17th International HIV Drug Resistance Workshop 10-14 June 2008, Sitges, Spain

Antivir Ther. 2008; 13(Suppl. 3):A5 (abstract no. 3)

J Meteer¹, D Koontz¹, KL Rapp², M Detorio², M Ruckstuhl², RF Schinazi² and JW Mellors^1
1University of Pittsburgh, Pittsburgh, PA, USA ²Emory University and VA Medical Center, Atlanta, GA, USA

BACKGROUND: Base modifications of nucleoside reverse transcriptase inhibitors (NRTI) influence their incorporation, excision, antiretroviral activity and resistance profile. For example, 3′-azido-2′,3′-dideoxyguanosine (AZG) exhibits potent activity against zidovudine (AZT)-resistant HIV-1 and AZG-5′-monophosphate is poorly excised from terminated primers by reverse transcriptase (RT) that readily excises AZT-5′-monophosphate. Mutations that confer resistance to AZG have not been identified. Here we selected AZG-resistant HIV-1 by serial in vitro passage.

METHODS: Wild-type HIV-1 was passaged in increasing concentrations of AZG in MT-2 and human peripheral blood mononuclear cells (PBMCs). Passaged virus was assayed for drug susceptibility to AZG and other NRTI in P4/R5 or PBMCs. The entire coding region of RT was amplified and sequenced from wild type and AZG-resistant virus to identify mutations that were selected.

RESULTS: After 90 passages in MT-2 cells, virus was 10.9-fold resistant to AZG (EC₅₀=9.2 µM) compared with wild type (0.84 µM). AZG-resistant virus showed reduced susceptibility to lamivudine (14.3-fold), AZT (5.6-fold), didanosine (4.3-fold), stavudine (3.5-fold), abacavir (3.4fold) and tenofovir (1.8-fold). Whole RT sequencing identified the following mutations compared with the starting wild-type virus: L74V, F77L, V106I, L214F and K476N. The K476N mutation is located in the RNase H primer grip of RT. After 70 weeks of AZG selection in PBMCs, virus was selected that encoded V75I, F77L and H221Y. Site-directed mutagenesis showed that the triple mutant exhibited 1.5-fold reduction in susceptibility to AZG.

CONCLUSIONS: AZG selects a novel combination of mutations in the polymerase and RNase H domains of RT that do not include classical thymidine analogue mutations (TAMs). These findings are consistent with the lack of cross-resistance of viruses containing TAMs to AZG and inefficient excision of AZG-5′-monophosphate by RT with TAMs. This study also provides further evidence that the primary determinant for the selection of TAMs is the base structure and not the 3′-azido group on the ribose sugar.

2008-06-10
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