Background:  2’,3’-Didehydro-3’-deoxy-4’-ethynylthymidine (4’-Ed4T), a novel thymidine analog is active against multi-drug-resistant HIV-1. Selection in vitro of HIV resistant to 4’-Ed4T revealed that the M184V mutation confers some resistance (3- to 10-fold) to 4’-Ed4T, yet additional mutations (P119S and T165A) were required to achieve phenotypically higher resistance (40-fold). In this study, we assessed the relative contribution of these mutations (ie, increasing replication capacity or resistance) to the evolution of 4’-Ed4T resistance. 

Methods:  The mutations were engineered by site directed mutagenesis into NL4-3 background. Standard growth assay was used to study the replication capacity. Viral RNA concentrations were determined by real time polymerase chain reaction (RT-PCR) starting on post infection day 4, and then plotted on a log scale. The HIV-1 RT region was amplified from culture supernatants obtained from day 8 post-infection, and then sequenced for analysis of changes at 119, 165, and 184 codons. For the antiviral susceptibility studies, 4 X 10⁴ TZM-bl cells (containing a firefly-luciferase reporter) were infected with the wild type NL4-3 or the 4’-Ed4T mutants at an multiplicity of infection of 0.01 in serial concentrations of 4’-Ed4T, stavudine (d4T), lamivudine (3TC), and zidovudine (AZT). The EC₅₀ (µM) of the various RT inhibitors were then calculated.

Results:  The 119S, 165A, 184V, and 119S/184V mutants replicated with kinetics similar to that of the wild type NL4-3. There was delay in replication of the double mutant 165A/184V, and the 119S/165A/184V triple mutant. The mutations of all the single mutants and double mutant (119S/184V) were intact at post-infection day 10. However, the 165A/184V mutant had reverted to wild type codon at the 184 locus and the triple mutant 119S/165A/184V had reverted to wild type codons at all loci. The reversion of the triple mutant coincided with increased replication at day 10. The reversion of the double mutant 165A/184V at the 184 locus did not increase the replication until day 12 when the 165A/184V acquired the 119S mutation. In general, 4’-Ed4T, had the lowest EC₅₀ for all the stains tested in comparison to d4T and 3TC. The EC₅₀ of 4’-Ed4T and 3TC for the 184V strain were 3.4 µM and >40 µM, respectively.

Conclusions:  Our data suggest that the 184V mutation is acting as a primary mutation (increasing resistance to 4’-Ed4T), and the 119S as a secondary mutation (increasing viral fitness). Addition of the 165A mutation to virus with the 184V mutation compromised the viral fitness.