Biochemical Mechanism of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Resistance to Stavudine

Abstract

We have found a close correlation between viral stavudine (d4T) resistance and resistance to d4T-triphosphate at the human immunodeficiency virus type 1 reverse transcriptase (RT) level. RT from site-directed mutants with 69S-XX codon insertions and/or conventional zidovudine resistance mutations seems to be involved in an ATP-dependent resistance mechanism analogous to pyrophosphorolysis, whereas the mechanism for RT with the Q151M or V75T mutation appears to be independent of added ATP for reducing binding to d4T-triphosphate.Although the use of nucleoside analog reverse transcriptase (RT) inhibitors (NRTIs) (in combination with nonnucleoside RT inhibitors and/or protease inhibitors) has proved highly successful in human immunodeficiency virus type 1 (HIV-1) therapy, this has not eliminated the selection of drug-resistant isolates (13,22,25). The mutational profile of these resistant viruses usually reflects which drugs have been used. For instance, the mutation M184V in RT results in specific and high-level resistance to lamivudine (24), and the changes at codons 41, 67, 70, 210, 215, and 219 contribute to high-level zidovudine (AZT) resistance (4,8,14). However, patient virus samples that are stavudine (d4T) resistant are also cross resistant to other nucleoside analogs rather than having d4T-specific resistance, and the magnitude of d4T resistance is usually low (6, 25). The biochemical mechanism of HIV-1 RT resistance to NRTIs is due in many cases to decreased binding of mutated RT to the respective NRTI-triphosphates (NRTITPs) (9). However, this does not appear to be the situation for AZT resistance, as no significant differences in discrimination between AZT-TP and dTTP in the polymerase reaction are seen when studying RTs with conventional AZT resistance the mutation (9,11,16). Recently, a new biochemical mechanism of AZT resistance has been reported where the RTs containing mutations D67N, K70R, T215F, or K219Q are able to remove AZT-monophosphate (AZT-MP) from blocked primers more efficiently than wild-type RT, thereby enabling resumption of DNA synthesis. This has been demonstrated to occur by either pyrophosphate-dependent pyrophosphorolysis (1) or by an ATP-mediated mechanism similar to pyrophosphorolysis, in which ATP at a physiological concentration (3.2 mM) acts as an acceptor to form, together with AZT-MP, a dinucleoside polyphosphate product (19). We have recently reported a close correlation between resistance to AZT-TP at the enzyme level and viral AZT resistance (16a). When we studied 11 mutants with either conventional AZT resistance mutations or multidrug resistance mutations (T69S-XX insertions or Q151M), we found this biochemical resistance mechanism to be mainly ATP dependent, with the exception of the Q151M mutant. Without added ATP, all the mutants (except the Q151M mutant) behaved similarly to wild-type RT. Mutations that appeared to be dominant in the ATP-dependent reaction were those in the ␤2-␤3 loop in the RT "fingers" domain, i.e., D67N, K70R, and insertions T69S-SG and T69S-AG...

Mechanistic Studies to Understand the Progressive Development of Resistance in Human Immunodeficiency Virus Type 1 Reverse Transcriptase to Abacavir

“…This makes sense, considering the intrinsic relationship between the two processes and would suggest that a mutant that mediates resistance by removal would not show dramatic reductions in the incorporation of an analog. In support of this hypothesis, studies on RT AZTR , which appears to confer resistance by removing chain-terminating AZTMP (34,36,37,40) and D4TMP (39,68,69), have shown very little or no reduction in the incorporation of AZTMP (25,35). Consistent with previous reports suggesting that AZT resistance mutations cause resistance by increasing the rate of dNMP removal a role in resistance to abacavir.…”

“…Interestingly, RT Q151M also showed an elevated rate of ATP-mediated removal of dGMP; however, it was slightly slower than RT WT at removing CBVMP. This suggests that removal may play a role in Q151M-mediated resistance to some compounds, although studies have suggested removal to be unimportant in its resistance to AZT (40) or D4T (68).…”

“…Despite a mechanistic understanding of M184V's resistance to 3TC (20,24,26,72) and a growing amount of information on the AZT resistance mutations, resistance to AZT (24, 34 -37, 40) and D4T (39,68,69), the mechanisms of many mutations and combinations of mutations are still poorly understood. In this report, we show that all but one of the tested mutations selected for by abacavir in cell culture increase selectivity against CBVTP.…”

Effects of the Δ67 Complex of Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Nucleoside Analog Excision

“…However, the level of resistance needed to have a clinical effect against d4T is small (1.4-fold) (32), and it has been reported that mutations causing resistance to AZT can cause cross-resistance to d4T (12,19,23,32,35). At high dNTP concentrations, which favor the formation of the ternary complex and inhibit the excision reaction, SSGR/T215Y excises d4TMP to a much greater extent than wild-type RT.…”

“…There is evidence to suggest that AZT resistance mutations cause low-level resistance to stavudine (d4T), which can be important clinically (19,23,32). We used the excision-extension assay to examine the ability of wild-type HIV-1 RT and the RT variants to excise d4TMP.…”