The Base Component of 3′-Azido-2′,3′-Dideoxynucleosides Influences Resistance Mutations Selected in HIV-1 Reverse Transcriptase

Abstract

We recently reported that HIV-1 resistant to 3-azido-3-deoxythymidine (AZT) is not cross-resistant to 3-azido-2,3-dideoxypurines. This finding suggested that the nucleoside base is a major determinant of HIV-1 resistance to nucleoside analogs. To further explore this hypothesis, we conducted in vitro selection experiments by serial passage of HIV-1 LAI in MT-2 cells in increasing concentrations of 3-azido-2,3-dideoxyguanosine (3-azido-ddG), 3-azido-2,3-dideoxycytidine (3-azido-ddC), or 3-azido-2,3-dideoxyadenosine (3-azidoddA). 3-Azido-ddG selected for virus that was 5.3-fold resistant to 3-azido-ddG compared to wild-type HIV-1 LAI passaged in the absence of drug. Population sequencing of the entire reverse transcriptase (RT) gene identified L74V, F77L, and L214F mutations in the polymerase domain and K476N and V518I mutations in the RNase H domain. However, when introduced into HIV-1 by site-directed mutagenesis, these 5 mutations only conferred ϳ2.0-fold resistance. Single-genome sequencing analyses of the selected virus revealed a complex population of mutants that all contained L74V and L214F linked to other mutations, including ones not identified during population sequencing. Recombinant HIV-1 clones containing RT derived from single sequences exhibited 3.2-to 4.0-fold 3-azido-ddG resistance. In contrast to 3-azido-ddG, 3-azido-ddC selected for the V75I mutation in HIV-1 RT that conferred 5.9-fold resistance, compared to the wild-type virus. Interestingly, we were unable to select HIV-1 that was resistant to 3-azido-ddA, even at concentrations of 3-azido-ddA that yielded high intracellular levels of 3-azido-ddA-5-triphosphate. Taken together, these findings show that the nucleoside base is a major determinant of HIV-1 resistance mechanisms that can be exploited in the design of novel nucleoside RT inhibitors.

Molecular mechanism of HIV-1 resistance to 3′-azido-2′,3′-dideoxyguanosine

“…L214F and R277K are polymorphisms that exist in both treatment-naïve and -experienced individuals, but both mutations have been associated with NRTI resistance (Puertas et al, 2009, Garriga et al, 2009 and Betancor et al, 2010). However, in antiviral drug-susceptibility assays the F77L and L214F mutations did not significantly appear to increase 3’-azido-ddG resistance in viruses containing L74V (Meteer et al, 2011). …”

“…As described in the Materials and Methods, we cloned a single-genome-derived full-length RT sequence (SGS3) derived from a 3’-azido-ddG resistant virus population selected in vitro (Meteer et al, 2011) that contained the L74V/F77L/V106I/L214F/R277K/K476N mutations into the p6HRT-PROT prokaryotic expression vector. We also generated 2 additional constructs that lacked either the L74V mutation (SGS3-L74V) or the K476N mutation (SGS3-K476N).…”

“…We previously reported that HIV-1 containing the L74V mutation, or L74V in combination with F77L, L214F, K476N and/or V518I, exhibited only a ~ 2.0-fold decrease in susceptibility to 3’-azido-ddG (Meteer et al, 2011). However, if we generated HIV-1 clones containing single-genome-derived full-length RT sequences from the 3’-azido-ddG resistant virus population selected in vitro (Meteer et al, 2011), the recombinant virus yielded higher levels of 3’-azido-ddG resistance (range 3.2 to 4.0-fold).…”

“…However, if we generated HIV-1 clones containing single-genome-derived full-length RT sequences from the 3’-azido-ddG resistant virus population selected in vitro (Meteer et al, 2011), the recombinant virus yielded higher levels of 3’-azido-ddG resistance (range 3.2 to 4.0-fold). Therefore, in this study we cloned into the p6HRT-PROT prokaryotic expression vector (Le Grice and Grüninger-Leitch, 1990) one of these single-genome-derived full-length RT sequences (SGS3) that contained L74V/F77L/V106I/L214F/R277K/K476N mutations.…”

“…This finding suggested that the 3’-azido-2’,3’-dideoxynucleoside base was a major determinant of HIV-1 resistance. To further explore this hypothesis, we conducted in vitro selection experiments by serial passage of HIV-1 LAI in MT-2 cells in the presence of increasing concentrations of 3’-azido-ddG (Meteer et al, 2011). 3’-Azido-ddG selected for virus that was 5.3-fold resistant to the nucleoside compared to wildtype (WT) HIV-1 LAI passaged in the absence of drug.…”