Characterization of novel non-nucleoside reverse transcriptase (RT) inhibitor resistance mutations at residues 132 and 135 in the 51 kDa subunit of HIV-1 RT

Several rare and novel nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) resistance mutations were recently detected at codons 132 and 135 in RTs from clinical isolates using the yeast-based chimeric Ty1/HIV-1 RT (TyHRT) phenotypic assay. I132 and I135 form part of the beta7-beta8 loop of HIV-1 RT (residues 132-140). To elucidate the contribution of these residues in RT structure-function and drug resistance, we constructed twelve recombinant enzymes harboring mutations at codons 132, 135, 136, 137, 138, 139 and 140. Several of the mutant enzymes exhibited reduced DNA polymerase activities. Using the yeast-two-hybrid assay for HIV-1 RT dimerization we show that in some instances this decrease in enzyme activity could be attributed to the mutations, in the context of the 51 kDA (p51) subunit of HIV-1 RT, disrupting the enzyme s subunit-subunit interactions. Drug resistance analyses using purified RT, the TyHRT assay and antiviral assays demonstrated that the I132M mutation conferred high-level resistance (> 10-fold) to nevirapine and delavirdine and low level resistance ( 2 to 3-fold) to efavirenz. The I135A and I135M mutations also conferred low level NNRTI resistance ( 2-fold). Subunit selective mutagenesis studies again demonstrated that resistance was conferred via the p51 subunit of HIV-1 RT. Taken together, our results highlight a specific role of residues 132 and 135 in NNRTI resistance and a general role for residues in the beta7-beta8 loop in the stability of HIV-1 RT.