Thymidine Analogue Resistance Suppression By V75i Of Hiv-1 Reverse Transcriptase: Effects Of Substituting Valine 75 On Stavudine Excision And Discrimination.

Abstract

Val75 of HIV-1 reverse transcriptase (RT) plays a role in positioning the template nucleotide +1, during the formation of the ternary complex. Mutations such as V75M and V75A emerge in patients infected with HIV-1 group M-subtype B and group O variants, after failing treatment with stavudine (d4T) and other nucleoside RT inhibitors. V75I is an accessory mutation of the Q151M multi-drug resistance complex of HIV-1 RT, and is rarely associated with thymidine analogue resistance mutations (TAMs). In vitro, it confers resistance to acyclovir. TAMs confer resistance to zidovudine (AZT) and d4T by increasing the rate of ATPmediated excision of the terminal nucleotide monophosphate (primer unblocking). In a wild-type HIV-1 group O RT sequence context, V75A and V75M conferred increased excision activity on d4T-terminated primers, in the presence of pyrophosphate (PPi). In contrast, V75I decreased the PPi-mediated unblocking efficiency on AZT and d4T-terminated primers, in different sequence contexts (i.e. wild-type group M-subtype B or group O RTs). Interestingly, in the sequence context of an excision-proficient RT (i.e., M41L/A62V/T69SSS/K70R/T215Y), the introduction of V75I led to a significant decrease of its ATP-dependent excision activity on AZT-, d4T-, and acyclovirterminated primers. The excision rate of d4T-monophosphate in the presence of ATP 3.2 mM was about 10 times higher for M41L/A62V/T69SSS/K70R/T215Y than for the mutant M41L/A62V/T69SSS/K70R/- V75I/T215Y RT. The antagonistic effect of V75I with TAMs was further demonstrated in phenotypic assays. Recombinant HIV-1 containing the M41L/A62V/T69SSS/- K70R/V75I/T215Y RT showed 18.3- and 1.5-fold increased susceptibility to AZT and d4T, respectively, in comparison with virus containing the M41L/A62V/T69SSS/- K70R/T215Y RT.