Background:  4’-Ethynyl D4T (4’-Ed4T), a novel thymidine analog, exhibits a 5~10-fold higher anti-HIV-1 activity and less cytotoxic than its progenitor (D4T). The triphosphate (4’-Ed4TTP) competitively inhibits HIV-1 reverse transcriptase (RT). 4’-Ed4TTP inhibits the RT M184V mutant with 3-fold less efficiency than the wild type. This is consistent with in vitro drug susceptibility studies, which showed that the M184V virus (IIIB strain) conferred 3- to 10-fold resistance to 4’-Ed4T. Upon long-term selection of resistant virus (IIIB strain) to 4’-Ed4T, the virus strain with P119S/T165A/M184V mutations in RT was isolated. In order to assess the role of P119S, T165A, and M184V mutations in the susceptibility of virus to 4’Ed4T, we engineered a series of RT and viral strains with these mutations in NL4-3 background instead of IIIB since both strains are sensitive to 4’-Ed4T. The contribution of these mutations to RT enzyme behavior, virus sensitivity to 4’-Ed4T and viral growth were examined.

Methods:  A steady-state single nucleotide incorporation assays was carried out with wild type and mutant RT to determine the K_m and k_cat values for dTTP incorporation as well as the inhibition constant K_i for 4’-Ed4TTP against dTTP. For antiviral susceptibility studies, TZM-bl cells were infected with the wt or mutant strains in serial concentrations of 4’-Ed4T to calculate the EC_50.  

Results:  The DNA polymerase activity of the mutant RT was comparable to that of wild type RT. The P119S, T165A, and P119S/T165A mutants showed no resistance to 4’-Ed4TTP, while the M184V mutant showed 3-fold resistance. The P119S/M184V and T165A/M184V mutants showed 8-, and 13-fold resistance, respectively. The P119S/T165A/M184V mutant conferred only 11-fold resistance, close to the double mutants. The P119S, T165A, and P119S/T165A viral strains had growth rates similar to wild type strain, and showed no resistance to 4’-Ed4T. The M184V strain conferred 3-fold resistance, while the combination of P119S and M184V doubled the M184V resistance. The growth of T165A/M184V and P119S/T165A/M184V strains was severely compromised.

Conclusions:  The RT enzymatic and growth assays demonstrated the resistance of P119S/M184V mutation to 4’-Ed4T. The T165A/M184V and P119S/T165A/M184V RT mutants showed increased resistance to 4’-Ed4TTP but the virus failed to grow. The ability of the triple mutant to grow in IIIB but not in NL4-3 background may suggest that the evolution to 4’-Ed4T resistance may be strain specific.