The L74V mutation in human immunodeficiency virus type 1 reverse transcriptase counteracts enhanced excision of zidovudine monophosphate associated with thymidine analog resistance mutations

Abstract

Thymidine analog mutations (TAMs) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) confer resistance to zidovudine (AZT) by increasing the rate of ATP-dependent phosphorolysis of the terminal nucleotide monophosphate (primer unblocking). By contrast, the L74V mutation, which confers resistance to didanosine, sensitizes HIV-1 to AZT and partially restores AZT susceptibility when present together with one or more TAMs. To compare rates of primer unblocking in RTs carrying different clusters of TAMs and to explore the biochemical mechanism by which L74V affects AZT susceptibility, ATP-mediated rescue of AZT-blocked DNA synthesis was assayed using a series of purified recombinant RTs. Rates of primer unblocking were higher in the 67N/70R/219Q RT than in the 41L/210W/215Y enzyme and were similar to rates observed with an RT carrying six TAMs (41L/67N/70R/210W/215Y/219Q). The presence of 74V in an otherwise wild-type RT reduced the rate of primer unblocking to a degree similar to that observed with the M184V mutation for lamivudine resistance, which also sensitizes HIV-1 to AZT. Introduction of 74V into RTs carrying TAMs partially counteracted the effect of TAMs on the rate of primer unblocking. The effect of 74V was less marked than that of the 184V mutation in the 67N/70R/219Q and 41L/210W/215Y RTs but similar in the RT carrying six TAMs. These results demonstrate that L74V enhances AZT susceptibility by reducing the extent of its removal by ATP-dependent phosphorolysis and provides further evidence for a common mechanism by which mutations conferring resistance to didanosine and lamivudine sensitize HIV-1 to AZT.

FIG. 1.

Effect of thymidine analog resistance mutations on ATP-mediated rescue of an AZT-blocked primer. A. Diagram depicting the three-step primer unblocking strategy used to monitor unblocking of an AZT-terminated primer. Z represents the incorporated AZT monophosphate at position +2 (terminated primer); +5 describes the position at which the unblocking reaction is terminated (rescued primer). In step I, the ³²P-labeled primer PPT-18 hybridized to the template. In step II, the primer is extended by 2 nucleotides until AZT-MP is incorporated. In step III, addition of ATP, dGTP, dTTP, and ddATP at the indicated concentrations results in excision of AZT and resumption of DNA synthesis until incorporation of ddATP at position +5. B. Representative sequencing gels showing the rescue of the AZT-terminated primer for wild-type (WT), 41L/210W/215Y, 67N/70R/219Q, and 6-TAM RTs at the indicated times; P shows the position of migration of the primer. C. The percentages of terminated primer over time were determined for the wild-type (•), 41L/210W/215Y (□), 67N/70R/219Q (▴), and 6-TAM (⋄) RTs. Means and standard deviations from three experiments are shown; curves connecting the points represent the best fit for exponential decay.

FIG. 2.

Effect of the L74V and M184V mutations on ATP-mediated rescue of an AZT-blocked primer. Reaction conditions were as in Fig. 1. A. Representative sequencing gels showing the rescue of the AZT-terminated primer for wild-type (WT), 74V, and 184V RTs at the indicated times. P shows the position for the migration of the primer. B. The percentages of terminated primer over time were determined for wild-type (•), 74V (○), and 184V (▴) RTs. Means and standard deviations from three experiments are shown; curves connecting the points represent the best fit for exponential decay. Some standard deviation bars are not visible due to small experimental variations.

FIG. 3.

Effect of 184V on ATP-mediated primer rescue in the context of TAMs. Representative sequencing gels showing the rescue of the AZT-terminated primer for wild-type (WT) and mutant RTs at the indicated times (P shows the position for the migration of the primer). Means and standard deviations from three experiments are shown; curves connecting the points represent the best fit for exponential decay. A. Wild-type (•) versus 41L/210W/215Y (▪) versus 41L/210W/215Y/184V (□) RTs; B. wild-type (•) versus 67N/70R/219Q (▴) versus 67N/70R/219Q/184V (▵) RTs; C. wild-type (•) versus 6-TAM (♦) versus 6-TAM plus 184V (⋄) RTs.

FIG. 4.

Effect of 74V on ATP-mediated primer rescue in the context of TAMs. Representative sequencing gels showing the rescue of the AZT-terminated primer for wild-type (WT) and mutant RTs at the indicated times (P shows the position for the migration of the primer). Means and standard deviations from three experiments are shown; curves connecting the points represent the best fit for exponential decay. A. Wild-type (•) versus 41L/210W/215Y (▪) versus 41L/210W/215Y/74V (□) RTs; B. wild-type (•) versus 67N/70R/219Q (▴) versus 67N/70R/219Q/74V (▵) RTs; C. wild-type (•) versus 6-TAM (♦) versus 6-TAM plus 74V (⋄) RTs.