Background:  Although several nucleoside reverse transcriptase inhibitors (NRTI) (notably didanosine [ddI] and abacavir [ABC]) select K65R and L74V mutations, the double mutant K65R+L74V is rare in clinics. The reason for this rarity is still debated. In order to gain further insight, we searched the Marseille database for the frequency of K65R/L74V pattern.

Methods:  Retrospective analysis of the Marseille database that contains 7151 sequences from 3201 patients. The pol gene from patients’ viruses harboring the double mutant (pure species or genotypic mixtures) was amplified, sequenced, and cloned in pGEM T easy.

Results:  The analysis revealed that 12 of 3201 patients (0.4%) harbored the double mutant either as K65R+L74V or as K65K/R+L74V/I/L by population-based sequencing. Among these 12 patients, we selected 4 patients harboring a wild-type protease devoid of resistance mutations. Two patients received ddI then tenofovir (TDF); the 2 other patients received TDF + ABC or ddI as part of the combination regimen. All 4 patients were virological non-responders (plasma viral load from 7130 to 1 x 10⁶ copies/mL). Cloning and sequencing (average of 18 clones: 12 to 27 clones) revealed notably the following:  Patient 1 harbored 100% clones: K65R+L74V and a K65R+L74V bulk sequence. Patient 2 harbored 15% clones:  K65R+L74I; 18% clones: K65R+L74I/L+T215Y, and a K65KR/L74IL/T215TNSY bulk sequence. Patient 3 harbored 46% clones:  K65R+L74V; 54% clones:  K65R+74L and a K65R+L74LV+215T bulk sequence. Patient 4 harbored no clone of the K65R/L74V double mutant, whereas 28.5% of the clones were K65R+T215Y+74L with a K65KR/L74LV/T215CY bulk sequence.

Conclusions:  Our data from a large database confirm that the presence of K65R+L74V double mutant is extremely rare in clinics (0.4% in the studied population). However, patients harboring this double mutant exhibit a high level plasma viral load (7130 to 1 x 10⁶ copies/mL) in the absence of protease resistance mutations, which is in disagreement with the in vitro studies reporting that this double mutant is highly attenuated for replication and renders the RT dysfunctional. Our findings suggest possible compensatory mechanisms allowing for the coexistence of K65R+L74V, which remain to be established. In addition, surprisingly, this clonal analysis revealed that in 2 patients, K65R+T215Y, known to be antagonistic, can in some rare cases co-exist in the same genome.

Keywords: HIV drug resistance; L74V; K65R