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. 2000 Apr;74(8):3579-85.
doi: 10.1128/jvi.74.8.3579-3585.2000.

The M184V mutation in the reverse transcriptase of human immunodeficiency virus type 1 impairs rescue of chain-terminated DNA synthesis

M Götte  1 , D Arion, M A Parniak, M A Wainberg
Affiliations

The M184V mutation in the reverse transcriptase of human immunodeficiency virus type 1 impairs rescue of chain-terminated DNA synthesis

M Götte et al. J Virol. 2000 Apr.

Abstract

Nucleoside analog chain terminators such as 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxy-3'-thiacytidine (3TC) represent an important class of drugs that are used in the clinic to inhibit the reverse transcriptase (RT) of human immunodeficiency virus type 1. Recent data have suggested that mutant enzymes associated with AZT resistance are capable of removing the chain-terminating residue with much greater efficiency than wild-type RT and this may, in turn, facilitate rescue of DNA synthesis; these experiments were performed using physiological concentrations of pyrophosphate or nucleoside triphosphates, respectively. The present study demonstrates that the M184V mutation, which confers high-level resistance to 3TC, can severely compromise the removal of chain-terminating nucleotides. Pyrophosphorolysis on 3TC-terminated primer strands was not detectable with M184V-containing, as opposed to wild-type, RT, and rescue of AZT-terminated DNA synthesis was significantly decreased with the former enzyme. Thus, mutated RTs associated with resistance to AZT and 3TC possess opposing, and therefore incompatible, phenotypes in this regard. These results are consistent with tissue culture and clinical data showing sustained antiviral effects of AZT in the context of viruses that contain the M184V mutation in the RT-encoding gene.

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Figures

FIG. 1
FIG. 1
Rescue of chain-terminated DNA synthesis via pyrophosphorolysis or nucleotide-dependent primer unblocking. Pathways of the forward and back-reactions catalyzed by HIV-1 RT are shown under different reaction conditions. In the absence of chain-terminating nucleotides, DNA synthesis is practically irreversible (14) and, therefore, the back-reaction plays only a minor role at physiological concentrations of PPi. In contrast, when chain-terminating nucleotides, such as AZT-MP, are incorporated into the growing strand, the forward reaction is blocked and pyrophosphorolysis can effectively occur. The addition of normal nucleotides may finally rescue the formerly blocked polymerization process. Removal of terminal nucleotides and rescue of DNA synthesis can, alternatively, be facilitated in the presence of nucleoside triphosphates such as ATP. Mutant enzymes that confer resistance to AZT showed an increase in both pyrophosphorolysis (2) and nucleotide-dependent primer unblocking (25), suggesting a possible rescue of DNA synthesis as a mechanism involved in such resistance.
FIG. 2
FIG. 2
Pyrophosphorolysis catalyzed by wt RT and RT-M184V. The time course of pyrophosphorolytic cleavage was analyzed with the same DNA primer-template substrate that was recently employed to study mechanistic aspects of initiation of HIV plus-strand synthesis (11). (A) Time course of removal of 3TC-MP (top) and AZT-MP (bottom). Chain-terminated primers are labeled (20D) DNA-AZT and (19D) DNA-3TC. Reaction products are indicated by (19D), (18D), and (17D), with numbers referring to the nucleotide length of the primer and D designating DNA. RT–primer-template complexes were incubated with 150 μM PPi as described in Materials and Methods. Reactions were stopped at 1, 3, 6, 10, 15, 22, 30, and 45 min and analyzed on 15% polyacrylamide gels. Lanes 1 to 8 represent these reactions, and lane C is a control performed in the absence of PPi. (B, C, and D) Graphic representation of the data shown at the bottom of panel A. Panel B shows the decrease in the amount of the uncleaved primer, while C and D show the formation of the primary product and subsequent reaction products, respectively.
FIG. 3
FIG. 3
Comparison of pyrophosphate- and nucleotide-dependent rescue of DNA synthesis. (A) Schematic representation of the assay used to study these events. RT–primer-template complexes were incubated with dATP, dCTP, and AZT-TP to generate an AZT-terminated primer (step 1). Reactions were allowed to proceed for 20 min to quantitatively convert the primer strand into an AZT-terminated product (step 2). Rescue of DNA synthesis requires removal of AZT-MP and incorporation of dTMP and at least an additional nucleotide. The AZT-terminated complex was therefore incubated simultaneously with dTTP, ddGTP, and PPi or ATP (step 3). The extent of rescued DNA synthesis is thus reflected by the efficiency with which ddGTP is incorporated at position +4. (B) Rescue of AZT-terminated DNA synthesis with wt RT, RT-AZTr, and RT-M184V. Reactions were performed with 150 μM PPi (top) and 3.5 mM ATP (bottom). Lane C shows the unextended primer, and lane 1 shows the AZT-terminated primer. Lanes 2 to 10 show reactions performed for 1, 3, 6, 10, 15, 22, 30, 45, and 60 min, respectively. (C) Graphic representation of data shown in panel B, including standard deviations. Rescue of AZT-terminated DNA synthesis was compared after a 60-min reaction with PPi (light bar) and ATP (dark bar), respectively.
FIG. 4
FIG. 4
Rescue of 3TC-terminated DNA synthesis. (A) Reactions performed in the presence of 150 μM pyrophosphate with wt RT, RT-AZTr, and RT-M184V. The reaction conditions were similar to those described in the legend to Fig. 3, except that chain-terminating 3TC-MP was incorporated at template position +2 and rescue of DNA synthesis was monitored by the addition of ddTMP at position +3. It is noteworthy that incorporation of 3TC-MP was not quantitatively achieved with wt RT and RT-AZTr (see Results). (B) Graphic representation of the data shown in panel A with standard deviations. Rescue of 3TC-terminated DNA synthesis was compared after a 60-min reaction.
FIG. 4
FIG. 4
Rescue of 3TC-terminated DNA synthesis. (A) Reactions performed in the presence of 150 μM pyrophosphate with wt RT, RT-AZTr, and RT-M184V. The reaction conditions were similar to those described in the legend to Fig. 3, except that chain-terminating 3TC-MP was incorporated at template position +2 and rescue of DNA synthesis was monitored by the addition of ddTMP at position +3. It is noteworthy that incorporation of 3TC-MP was not quantitatively achieved with wt RT and RT-AZTr (see Results). (B) Graphic representation of the data shown in panel A with standard deviations. Rescue of 3TC-terminated DNA synthesis was compared after a 60-min reaction.
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