Implication of the tRNA Initiation Step for Human Immunodeficiency Virus Type 1 Reverse Transcriptase in the Mechanism of 3‘-Azido-3‘-deoxythymidine (AZT) Resistance

Abstract

There is a lack of correlation between biochemical studies and the observed clinical resistance of AIDS patients on long-term AZT therapy. Mutant HIV-1 reverse transcriptase in the viral isolates from these patients shows a 100-fold decrease in sensitivity to AZT whereas little or no difference is observed in kinetic parameters in vitro using steady-state kinetic analysis. A pre-steady-state kinetic analysis was used to examine the binding and incorporation of 2‘-deoxythymidine 5‘-triphosphate (dTTP) and 3‘-azido-3‘-deoxythymidine 5‘-triphosphate (AZTTP) by wild-type HIV-1 reverse transcriptase and a clinically important AZT-resistant mutant form of the enzyme (D67N, K70R, T215Y, K219Q) utilizing a physiologically relevant RNA 18-mer/RNA 36-mer primer-template substrate. It was determined that with this RNA/RNA substrate there is a 2.6-fold increase in the selection for incorporation of the natural nucleotide dTTP over the unnatural nucleoside analogue AZTTP by AZT-resistant reverse transcriptase as compared to its wild-type form. This observation indicates that the tRNA^Lys initiation step plays an important role in the development of drug resistance. Furthermore, this result implies that the structural basis of AZT resistance in HIV-1 reverse transcriptase involves the conformation of the RNA−DNA junction (formed upon attachment of a deoxynucleotide to the RNA primer). Taken together, these observations suggest a new pharmacological basis for the development of more effective and novel AIDS drugs.