Background:  The K65R mutation in HIV-1 reverse transcriptase (RT) can be selected by abacavir (ABC), didanosine (ddI), tenofovir (TDF), and stavudine (4dT) in vivo. HIV-1 with K65R is associated with reduced susceptibility to these drugs, as well as decreased replication capacity.  Although K65R is a relatively rare mutation, when present it is often accompanied by the A62V and S68G RT mutations in HIV-1 sequence databases and in patients failing tenofovir therapy. We assessed the effects of these additional mutations on nucleoside reverse transcriptase inhibitor (NRTI) drug susceptibilities, viral replication capacity and RT enzymatic function.   

Methods:  We developed a multi-cycle growth competition assay in MT-2 cells to determine the relative replication capacity of site-directed K65R mutant HIV-1 alone or in combination with the A62V and S68G mutations. Site-directed mutant viruses were subcloned into a pair of LAI-based HIV-1 vectors with markers consisting of silent mutations in the RT gene, which were detected by allele-specific real-time polymerase chain reaction (AS-PCR). Relative fitness was assessed in the presence or absence of TDF. We also assessed NRTI susceptibilities of the mutant HIV-1. Finally, using pre-steady state analysis, we studied the incorporation kinetics of natural dNTP and NRTI di- or triphosphates using wild type and mutant RT.

Results:  The addition of A62V and S68G to K65R caused no significant change in HIV-1 resistance for TDF, ABC, ddI, or 3dT (<1.5-fold changes relative to K65R). In head-to-head growth competition assays, K65R was found to strongly impair viral replication in comparison to wild type virus. The fitness defect of K65R was partially compensated by addition of either A62V or S68G alone or in combination; however, the triple mutant K65R+A62V+S68G still showed replication defect compared to wild type. Pre-steady state kinetic analysis demonstrated that K65R resulted in a decreased rate of incorporation (K_pol) for all natural dNTP. These K_pol defects were partially restored to levels observed with wild type RT by addition of the A62V and S68G mutations with no further increase in TDF resistance observed enzymatically. 

Conclusions:  Our findings demonstrate that A62V and S68G are partial compensatory mutations for the K65R mutation in RT by improving the viral replication capacity, which is likely due to the increased incorporation efficiency of natural dNTP. Relative to K65R, resistance levels remain unchanged for the triple mutant virus and it still remains replication impaired relative to wild type HIV-1.