Mechanisms Associated with HIV-1 Resistance to Acyclovir by the V75I Mutation in Reverse Transcriptase

Egor P Tchesnokov,Aleksandr Obikhod,Ivana Massud,Andrea Lisco,Christophe Vanpouille,Beda Brichacek,Jan Balzarini,Christopher Mcguigan,Marco Derudas,Leonid Margolis,Raymond F Schinazi,Matthias Götte

doi:10.1074/jbc.m109.024026

Abstract

It has recently been demonstrated that the anti-herpetic drug acyclovir (ACV) also displays antiviral activity against the human immunodeficiency virus type 1 (HIV-1). The triphosphate form of ACV is accepted by HIV-1 reverse transcriptase (RT), and subsequent incorporation leads to classical chain termination. Like all approved nucleoside analogue RT inhibitors (NRTIs), the selective pressure of ACV is associated with the emergence of resistance. The V75I mutation in HIV-1 RT appears to be dominant in this regard. By itself, this mutation is usually not associated with resistance to currently approved NRTIs. Here we studied the underlying biochemical mechanism. We demonstrate that V75I is also selected under the selective pressure of a monophosphorylated prodrug that was designed to bypass the bottleneck in drug activation to the triphosphate form (ACV-TP). Pre-steady-state kinetics reveal that V75I discriminates against the inhibitor at the level of catalysis, whereas binding of the inhibitor remains largely unaffected. The incorporated ACV-monophosphate (ACV-MP) is vulnerable to excision in the presence of the pyrophosphate donor ATP. V75I compromises binding of the next nucleotide that can otherwise provide a certain degree of protection from excision. Collectively, the results of this study suggest that ACV is sensitive to two different resistance pathways, which warrants further investigation regarding the detailed resistance profile of ACV. Such studies will be crucial in assessing the potential clinical utility of ACV and its derivatives in combination with established NRTIs.

Highlights

Acyclovir (ACV)4 (Fig. 1, right) was developed decades ago as one of the first selective antiviral agents, and it is still used in the
Mutations M184V and T69N are other previously known resistance-conferring mutations that emerged under the selective pressure of ACV; V75I outgrew the culture over protracted periods of time, suggesting that this mutation is strongly associated with ACV resistance
Sequencing of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) revealed the emergence of V75I, and phenotypic resistance measurements revealed a 15-fold increase in the EC50 value (Table 1)

Summary

Introduction

Acyclovir (ACV) (Fig. 1, right) was developed decades ago as one of the first selective antiviral agents, and it is still used in the. Like most other nucleoside analogues (14 –16), the incorporated ACV-MP can be excised from the 3Ј-end of the primer in the presence of PPi or the PPi-donor ATP [10] This reaction can reduce the overall inhibitory effect; the removal of the chain terminator can be blocked through formation of a DEC [17]. The first mechanism is based on substrate discrimination In this case the mutant enzyme can selectively diminish binding and/or incorporation of the nucleotide analogue, whereas the properties of the natural counterpart remain largely unaffected. The second major resistance mechanism associated with NRTIs is based on excision In this case, the mutant enzyme can increase the rate of excision of the incorporated inhibitor. Thymidine analogue-associated mutations (TAMs) were shown to be able to recruit ATP as a PPi donor and increase excision of incorporated AZT-MP [17]. The data suggest that ACV is vulnerable to both major resistance mechanisms

Results

Discussion

Conclusion