Two distinct mechanisms can be envisioned for resistance of HIV-1 RT to nucleoside analogs: one in which the mutations interfere with the ability of HIV-1 RT to incorporate the analog, and the other in which the mutations enhance the excision of the analog after it has been incorporated. It has been clear for some time that there are mutations that selectively interfere with the incorporation of nucleoside analogs; however, it has only recently been proposed that AZT resistance can involve the excision of the nucleoside analog after it has been incorporated into viral DNA (1
). Although this proposal resolves some important issues, it leaves other questions unanswered. In particular, how do the AZT resistance mutations enhance excision, and what mechanism(s) causes the excision reaction to be relatively specific for AZT? We have used both structural and biochemical data to develop a model. In this model, several of the mutations associated with AZT resistance act primarily to enhance the binding of ATP, which is the most likely pyrophosphate donor in the in vivo excision reaction. These mutations serve to increase the affinity of RT for ATP so that, at physiological ATP concentrations, excision is reasonably efficient.
So far as we can determine, the specificity of the excision reaction for an AZT-terminated primer is not due to the mutations that confer resistance, but depends instead on the structure of the region around the HIV-1 RT polymerase active site and on its interactions with the azido group of AZT. The azido group of AZT appears to not interfere substantially with the binding of AZTTP at the N site or its incorporation into DNA. If the end of the primer is translocated to the P site, which would allow the incoming dNTP to bind at the N site, the azido group would have unfavorable interactions with D185. This steric clash could distort the end of the primer, which would also interfere with the binding of the incoming dNTP. There is biochemical support for this idea; a primer with an AZT-terminated end interferes with the ability of wild-type HIV-1 RT to form the closed complex with an incoming dNTP (15
). This could be either an effect of the azido group (the AZT end could be in the P site, but the azido group could block the incoming dNTP from binding) or it could be the consequence of the AZT-terminated 3′ end preferentially occupying the N site, which would interfere with the binding of the incoming dNTP. Whether the effect is direct or indirect, both the model and the data support the idea that, in the presence of dNTPs, an AZT-terminated primer is much more likely to be bound at the N site than is a dideoxy-terminated primer for which there is no steric hindrance. We tested this idea directly by measuring the effects of dNTPs on the excision of either a dideoxy nucleoside or AZT. The presence of the appropriate incoming dNTP interferes with the excision of a dideoxy nucleoside, but it does not interfere with the excision of AZT. This is the basis of the selectivity of resistance for AZT; as has already been mentioned, this selectivity does not appear to be caused by the mutations that cause AZT resistance, but it appears to be an inherent property of the active site of HIV-1 RT. This effect occurs at normal levels of dNTPs (micromolar). However, ATP is present at much higher concentrations (millimolar). Although ATP does not bind to the active site of RT nearly as well as the incoming dNTP does, high concentrations of ATP can affect the excision of a dideoxy nucleoside from the 3′ end of the primer.
Under selective pressure from different nucleoside analogs, HIV-1 RT can develop resistance to multiple drugs. In some cases, it appears that a single RT can carry mutations that interfere with the incorporation of certain nucleoside analogs (the lamivudine [3TC] resistance mutation, M184V, for example), as well as the mutations (M41, D67N, K70R, L210W, and T215) that specifically facilitate the excision of AZT. When the M184V mutation is introduced into an RT in either the presence or the absence of the classical AZT resistance mutations, the enzyme becomes 3TC resistant. There is also a modest increase (ca. 5- to 10-fold) in the sensitivity of the enzyme to AZT (21
); the explanation is that the M184V mutation modestly interferes with AZT excision. We propose that this is the result of an effect of the mutations at position 184 on the ability of the AZT-terminated primer to occupy the P site. As has already been discussed, if the primer has an AZT end, the incoming dNTP does not bind appropriately. Introducing an I or a V at position 184 not only changes the protein; the position of the template-primer is also altered by these mutations (18
). As described above, we believe that there is steric hindrance for an AZTMP at the end of the primer with amino acid 185. Introducing either V or I at position 184 relaxes the constraints that prevent the formation of the closed complex with the incoming dNTP when the end of the primer is AZTMP, and it decreases the unfavorable interactions with the aspartic acid at position 185. This means that the 3TC resistance mutations reduce the amount of AZT excision. The fact that the introduction of the 3TC resistance mutations affects the AZT sensitivity of HIV-1 viruses that do, or do not, carry the classical AZT resistance mutations to approximately the same degree (about 5- to 10-fold) suggests that the wild-type HIV-1 RT carries out sufficient AZT excision to significantly affect the susceptibility of the wild-type virus to AZT. These data, taken in the context of the model, also suggest that, for the wild-type enzyme, the efficiency of the excision reaction is primarily limited by the relatively weak binding of ATP; the AZT resistance mutations resolve this deficiency by enhancing ATP binding.
Both wild-type HIV-1 RT and the AZT-resistant variants can excise AZT from the 3′ end of a primer both in vitro and in vivo, which raises a question: can these enzymes also excise misincorporated nucleotides? HIV-1 RT can add an untemplated base to the primer strand after it has completely copied a template (16
). Although the addition of a nontemplated base is efficient in vitro, strand transfer points do not seem to be sites of increased mutation in vivo (24
). In agreement with published data, we found that both wild-type HIV-1 RT and the AZT-resistant mutants efficiently add a nontemplated base. However, if there is sufficient ATP present, this nontemplated base can also be removed. This suggests that the excision reaction can correct certain DNA synthesis errors, and it raises the possibility that other types of incorporation errors could be corrected by an excision mechanism. However, the in vivo mutation rate of AZT-resistant HIV-1 is slightly higher than that of wild-type HIV-1 (13
). It is possible, based on this information, that the RT excision reaction does not make a significant contribution to the fidelity of HIV-1 RT in vivo. However, it is also possible that the host DNA-dependent RNA polymerase plays a major role in determining the overall mutation rate in the HIV-1 life cycle.
There are RTs, typically from HIV-1 from patients who have been extensively treated with several nucleoside analogs, that have a large number of mutations in RT, and these viruses are broadly resistant to a variety of nucleoside analogs. In some patients, the RT has the suite of mutations associated with AZT resistance which we now believe cause excision and, in addition, it has a number of other mutations known to interfere with the incorporation of nucleoside analogs. Because some of these RTs have a large number of mutations, it is not a simple matter to predict the precise mechanism(s) of resistance associated with individual nucleoside analogs; however, it is possible, at least for some nucleoside analogs, that resistance is the result of the combined effects of a decreased ability to incorporate the analog and an enhanced ability to excise the analog after it has been incorporated.