699    

Background:  Thymidine analog resistance mutations (TAM) in HIV-1 reverse transcriptase (RT) confer resistance to zidovudine (AZT) by increasing the rate of ATP-dependent phosphorolysis of the terminal nucleotide monophosphate (primer unblocking). These mutations occur in one of 2 clusters: M41L/L210W/T215Y, or D67N/K70R/K219Q (or E); presence of all six mutations confers high-level AZT resistance. The didanosine resistance mutation (L74V) partially restores AZT susceptibility in HIV-1 carrying TAM. To determine the extent to which the 74V mutation reverses the effect of TAM on primer unblocking, we studied purified recombinant RT carrying various combinations of TAM, and compared the effect of L74V to that of the M184V mutation for lamivudine (3TC) resistance.

Methods:  TAM (41L, 67N, 70R, 210W, 215Y, and 219Q), 74V, and 184V were introduced into p51 and p66 subunits of wild type HIV-1 RT (HXB2) by site-directed mutagenesis. Recombinant RT was purified using a double-tag strategy. ATP-dependent primer unblocking was assayed using an AZT-terminated ³²P-labeled primer (PPT-18) hybridized to a DNA template (PPT-57). Rescue of AZT-terminated DNA was performed in the presence of 3.5 mM ATP and 100 µM dTTP, dGTP, and ddATP. Reaction products were resolved on 8% polyacrylamide-7M urea gels, analyzed on a phosphorimager, and bands quantified using ImageQuant software. All experiments were performed in triplicate.

Results:  Presence of L74V in an otherwise wild type RT reduced ATP-mediated primer unblocking to a degree similar to that observed with the M184V mutation. RT carrying different TAM (41L/210W/215Y, 67N/70R/219Q, or 41L/67N/70R/210W/219Q) showed increased primer unblocking as expected. Introduction of 74V into RT carrying 67N/70R/219Q significantly reduced primer unblocking, partially reversing the effect of TAMs; similar effects were observed when 184V was introduced into the 67N/70R/219Q enzyme. Smaller effects of 74V were observed in RT with 41L/210W/215Y and in RT with all 6 TAM. By contrast, 184V reduced the rate of ATP-mediated primer unblocking to wild-type levels when introduced into the 41L/210W/215Y enzyme; the effect of 184V on reducing primer unblocking in RT carrying all 6 TAM was comparable to that of 74V.  

Conclusions:  Both 74V and 184V mutations reverse the effect of TAM on ATP-mediated primer unblocking in HIV-1 RT. The effect of 184V was greatest in viruses carrying the 41L/210W/215Y combination of TAM.

Keywords: HIV-1 Drug Resistance; TAMs; Primer unblocking