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Antimicrob. Agents Chemother. doi:10.1128/AAC.00314-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

EFFECTS OF THE TRANSLOCATION STATUS OF HIV-1 REVERSE TRANSCRIPTASE ON THE EFFICIENCY OF EXCISION OF TENOFOVIR

From the Departments of Microbiology & Immunology and Medicine, and Biochemistry McGill University, Montréal, Québec, Canada; Gilead Sciences, Inc., Foster City, CA 94404, USA

^* To whom correspondence should be addressed. Email: matthias.gotte{at}mcgill.ca.

	Abstract

The ATP-dependent phosphorolytic excision of nucleoside analogue reverse transcriptase inhibitors can diminish their inhibitory effects on human immunodeficiency virus replication. Previous studies have shown that excision can only occur when the reverse transcriptase complex exists in its pre-translocational state. Binding of the next complementary nucleotide causes the formation of a stable dead-end complex in the post-translocational state, which blocks the excision reaction. To provide mechanistic insight into the excision of the acyclic phosphonate nucleotide analog tenofovir, we compared the efficiency of the reaction with changes in the translocation status of the enzyme. We found that rates of excision of tenofovir with wild-type reverse transcriptase can be as high as seen with 3'-azido-3'-deoxythymidine monophosphate (AZT-MP). Thymidine-associated mutations, which confer >100-fold and 3-fold decreased susceptibility to AZT and tenofovir, respectively, caused substantial increases in the efficiency of excision of both inhibitors. However, in contrast to AZT-MP, the removal of tenofovir is highly sensitive to dead-end complex formation. Site-specific footprinting experiments revealed that complexes with AZT-terminated primers exist predominantly pre-translocation. In contrast, complexes with tenofovir-terminated primers are seen in both configurations. Low concentrations of the next nucleotide are sufficient to trap the complex post-translocation despite the flexible, acyclic character of the compound. Thus, the relatively high rate of excision of tenofovir is partially neutralized by the facile switch to the post-translocational state and dead-end complex formation, which provides a degree of protection from excision in the cellular environment.

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