Selective Excision of AZTMP by Drug-Resistant Human Immunodeficiency Virus Reverse Transcriptase

Abstract

Two distinct mechanisms can be envisioned for resistance of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (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 zidovudine (AZT) resistance can involve the excision of the nucleoside analog after it has been incorporated into viral DNA. Although this proposal resolves some important issues, it leaves some 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. The AZT resistance 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. Steric constraints involving the azido group cause the end of an AZT 5-monophosphate-terminated primer to preferentially reside at the nucleotide binding site, which favors excision.

Molecular Mechanism by Which the K70E Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Resistance to Nucleoside Reverse Transcriptase Inhibitors

“…RT containing thymidine analog mutations (TAMs) shows an increased capacity to unblock AZT-MP-terminated primers in the presence of physiological concentrations of ATP (3,21). Since the K70E mutation reduced the ability of RT to excise AZT-MP, we hypothesized that this mutation may antagonize excision by TAMs.…”

“…HIV-1 RT has the innate capacity to unblock NRTI-MPterminated primers in the presence of physiological concentrations of ATP or PPi (1,22). Biochemical studies show that TAMs confer resistance to AZT and other NRTI by accelerating the rate at which the terminal AZT-MP (or other NRTI-MP) is removed through phosphorolytic cleavage (3,21). Both the K65R (this study; 25,29,35) and K70E (this study) mutant enzymes exhibit rates of ATP-or PPi-mediated phosphorolytic excision that are either significantly reduced or similar (in the case or PPi) to the WT enzyme.…”

Site-specific Footprinting Reveals Differences in the Translocation Status of HIV-1 Reverse Transcriptase

“…Previous biochemical studies have shown that the incorporated chain terminator can be removed from the primer terminus through phosphorolytic cleavage in the presence of either pyrophosphate (PP i ) (1) or in the presence of ribonucleotide triphosphates (NTPs) (2), which were shown to act as pyrophosphate donors. Increasing evidence suggests that the latter reaction pathway provides an important mechanism for HIV resistance to AZT and, to a certain degree, also to other NRTIs (3)(4)(5)(6)(7)(8)(9)(10)(11).…”

Pharmacokinetic–pharmacodynamic relationship of NRTIs and its connection to viral escape: An example based on zidovudine

“…Resistance against AZT-TP was attributed to two distinct mechanisms (Clavel and Hance, 2004;Boyer et al, 2001;Ray et al, 2003): (i) excision of AZT-TP from the DNA chain, and (ii) decrease in affinity of RT to AZT-TP. Both processes can be incorporated into our effect model by increasing K m .…”

“…HIV develops resistance against zidovudine by two distinct mechanisms: (i) lowering the affinity of AZT-TP to RT, and (ii) enhancing the ATP-dependent excision of incorporated AZT-TP (Clavel and Hance, 2004;Boyer et al, 2001;Ray et al, 2003). In terms of our mechanistic effect model, both processes can be accounted for by increasing K RT m,TP .…”