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 kpol 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 kpol, 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.
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