HIV-1 susceptibility studies from our laboratory have identified bidirectional, phenotypic antagonism between K65R and TAMs [22,27]. The current work provides detailed kinetic insights into the mechanisms involved in this antagonism at the enzyme level. In particular, we show that K65R antagonizes TAMs by significantly reducing the ability of RT to excise all chain-terminating NRTI monophosphate, and that TAMs antagonize K65R by diminishing the extent to which it can discriminate against D- (but not L) nucleotide analogs. Unexpectedly, we also found that TAM67 RT can confer resistance to lamivudine triphosphate by discriminating against its incorporation.
Previous studies have shown that RT containing the K65R mutation alone has a lower capacity to excise NRTI monophosphate using ATP as the excision substrate . In addition, White et al.  recently showed that the introduction of K65R into RT containing M41L, D67N, L210W and T215Y significantly reduced the enzyme's ability to excise zidovudine monophosphate, thereby providing a mechanism by which K65R can antagonize TAMs. Our data confirm the findings of White et al. , and expand on them by showing that K65R inhibits the ability of RT containing TAMs to excise all of the US Food and Drug Administration-approved D-nucleoside analogs (stavudine monophosphate, tenofovir, ddA monophosphate, carbovir monophosphate, zalcitabine monophosphate). In general, this effect was caused by decreases in the rate of ATP-mediated NRTI monophosphate excision. We were, however, unable to determine the Kd for ATP for either wild-type or mutant enzymes because of experimental limitations, and thus we can not rule out the possibility that K65R might also affect ATP binding. Interestingly, the burst amplitude (the total amount of product generated in the excision assay) was significantly decreased when the ATP-mediated excision rate constant for wild-type or mutant RT was less than 0.30 min−1 (Table 1). This observed decrease in burst amplitude may result from the NRTI monophosphate excision rate approximating the rate of RT T/P dissociation, which is the overall rate-limiting step in polymerization and excision reactions [30,37].
The K70E, L74V and M184V mutations, alone or in combination with TAMs, have also been shown to impair the rescue of NRTI monophosphate chain-terminated DNA [13–15,38,39]. This supports the notion that mutations associated with the NRTI discrimination phenotype are, in general, antagonistic toward TAMs. TAMs do not, however, appear to compromise the resistance phenotypes of K70E, L74V or M184V. In the current study, we were provided with a unique opportunity to define a biochemical mechanism by which TAMs antagonize the discrimination phenotype of K65R. Consistent with previously published data , our results show that K65R increases the selectivity of RT for dGTP and dATP versus carbovir triphosphate and tenofovir diphosphate, respectively (Table 2), and that this selectivity could primarily be attributed to decreased k pol rates. When K65R RT is combined with TAMs, the enzyme is less able to incorporate the natural substrate selectively over the NRTI triphosphate (Table 2). Interestingly, this decrease in NRTI triphosphate selectivity is primarily caused by the TAM compensating for the decreased maximum rate of NRTI triphosphate incorporation mediated by the K65R mutation. In the crystal structures of the ternary HIV-1 RT T/P–dNTP complexes, the ε-amino group of K65 interacts with the γ-phosphate of the bound nucleotide substrate [40,41]. Introduction of the K65R mutation into these structures slightly alters the spatial arrangement of the nucleotide phosphate backbone [16,42] (Fig. 4a), and results in the formation of additional hydrogen bonds between R65 and the backbone N–H of D113 and W71  (Fig. 4a). As described in this study and others [9–12], K65R negatively impacts k pol, a rate-constant that defines a rate-limiting conformational change that precedes phosphodiester bond formation . Molecular dynamic simulation studies revealed that K65R reduces the mobility of the β3–β4 loop (as a result of the additional hydrogen bond interactions with D113 and W71) . This decreased active site flexibility may be responsible for the decreased incorporation and decreased excision rates observed K65R . Additional modeling analyses show that the M41L, D67N, K70R and L210W mutations do not directly alter the DNA polymerase active site structure of HIV-1 RT (data not shown). The T215F/Y mutations distort the precise positioning of residues D113 and A114 (Fig. 4b). Both of these residues display critical interactions with the phosphate backbone of the bound nucleotide substrate [40,41]. We hypothesize that these additional conformational changes induced by the T215F/Y mutations may partly compensate for the decreased active site flexibility conferred by the K65R mutation.
Interestingly, our data also demonstrate that TAM67 RT, but not TAM41 RT, confers resistance to lamivudine triphosphate via a nucleotide discrimination phenotype as opposed to an excision phenotype. These data, which are entirely consistent with the observed viral susceptibility data , suggest that the D67N, K70N or K219Q mutations are responsible for the lamivudine triphosphate discrimination phenotype. In addition, our data also clearly demonstrate that TAMs do not antagonize the capacity of K65R to discriminate between dCTP and lamivudine triphosphate. Therefore, the antagonism between TAMs and K65R appears to be specific towards D-nucleosides (abacavir, zidovudine, didanosine, zalcitabine, stavudine and tenofovir) and not L-nucleosides (lamivudine and emtricitabine). This specificity is probably caused by differences in the binding interactions between D- and L-NRTI triphosphate with the active site of RT.
As described previously, NRTI mutations such as L74V and M184V have also been shown to be antagonistic to TAMs [17,18]. HIV-1 isolates that exhibit dual resistance to zidovudine plus didanosine or zidovudine plus lamivudine and contain both TAMs and L74V or TAMs and M184V have been identified [18,19,43]. In these isolates, dual NRTI resistance was associated with the acquisition of either additional TAMs  or novel resistance mutations [19,43]. In comparison, K65R and multiple TAMs are rarely detected (< 0.1%) in the same plasma sample. If they are, DNA sequencing of single viral genomes has shown that K65R is not found on the same genome with T215F/Y and two or more TAMs except in the presence of the Q151M multi-NRTI resistance complex. The acquisition of the Q151M complex in clinical isolates containing K65R and TAMs highlights the difficulty that HIV-1 has in evolving multi-NRTI resistance with both set of mutations . Taken together, these studies clearly demonstrate that there is no net advantage for HIV-1 simultaneously to evolve both the K65R and TAMs resistance pathways. Therefore, concomitant therapy with NRTI that selects for TAMs and K65R may forestall the development of NRTI resistance. Clinical trials to assess this possibility are in progress.
1. Parniak MA, Sluis-Cremer N. Inhibitors of HIV-1 reverse transcriptase. Adv Pharmacol 2000; 49:67–109.
2. Sluis-Cremer N, Arion D, Parniak MA. Molecular mechanisms of HIV-1 resistance to nucleoside reverse transcriptase inhibitors (NRTIs). Cell Mol Life Sci 2000; 57:1408–1422.
3. Selmi B, Deval J, Boretto J, Canard B. Nucleotide analogue binding, catalysis and primer unblocking in the mechanisms of HIV-1 reverse transcriptase-mediated resistance to nucleoside analogues. Antivir Ther 2003; 8:143–154.
4. Deval J, Courcambeck J, Selmi B, Boretto J, Canard B. Structural determinants and molecular mechanisms for the resistance of HIV-1 RT to nucleoside analogues. Curr Drug Metab 2004; 5:305–316.
5. Arion D, Kaushik N, McCormick S, Borkow G, Parniak MA. Phenotypic mechanism of HIV-1 resistance to 3′-azido-3′-deoxythymidine (AZT): increased polymerization processivity and enhanced sensitivity to pyrophosphate of the mutant viral reverse transcriptase. Biochemistry 1998; 37:15908–15917.
6. Meyer PR, Matsuura SE, Mian AM, So AG, Scott WA. A mechanism of AZT resistance: an increase in nucleotide-dependent primer unblocking by mutant HIV-1 reverse transcriptase. Mol Cell 1999; 4:35–43.
7. Boyer PL, Sarafianos SG, Arnold E, Hughes SH. Selective excision of AZTMP by drug-resistant human immunodeficiency virus reverse transcriptase. J Virol 2001; 75:4832–4842.
8. Feng JY, Anderson KS. Mechanistic studies examining the efficiency and fidelity of DNA synthesis by the 3TC-resistant mutant (184V) of HIV-1 reverse transcriptase. Biochemistry 1999; 38:9440–9448.
9. Selmi B, Boretto J, Sarfati SR, Guerreiro C, Canard B. Mechanism-based suppression of dideoxynucleotide resistance by K65R human immunodeficiency virus reverse transcriptase using an alpha-boranophosphate nucleoside analogue. J Biol Chem 2001; 276:48466–48472.
10. Deval J, Selmi B, Boretto J, Egloff MP, Guerreiro C, Sarfati S, Canard B. The molecular mechanism of multidrug resistance by the Q151M human immunodeficiency virus type 1 reverse transcriptase and its suppression using alpha-boranophosphate nucleotide analogues. J Biol Chem 2002; 277:42097–42104.
11. Deval J, White KL, Miller MD, Parkin NT, Courcambeck J, Halfon P, et al
. Mechanistic basis for reduced viral and enzymatic fitness of HIV-1 reverse transcriptase containing both K65R and M184V mutations. J Biol Chem 2004; 279:509–516.
12. Deval J, Navarro JM, Selmi B, Courcambeck J, Boretto J, Halfon P, et al
. A loss of viral replicative capacity correlates with altered DNA polymerization kinetics by the human immunodeficiency virus reverse transcriptase bearing the K65R and L74V dideoxynucleoside resistance substitutions. J Biol Chem 2004; 279:25489–25496.
13. Naeger LK, Margot NA, Miller MD. Increased drug susceptibility of HIV-1 reverse transcriptase mutants containing M184V and zidovudine-associated mutations: analysis of enzyme processivity, chain-terminator removal and viral replication. Antivir Ther 2001; 6:115–126.
14. Boyer PL, Sarafianos SG, Arnold E, Hughes SH. The M184V mutation reduces the selective excision of zidovudine 5′-monophosphate (AZTMP) by the reverse transcriptase of human immunodeficiency virus type 1. J Virol 2002; 76:3248–3256.
15. Miranda LR, Gotte M, Liang F, Kuritzkes DR. The L74V mutation in human immunodeficiency virus type 1 reverse transcriptase counteracts enhanced excision of zidovudine monophosphate associated with thymidine analog resistance mutations. Antimicrob Agents Chemother 2005; 49:2648–2656.
16. White KL, Chen JM, Feng JY, Margot NA, Ly JK, Ray AS, et al
. The K65R reverse transcriptase mutation in HIV-1 reverses the excision phenotype of zidovudine resistance mutations. Antivir Ther 2006; 11:155–163.
17. Larder BA, Kemp SD, Harrigan PR. Potential mechanism for sustained antiretroviral efficacy of AZT–3TC combination therapy. Science 1995; 269:696–699.
18. Shafer RW, Kozal MJ, Winters MA, Iversen AK, Katzenstein DA, Ragni MV, et al
. Combination therapy with zidovudine and didanosine selects for drug-resistant human immunodeficiency virus type 1 strains with unique patterns of pol gene mutations. J Infect Dis 1994; 169:722–729.
19. Stoeckli TC, MaWhinney S, Uy J, Duan C, Lu J, Shugarts D, Kuritzkes DR. Phenotypic and genotypic analysis of biologically cloned human immunodeficiency virus type 1 isolates from patients treated with zidovudine and lamivudine. Antimicrob Agents Chemother 2002; 46:4000–4003.
20. Parikh UM, Koontz DL, Chu CK, Schinazi RF, Mellors JW. In vitro activity of structurally diverse nucleoside analogs against human immunodeficiency virus type 1 with the K65R mutation in reverse transcriptase. Antimicrob Agents Chemother 2005; 49:1139–1144.
21. Winston A, Mandalia S, Pillay D, Gazzard B, Pozniak A. The prevalence and determinants of the K65R mutation in HIV-1 reverse transcriptase in tenofovir-naive patients. AIDS 2002; 16:2087–2089.
22. Parikh UM, Bacheler L, Koontz D, Mellors JW. The K65R mutation in human immunodeficiency virus type 1 reverse transcriptase exhibits bidirectional phenotypic antagonism with thymidine analog mutations. J Virol 2006; 80:4971–4977.
23. Miller MD. K65R, TAMs and tenofovir. AIDS Rev 2004; 6:22–33.
24. Valer L, Martin-Carbonero L, de Mendoza C, Corral A, Soriano V. Predictors of selection of K65R: tenofovir use and lack of thymidine analogue mutations. AIDS 2004; 18:2094–2096.
25. Segondy M, Montes B. Prevalence and conditions of selection of the K65R mutation in the reverse transcriptase gene of HIV-1. J Acquir Immune Defic Syndr 2005; 38:110–111.
26. Wirden M, Marcelin AG, Simon A, Kirstetter M, Tubiana R, Valantin MA, et al
. Resistance mutations before and after tenofovir regimen failure in HIV-1 infected patients. J Med Virol 2005; 76:297–301.
27. Parikh UM, Barnas DC, Faruki H, Mellors JW. Antagonism between the HIV-1 reverse-transcriptase mutation K65R and thymidine-analogue mutations at the genomic level. J Infect Dis 2006; 194:651–660.
28. Le Grice SF, Gruninger-Leitch F. Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. Eur J Biochem 1990; 187:307–314.
29. Le Grice SF, Cameron CE, Benkovic SJ. Purification and characterization of human immunodeficiency virus type 1 reverse transcriptase. Methods Enzymol 1995; 262:130–144.
30. Kati WM, Johnson KA, Jerva LF, Anderson KS. Mechanism and fidelity of HIV reverse transcriptase. J Biol Chem 1992; 267:25988–25997.
31. Sluis-Cremer N, Arion D, Parikh U, Koontz D, Schinazi RF, Mellors JW, Parniak MA. The 3′-azido group is not the primary determinant of 3′-azido-3′-deoxythymidine (AZT) responsible for the excision phenotype of AZT-resistant HIV-1. J Biol Chem 2005; 280:29047–29052.
32. Arion D, Sluis-Cremer N, Parniak MA. Mechanism by which phosphonoformic acid resistance mutations restore 3′-azido-3′-deoxythymidine (AZT) sensitivity to AZT-resistant HIV-1 reverse transcriptase. J Biol Chem 2000; 275:9251–9255.
33. Radzio J, Sluis-Cremer N. Stereo-selectivity of HIV-1 reverse transcriptase toward isomers of thymidine-5(-O-1-thiotriphosphate. Protein Sci 2005; 14:1929–1933. E-pub 3 June 2005.
34. Johnson KA. Rapid quench kinetic analysis of polymerases, adenosinetriphosphatases, and enzyme intermediates. Methods Enzymol 1995; 249:38–61.
35. Whitcomb JM, Parkin NT, Chappey C, Hellmann NS, Petropoulos CJ. Broad nucleoside reverse-transcriptase inhibitor cross-resistance in human immunodeficiency virus type 1 clinical isolates. J Infect Dis 2003; 188:992–1000.
36. White KL, Margot NA, Ly JK, Chen JM, Ray AS, Pavelko M, et al
. A combination of decreased NRTI incorporation and decreased excision determines the resistance profile of HIV-1 K65R RT. AIDS 2005; 19:1751–1760.
37. Ray AS, Murakami E, Basavapathruni A, Vaccaro JA, Ulrich D, Chu CK, et al
. Probing the molecular mechanisms of AZT drug resistance mediated by HIV-1 reverse transcriptase using a transient kinetic analysis. Biochemistry 2003; 42:8831–8841.
38. Gotte M, Arion D, Parniak MA, Wainberg MA. The M184V mutation in the reverse transcriptase of human immunodeficiency virus type 1 impairs rescue of chain-terminated DNA synthesis. J Virol 2000; 74:3579–3585.
39. Frankel FA, Marchand B, Turner D, Gotte M, Wainberg MA. Impaired rescue of chain-terminated DNA synthesis associated with the L74V mutation in human immunodeficiency virus type 1 reverse transcriptase. Antimicrob Agents Chemother 2005; 49:2657–2664.
40. Tuske S, Sarafianos SG, Clark AD Jr, Ding J, Naeger LK, White KL, et al
. Structures of HIV-1 RT-DNA complexes before and after incorporation of the anti-AIDS drug tenofovir. Nat Struct Mol Biol 2004; 11:469–474.
41. Huang H, Chopra R, Verdine GL, Harrison SC. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science 1998; 282:1669–1675.
42. Sluis-Cremer N, Arion D, Kaushik N, Lim H, Parniak MA. Mutational analysis of Lys65 of HIV-1 reverse transcriptase. Biochem J 2000; 348:77–82.
43. Kemp SD, Shi C, Bloor S, Harrigan PR, Mellors JW, Larder BA. A novel polymorphism at codon 333 of human immunodeficiency virus type 1 reverse transcriptase can facilitate dual resistance to zidovudine and L-2′,3′-dideoxy-3′-thiacytidine. J Virol 1998; 72:5093–5098.