Background:  We recently reported that I132M in the b7-b8 loop of the p51 subunit of HIV-1 reverse transcriptase (RT) confers significant resistance (>20-fold) to the NNRTI, nevirapine and delavirdine. However, this mutation is only rarely detected in NNRTI-experienced HIV-infected individuals. Accordingly, in the current study we have carried out in-depth virological and biochemical analyses to further characterize this mutation.

Methods:  Replication kinetics and drug susceptibility analyses of HIV-1_LAI containing I132M were evaluated in cellular assays. Recombinant HIV-1 RT containing I132M was also generated and subjected to detailed steady-state and pre-steady-state kinetic analyses.

Results:  The replication kinetics of HIV-1 containing I132M was severely impaired in T cells, as was the DNA polymerase activity of recombinant I132M HIV-1 RT. However, the acquisition of the A62V or L214I mutations partially or completely compensated for the observed decrease in the viral replication kinetics of I132M HIV-1. Surprisingly, we also found that I132M HIV-1 conferred marked hypersusceptibility to the nucleoside analogs (NRTI), tenofovir and lamivudine, both at the virus and enzyme level. Subunit specific mutagenesis experiments revealed that I132M in the p51 subunit of RT was responsible for the observed NRTI hypersusceptibility and NNRTI resistance. Pre-steady-state kinetic experiments demonstrated that I132M HIV-1 RT bound the natural dCTP substrate with less affinity, but lamivudine-triphosphate with increased affinity, in comparison with the wild type enzyme, thus providing a mechanistic explanation for the observed NRTI hypersusceptibility.

Conclusions:  Taken together, these data help to explain why I132M is infrequently selected by either nevirapine- or delavirdine-containing regimens, and furthermore demonstrate that a single mutation outside of the polymerase active site and in the p51 subunit of HIV-1 RT can significantly influence nucleotide selectivity.