Background:  The K65R and L74V mutations in HIV-1 RT share several characteristics, i.e. diminished incorporation of relevant nucleoside reverse transcriptase inhibitors (NRTI), impaired rescue of zidovudine (ZDV)-terminated DNA primers and decreased viral replication capacity. However, the occurrence of viruses containing both K65R and L74V is extremely infrequent, possibly due to a poor ability of K65R/L74V RT to use natural deoxyribonucleotide triphosphates (dNTP) and the rapidity of  65R ® K reversion in the absence of selection pressure. We have studied purified RT containing K65R, L74V, or both to try to understand resistance to tenofovir diphosphate (TDF-DP) and ddATP. We also studied the efficiency of excision of ZDV-terminated primers and initiation of synthesis of (-)ssDNA.

Methods:  IC₅₀ and fold-resistance (FR) for TDF-DP and ddATP were determined for recombinant wild type, L74V, M184V, K65R RTs and related combinations in cell-free assays. ATP-dependent excision of ZDV-terminated DNA primers was studied at a single template position with either wt or mutated RT. Initiation of (-)ssDNA synthesis was performed in the presence of tRNA₃^Lys/HIV-1 RNA, wild type or mutated RT, dNTP/ddATP and resolved in gel-based assays.

Results:  ATP-dependent excision of ZDV-terminated primer was:  wild type > L74V > K65R = K65R/L74V > K65R/L74V/M184V RT. Efficiency of initiation of (-)ssDNA synthesis was: wt > L74V ≈ M184V > K65R ≥ K65R/L74V > K65R/M184V ≈ K65R/L74V/M184V RTs. The latter 2 RT showed the maximum decrease in product formation and accumulation of DNA at the +3 pausing site. However, the efficiency of initiation of K65R/L74V RT was slightly lower than that of  K65R RT.

Conclusions:  The simultaneous presence of K65R and L74V may have an additive effect in regard to resistance to TDF-DP. Addition of M184V yielded resensitization to TDF-DP, but not to ddATP. K65R/L74V/M184V RT displayed the highest decrease in susceptibility to ddATP. Although K65R/L74V RT is found infrequently in clinical isolates, this doubly mutated enzyme displays high level resistance to both TDF-DP and ddATP in cell-free assays, severely impairs rescue of ZDV-terminated primers and is inefficient in synthesis of (-)ssDNA.