13th International HIV Drug Resistance Workshop 8–12 June 2004, Tenerife Sur-Costa Adeje, Canary Islands, Spain

BACKGROUND: In vivo, the growth of HIV-1 in the presence of chain-terminating drugs leads to the appearance of resistance mutations in reverse transcriptase (RT), such as the thymidine analogue mutations (TAMs). In vitro, TAMs are associated with increased RT-dependent removal of chain terminators. In this study, we wanted to define conditions where TAMs would provide an enzymatic advantage over WT. Experiments were carried out using immune cell extracts or mixtures of ATP and PP_i to compare the excision by 67N/70R/215Y/219Q RT (RT^AZT) and wild-type RT (RT^WT).

METHODS: Cell extracts were prepared from purified primary CD4⁺ T cells. Removal of 2', 3'-dideo- xyadenosine monophosphate (ddAMP) or AZTMP was assessed by incubating 3' [³²P]-labelled ddAMP or AZTMP-terminated primer/template with either RT^AZT or RT^WT and cell extract or mixtures of ATP and PP_i.

RESULTS: The rate of excision of [³²P]ddAMP or [³²P]AZTMP from terminated primer/templates by HIV- 1 RT^AZT was 3–6-fold greater than RT^WT when the acceptor substrates were provided from an extract of unstimulated CD4⁺ T cells (PP_i, 8 µM). In contrast, no difference was observed in excision rates for RT^WT and RT^AZT when the acceptor substrates were provided by an extract of highly stimulated CD4⁺ T cells (PP_i, 79 µM). To more specifically define the ATP/PP_i conditions required for an excision advantage with the mutant enzyme, mixtures of ATP and PP_i were used as the acceptor substrates. In a mixture of 2.9 mM ATP and submicromolar concentrations of PP_i, excision of the drugs by RT^AZT occurred at about 10 times the rate of RT^WT. This difference between enzymes diminished as the PP_i levels were increased; no difference was observed when PP_i was greater than 35 µM.

CONCLUSIONS: The most significant biochemical alteration in RT^AZT in comparison with RT^WT is enhanced excision activity, yet this difference is not manifested in a setting where the PP_i-dependent reaction is greater than 35 µM. This suggests that the selection of AZT resistance occurs when PP_i concentrations are low, such as in unstimulated CD4⁺ T cells or macrophages, or under conditions that restrict the ability of PP_i to participate in the excision reaction at the site of reverse transcription.

PRESENTING AUTHOR:AJ Smith

Supported by NIH grant AI-39973 and American Heart Association Predoctoral Fellowship 0215087B.

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2004-06-08
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