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

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

doi:10.1074/jbc.m109.024026

Cited by 25 publications

(19 citation statements)

References 63 publications

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“…Pre-steady-state kinetics. Assays were performed as described previously (9,14,31). 5Ј-labeled 20Dϩ3 primer (100 nM) was heat annealed with 200 nM 42D template.…”

Section: Methodsmentioning

confidence: 99%

Mechanism of Resistance to GS-9148 Conferred by the Q151L Mutation in HIV-1 Reverse Transcriptase

Scarth

White

Chen

et al. 2011

Antimicrob Agents Chemother

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GS-9148 is an investigational phosphonate nucleotide analogue inhibitor of reverse transcriptase (RT) (NtRTI) of human immunodeficiency virus type 1 (HIV-1). This compound is an adenosine derivative with a 2,3-dihydrofuran ring structure that contains a 2-fluoro group. The resistance profile of GS-9148 is unique in that the inhibitor can select for the very rare Q151L mutation in HIV-1 RT as a pathway to resistance. Q151L is not stably selected by any of the approved nucleoside or nucleotide analogues; however, it may be a transient intermediate that leads to the related Q151M mutation, which confers resistance to multiple compounds that belong to this class of RT inhibitors. Here, we employed pre-steady-state kinetics to study the impact of Q151L on substrate and inhibitor binding and the catalytic rate of incorporation. Most importantly, we found that the Q151L mutant is unable to incorporate GS-9148 under single-turnover conditions. Interference experiments showed that the presence of GS-9148-diphosphate, i.e., the active form of the inhibitor, does not reduce the efficiency of incorporation for the natural counterpart. We therefore conclude that Q151L severely compromises binding of GS-9148-diphosphate to RT. This effect is highly specific, since we also demonstrate that another NtRTI, tenofovir, is incorporated with selectivity similar to that seen with wild-type RT. Incorporation assays with other related compounds and models based on the RT/DNA/GS-9148-diphosphate crystal structure suggest that the 2-fluoro group of GS-9148 may cause steric hindrance with the side chain of the Q151L mutant.The DNA polymerase activity of HIV-1 reverse transcriptase (RT) is targeted by nucleos(t)ide analogue RT inhibitors [N(t)RTIs], which represent the backbone of frequently used drug regimens. N(t)RTIs compete with natural deoxynucleoside triphosphate (dNTP) pools for incorporation and cause chain termination. There are currently seven approved NRTIs available for the treatment of HIV-1 infection (7). The only approved NtRTI, i.e., tenofovir, is a phosphonate with an acyclic linker attached to the adenine base (7). An investigational NtRTI, GS-9148, has recently been shown to be active against HIV-1 in cell culture and to possess a promising resistance profile in addition to a low nephrotoxic potential (4, 5). GS-9148 is the intracellularly metabolized form of the orally bioavailable phosphonate GS-9131 (29) (Fig. 1). GS-9148 undergoes two phosphorylations to become GS-9148-diphosphate (GS-9148-DP), which, like tenofovir-DP, is the active metabolite that is incorporated by HIV-1 RT. In contrast to tenofovir, GS-9148 is composed of a 2Ј,3Ј-dihydrofuran that contains a 2Ј-fluoro group (2) (Fig. 1).In vitro selection experiments in cell culture revealed the emergence of two different resistance pathways. The compound selects either for Q151L, with high-level resistance to GS-9148 (16), or for a combination of K70E, D123N, and T165I, which shows low-level resistance to . Q151L is a potential intermediate in the development of...

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“…Pre-steady-state kinetics. Assays were performed as described previously (9,14,31). 5Ј-labeled 20Dϩ3 primer (100 nM) was heat annealed with 200 nM 42D template.…”

Section: Methodsmentioning

confidence: 99%

Mechanism of Resistance to GS-9148 Conferred by the Q151L Mutation in HIV-1 Reverse Transcriptase

Scarth

White

Chen

et al. 2011

Antimicrob Agents Chemother

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“…In addition, V75I has been recently identified as a major acyclovir resistance mutation in cells co-infected with HIV-1 and herpes simplex virus (24,25). Structural analysis of HIV-1 RT showed that Val 75 plays a role in positioning the template nucleotide ϩ1, during the formation of the ternary complex (26).…”

Section: * This Work Was Supported By Spanish Ministry Of Science Andmentioning

confidence: 99%

“…impact on ATP-dependent phosphorolytic activity on primers blocked with several chain-terminating inhibitors. However, it should be noted that V75I impairs the incorporation of acyclovir-TP (25), and therefore, acyclovir excision is not expected to be relevant in vivo.…”

Section: Effects Of Substituting Val 75 In Wild-type Hiv-1 Rts On Thementioning

confidence: 99%

Thymidine Analogue Resistance Suppression by V75I of HIV-1 Reverse Transcriptase

Matamoros

Nevot

Martı́nez

et al. 2009

Journal of Biological Chemistry

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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 multidrug 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 ATP-mediated 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 PP i . In contrast, V75I decreased the PP i -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 acyclovir-terminated 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.

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“…Biochemical studies showed that mutant V75I RT did not alter binding efficiency of ACV-triphosphate but increased incorporation of dGTP versus ACV-5Ј-triphosphate through a discrimination mechanism (28). This suggests that a discrimination mechanism may also be respon- sible for the observed HIV-1 resistance to 3Ј-azido-ddC.…”

Section: Discussionmentioning

confidence: 86%

“…The V75I mutation identified in the 3Ј-azido-ddC-resistant HIV-1 has also been observed in selection experiments with acyclovir (ACV) and a monophosphorylated prodrug of ACV (9,28). Biochemical studies showed that mutant V75I RT did not alter binding efficiency of ACV-triphosphate but increased incorporation of dGTP versus ACV-5Ј-triphosphate through a discrimination mechanism (28).…”

Section: Discussionmentioning

confidence: 96%

The Base Component of 3′-Azido-2′,3′-Dideoxynucleosides Influences Resistance Mutations Selected in HIV-1 Reverse Transcriptase

Meteer¹,

Koontz²,

Asif³

et al. 2011

Antimicrob Agents Chemother

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We recently reported that HIV-1 resistant to 3-azido-3-deoxythymidine (AZT) is not cross-resistant to 3-azido-2,3-dideoxypurines. This finding suggested that the nucleoside base is a major determinant of HIV-1 resistance to nucleoside analogs. To further explore this hypothesis, we conducted in vitro selection experiments by serial passage of HIV-1 LAI in MT-2 cells in increasing concentrations of 3-azido-2,3-dideoxyguanosine (3-azido-ddG), 3-azido-2,3-dideoxycytidine (3-azido-ddC), or 3-azido-2,3-dideoxyadenosine (3-azidoddA). 3-Azido-ddG selected for virus that was 5.3-fold resistant to 3-azido-ddG compared to wild-type HIV-1 LAI passaged in the absence of drug. Population sequencing of the entire reverse transcriptase (RT) gene identified L74V, F77L, and L214F mutations in the polymerase domain and K476N and V518I mutations in the RNase H domain. However, when introduced into HIV-1 by site-directed mutagenesis, these 5 mutations only conferred ϳ2.0-fold resistance. Single-genome sequencing analyses of the selected virus revealed a complex population of mutants that all contained L74V and L214F linked to other mutations, including ones not identified during population sequencing. Recombinant HIV-1 clones containing RT derived from single sequences exhibited 3.2-to 4.0-fold 3-azido-ddG resistance. In contrast to 3-azido-ddG, 3-azido-ddC selected for the V75I mutation in HIV-1 RT that conferred 5.9-fold resistance, compared to the wild-type virus. Interestingly, we were unable to select HIV-1 that was resistant to 3-azido-ddA, even at concentrations of 3-azido-ddA that yielded high intracellular levels of 3-azido-ddA-5-triphosphate. Taken together, these findings show that the nucleoside base is a major determinant of HIV-1 resistance mechanisms that can be exploited in the design of novel nucleoside RT inhibitors.

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

Cited by 25 publications

References 63 publications

Mechanism of Resistance to GS-9148 Conferred by the Q151L Mutation in HIV-1 Reverse Transcriptase

Mechanism of Resistance to GS-9148 Conferred by the Q151L Mutation in HIV-1 Reverse Transcriptase

Thymidine Analogue Resistance Suppression by V75I of HIV-1 Reverse Transcriptase

The Base Component of 3′-Azido-2′,3′-Dideoxynucleosides Influences Resistance Mutations Selected in HIV-1 Reverse Transcriptase

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