Background:  We recently reported that two novel mutations, A371V and Q509L, were selected on the same viral genome with thymidine analog mutations (TAM) D67N, K70R, and T215I/F by 3’-azido-3’-dideoxythymidine (AZT) in vitro and were associated with ~1000-fold resistance to AZT. We have now clarified the effect of these mutations on AZT resistance (at the viral and enzyme level) and nucleoside reverse transcriptase inhibitor (NRTI) cross-resistance.

Methods:  Recombinant xxLAI viruses, containing the mutational patterns selected in vitro, were generated by site-specific mutagenesis and were used to determine AZT susceptibility and NRTI cross-resistance in a single cycle assay using P4/R5 cells. The ATP-mediated AZT-monophosphate (AZT-MP) excision activity for each enzyme was determined using a DNA/DNA template/primer. The relative rate of RNase H activity for each of the enzymes was also investigated.

Results:  The order in which the mutations emerged during in vitro selection and the AZT fold-resistance (fold-R) of recombinant viruses compared to wild type were: D67N/K70R (4.6 fold-R), D67N/K70R/A371V/Q509L (39 fold-R), D67N/K70R/T215I/A371V/Q509L (41 fold-R), and D67N/K70R/T215F/A371V/Q509L (934 fold-R). These data confirm that A371V and Q509L increase AZT resistance in combination with TAM. In comparison with the D67N/K70R/T215F mutant, the D67N/K70R/T215F/A371V/Q509L mutant exhibited greater cross-resistance to lamivudine (3TC) (15- vs 7-fold), abacavir (ABC) (3.0- vs 2.4-fold), and tenofovir (TDF) (2.7- vs 1.5-fold), but not to stavudine (d4T) or didanosine (ddI). The addition of T215F to RT containing D67N/K70R improved the enzyme’s ability to bind ATP (~3-fold) without changing the rate of excision. The addition of A371V/Q509L to TAM did not further influence the rates of AZT-MP excision or the enzyme’s affinity for ATP.  However, the DNA-dependent RNase H cleavage activities of enzymes containing A371V/Q509L were slowed compared with enzymes that lacked these mutations.  

Conclusions:  The A371V and Q509L mutations are co-selected on the same genome as TAM and increase AZT resistance when combined with TAM. They also confer greater cross-resistance to 3TC, ABC, and TDF. A371V and Q509L do not affect the efficiency of ATP-mediated excision reactions carried out on DNA/DNA template/primer, but do affect the rate of DNA-dependent RNase H cleavage, suggesting a possible role for A371V/Q509L in AZT-MP excision from RNA/DNA template/primer.