–73] Although the frequency of K65R had been very low among therapy-failure cases before the approval of tenofovir, this mutation was recently reconfirmed as a primary mutation conferring tenofovir resistance. In a large clinical trial of 299 therapy-naive patients on a tenofovir/lamivudine/efavirenz regimen, the K65R mutation appeared to be the only pathway to tenofovir resistance (17%, 8/47 therapy-failure cases).[74] By contrast, no K65R mutation was detected in any of the 12 therapy-failure cases reported in another large clinical trial of 258 therapy-naive patients on a tenofovir/emtricitabine/efavirenz regimen.[75] Since RT residue 65 is located at the tip of the fingers subdomain, which functions as a crucial part of the dNTP-binding site (Figure 3C), the K65R mutation enables HIV-1 RT to discriminate against NRTIs by decreasing the incorporation rate of their 5'-triphosphate forms more than that of natural dNTP substrates.[49,76–79] Interestingly, K65R inhibited TAM-mediated excision, and the decreased excision rate was responsible for the hypersusceptibility of K65R-containing RT to zidovudine.[73,79–82] In fact, TAMs demonstrated a lower prevalence in viral genotypes containing the K65R mutation.[40,82–86] K65R-containing RT was shown in a recent structural study to be able to discriminate drugs from natural substrates owing to a molecular platform formed by two arginines at positions 65 and 72 that force a conformational restriction on TAM-mediated excision.[87]

The complex of TAMs described above is one of three patterns resulting in multi-NRTI resistance; the remaining two patterns are termed the '69 insertion complex' and 'Q151M complex'. The insertion mutation at position 69 was first identified in a zidovudine-experienced patient receiving subsequent combination therapy with didanosine and hydroxyurea.[88] The prevalence of insertion mutations at position 69 was recently reported to be low (0–0.6%).[37,38,40] The insertion mutations consist of one to three amino acids, and a two-amino acid insert is the most frequently observed pattern. The region with the 69th amino acid, the tip of the fingers subdomain, appears to be structurally vulnerable for acquiring insertion mutations. Interestingly, higher numbers of insertions have been reported, ranging from four to 11 amino acids.[89,90] Usually, the insertion mutation accompanies other NRTI-resistance mutations, such as M41L, A62V, K70R, L210W, T215Y/F and K219Q/E. This 69 insertion complex confers cross-resistance to all approved NRTIs.[57,62,91–95] RT with the 69 insertion complex had a decreased binding or incorporation rate for some NRTI triphosphate forms, with simultaneously enhanced activity for removing a chain terminator from the end of DNA.[64,96–100] Therefore, the key mutation essential for ATP-mediated excision, T215Y/F, was found in most viruses with the 69 insertion complex (97%).[96]

A complex of the Q151M mutation with four other mutations (A62V, V75I, F77L and F116Y) was first identified in a patient receiving long-term therapy with an alternating regimen of zidovudine and zalcitabine.[101] Soon after this identification, Q151M complex-mediated cross-resistance to zidovudine, didanosine, zalcitabine and stavudine was well characterized.[102,103] The Q151M complex alone still retained moderate susceptibility to lamivudine, emtricitabine and tenofovir (<2.7-fold change), but the Q151M complex combined with the K65R mutation dramatically decreased susceptibility to drugs (>13.5-fold change).[57,63] In fact, although the Q151M mutation had a low prevalence (0.9–1.4%),[37,38,40] it tended to emerge more frequently in the viral genotype with the K65R mutation.[82,84–86] Concerning the mechanism of multi-NRTI resistance, the Q151M complex is unlike the other two patterns (TAMs and 69 insertion complexes) because it does not rely on ATP-mediated excision of chain terminators.[64,104] The Q151M complex did not change RT binding affinities for NRTI triphosphate forms, but selectively reduced NRTI incorporation rates at the catalytic step.[104,105] To date, the crystal structure of RT with either the Q151M or the 69 insertion complex has not been published; such a structure would provide valuable information for understanding the molecular mechanism of multi-NRTI resistance.

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