XI International HIV Drug Resistance Workshop: Basic Principles and Clinical Implications


Seville, Spain, 2–5 July 2002


MECHANISMS OF NUCLEOSIDE ANALOGUE RESISTANCE

Antivir Ther. 2002;7(Suppl 1):S25 (abstract no. 27)

PL Boyer1, SG Sarafianos2, E Arnold2 and SH Hughes1
1HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, Md., USA; and 2Center for Advanced Biotechnology and Medicine (CABM) and Rutgers University Chemistry and Chemical Biology Department, Piscataway, NJ, USA

BACKGROUND: HIV-1 resistance to nucleoside analogues requires increased discrimination between normal nucleosides and the analogues. This enhanced discrimination can occur either at the time of incorporation, which is the mechanism of lamivudine resistance, or can involve enhanced excision of an incorporated analogue, which is the basis of zidovudine resistance. We developed a model to explain the enhanced excision of zidovudine-MP. In the model, ATP is the pyrophosphate donor and several of the well-known zidovudine-resistance mutations enhance ATP binding, thus promoting excision. With the classic zidovudine resistance mutations, resistance is specific for zidovudine; however, insertions in the fingers subdomain cause resistance to a number of nucleoside analogues.

METHODS/RESULTS: In the model we proposed for zidovudine resistance, zidovudine-MP is selectively excised because steric hindrance with the azido group interferes with the formation of a stable closed ternary complex. We used X-ray crystallography to solve the structures of two HIV-1 reverse transcriptase (RT)/DNA complexes which have zidovudine-MP at the 5´ end of the primer strand. These structures have the zidovudine-MP terminated primer bound either at: 1) at the P (or priming) site (3.1 Å resolution), or 2) at the N (or nucleoside binding) site (3.0 Å resolution). We also measured the effects of insertions in the fingers subdomain on the incorporation of a number of triphosphate analogues and their subsequent excision using in vitro assays. The fingers insertions cause resistance to some analogues by interfering with their incorporation (for example, lamivudine-TP). However, in concert with the T215Y mutation, the fingers insertions also lead to the enhanced excision of a number of nucleoside analogues.

CONCLUSIONS: The structures of HIV-1 RT in complex with a DNA with an zidovudine-MP terminated primer at either the N or the P site have allowed us to refine our model for zidovudine excision. The long azido group does not block the translocation of an zidovudine-MP terminated primer to the P site, but does interfere with the formation of the closed complex. In contrast to the classic zidovudine resistance mutations, which are quite selective for zidovudine- MP excision, the fingers insertions (together with T215Y) show enhanced excision with a number of nucleoside analogues. This is due, at least in part, to an overall decrease in the stability of the closed complex, which gives the end of the primer better access to the N site, favouring excision.

PRESENTING AUTHOR: SH Hughes

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2002-07-02
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