The lack of correlation between changes in inhibitor binding for AZT and the significant changes in AZT susceptibility for viruses with thymidine analog resistance mutations has led to research into other resistance mechanisms. Recent work has suggested that an increased rate of AZT excision by pyrophosphorolysis (reverse nucleotide polymerization) or a similar mechanism using ATP instead of pyrophosphate could account for part of the AZT resistance resulting from thymidine analog resistance mutations (1
). Our results confirm that HIV-1 RT can remove chain-terminating RT inhibitors in the presence of physiological concentrations of ATP. A comparison of levels of ATP-dependent chain terminator removal of NRTIs by a mutant RT containing common thymidine analog resistance mutations D67N, K70R, and T215Y demonstrated that the thymidine analogs, AZT and d4T, were the most efficiently removed. This mutant RT showed intermediate levels of removal of ddC, carbovir, and DXG, followed by minimal removal of 3TC, ddA, and tenofovir. The inefficient removal of ddC, carbovir, DXG, ddA, 3TC, and tenofovir by the RT mutant is consistent with the minor in vitro changes in the susceptibility of the mutant virus to these inhibitors.
The addition of ATP and the next complementary dNTP reduced ATP-mediated removal of d4T, ddC, and DXG, while AZT and carbovir removal was unaffected. Tenofovir, ddA, and 3TC were minimally removed in the presence or absence of the next complementary dNTP. The catalytic efficiency of tenofovir removal by the mutant RT enzyme was 35- and 22-fold less efficient than removal of AZT and d4T, respectively. Furthermore, mutant RT showed a less-than-twofold increase in tenofovir removal compared to wild-type RT, consistent with the twofold changes in tenofovir susceptibility for the mutant virus. AZT removal by the mutant RT remained four- to fivefold more efficient than that by wild-type RT even in the presence of the next dNTP, consistent with susceptibility changes for HIV mutants with thymidine analog resistance mutations and showing AZT resistance. A mutant containing thymidine analog mutations M41L and K219Q in addition to D67N, K70R, and T215Y has been analyzed and has been shown to have a 131-fold decrease in tenofovir removal compared to AZT removal. Tenofovir removal by the mutant RT with M41L, D67N, K70R, T215Y, K219Q mutations is not significantly different from that of wild-type RT, but AZT removal is increased by sevenfold compared to that by wild-type RT. These results are consistent with the changes in the susceptibility of the mutant viruses to these inhibitors (L. K. Naeger, unpublished results).
While d4T is readily removed by the RT mutant with D67N, K70R, T215Y mutations, the next incoming dNTP can inhibit the efficiency of d4T removal by the mutant to levels comparable to those for wild-type RT. These findings with d4T suggest that current in vitro antiviral phenotypic assays may not adequately measure susceptibility to d4T. The higher dNTP concentrations of the activated peripheral blood lymphocytes and transformed human cells used in in vitro phenotypic assays (11
) may inhibit removal of d4T. Cell culture results for HIV-1 containing thymidine analog resistance mutations commonly demonstrate resistance to AZT and sensitivity to d4T, but the true cross-resistance of d4T in these cells may be masked by high dNTP concentrations. Under conditions of low dNTP levels (e.g., quiescent primary T cells or macrophages), d4T removal would be expected to occur at a higher efficiency, thus possibly explaining the observed in vivo cross-resistance to d4T of HIV-1 mutants containing thymidine analog resistance mutations. The inhibition of ddC and DXG removal by the next complementary dNTP also suggests that current in vitro phenotypic assays showing less-than-twofold changes in ddC and DXG susceptibility for viruses producing RT mutants with thymidine analog resistance mutations may not adequately reflect their in vivo susceptibilities.
The modeling of an AZT-terminated primer/template with HIV-1 RT has shown that translocation of the terminating AZT to the P site (primer site) from the N site (incoming dNTP site) would result in the azide group of AZT having unfavorable interactions (steric clash) with amino acid D185, suggesting that the AZT-terminated end of the primer would preferentially occupy the N site and block the incoming dNTP (5
). Furthermore, if the terminated AZT primer end translocated to the P site, steric hindrance from the azide group would interfere with the binding of the incoming dNTP. Therefore, AZT is more likely to occupy the N position, even when dNTPs are present, compared to other dideoxynucleoside triphosphates, resulting in AZT being in a favorable position for removal by ATP. Conversely, dideoxynucleotides, such as d4T, where there is no steric hindrance, would likely favor the P position rather than N position so that the incoming dNTP could interfere with their removal (5
). These results support the biochemical data of our group and others showing that removal of AZT is not inhibited by the next incoming dNTP, whereas removal of d4T is inhibited (5
). Furthermore, the modeling performed by Boyer et al. proposes that mutations M41L, D67N, K70R, T215Y, and K219Q enhance ATP binding to HIV-1 RT so that, at physiological concentrations of ATP, removal would be expected to be more efficient regardless of the nucleotide (5
). Consequently, the specificity of the removal reaction is not dictated by the mutations conferring resistance but rather depends on the structure of the region around the active site of HIV-1 RT and the structure of the nucleoside analog.
The reduced removal of tenofovir might result from the unique phosphonate bond of tenofovir or the unique acyclic structure of tenofovir. The incorporation of tenofovir into the primer may favor the P site rather than the N site, resulting in tenofovir being less accessible to removal by ATP, opposite to what was proposed for AZT. Analyses of crystal structures of tenofovir-terminated primers/templates with HIV-1 RT to address this possibility are under way. Similarly, inefficient removal of 3TC and ddA may also occur because these nucleoside analogs favor the P site rather than the N site. It is also interesting that both tenofovir and ddA are inefficiently removed by ATP, suggesting that adenosine analogs are less likely to be removed by this mechanism than other nucleoside analogs. Our in vitro results reported here showing reduced tenofovir removal by a mutant RT with thymidine analog resistance mutations are consistent with clinical results that have demonstrated significant anti-HIV activity of tenofovir disoproxil fumarate in antiretroviral-experienced patients with extensive resistance mutations, including thymidine analog resistance mutations, in their HIV (22
; R. Schooley, R. Myers, P. Ruane, G. Beall, H. Lampiris, M. Miller, R. Mills, and I. McGowan, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 692, p. 293, 2000).