A novel point mutation at position 156 of reverse transcriptase from feline immunodeficiency virus confers resistance to the combination of (-)-beta-2',3'-dideoxy-3'-thiacytidine and 3'-azido-3'-deoxythymidine

Abstract

Mutants of feline immunodeficiency virus (FIV) resistant to (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC) were selected by culturing virus in the presence of increasing stepwise concentrations of 3TC. Two plaque-purified variants were isolated from the original mutant population, and both of these mutants were resistant to 3TC. Surprisingly, these mutants were also phenotypically resistant to 3'-azido-3'-deoxythymidine (AZT) and to the combination of 3TC and AZT. Purified reverse transcriptase (RT) from one of these plaque-purified mutants was resistant to the 5'-triphosphates of 3TC and AZT. DNA sequence analysis of the RT-encoding region of the pol gene amplified from the plaque-purified mutants revealed a Pro-to-Ser mutation at position 156 of RT. A site-directed mutant of FIV engineered to contain this Pro-156-Ser mutation was resistant to 3TC, AZT, and the combination of 3TC and AZT, confirming the role of the Pro-156-Ser mutation in the resistance of FIV to these two nucleoside analogs. This represents the first report of a lentiviral mutant resistant to the combination of AZT and 3TC due to a single, unique point mutation.

FIG. 1

Susceptibility to inhibition by 3TC of FIV 34TF10 (▪) and the plaque-purified 3TC-resistant mutants 3TR-3c (▵) and 3TR-7c (○). Results are from three or more experiments, with four determinations per experiment. Bars represent standard errors of the means.

FIG. 2

Nucleotide and deduced amino acid sequences of the region of the FIV pol gene surrounding position 2801. The corresponding amino acid sequence from HIV-1 is also shown for comparison. Note that HIV-1 and FIV RTs exhibit extensive homology in this region and that the FIV sequence is displaced by one residue relative to the HIV-1 sequence. Nucleotide sequence data for 3TR-3c and 3TR-7c were compared to the sequence data for FIV 34TF10. Mutations are shown in boldface.

FIG. 3

Susceptibility to inhibition by the combination of 3TC and AZT of 34TF10 (□), 3TR-3c (▪), 3TR-7c (•), FIVMet183Val (○), FIVMet183Thr (▴), and FIVPro156Ser (▵). Values in the plot represent equimolar concentrations of 3TC plus AZT. For example, a 1 μM value for the drug combination represents 1 μM 3TC plus 1 μM AZT. Results are from three experiments with four determinations per experiment. Bars represent standard errors of the means and are omitted when the error is too small to be shown.