Interaction of tRNA-derivatives and oligonucleotide primers with AZT-resistant mutants of HIV-1 reverse transcriptase

While the molecular basis of HIV-1 AZT resistance has been widely studied, a biochemical explanation of this process is not well known. No significant changes in the binding affinity of reverse transcriptase (RT) mutants for AZT-triphosphate has been found. Here we analyzed the interaction of wild t ype and AZT-resistant mutant forms of HIV-1 RT with different primers. Site -directed mutagenesis was used to introduce point mutations on the retrovir al enzyme. Primers were either synthetic oligonucleotides or tRNA(Lys3) der ivatives containing d(pT)(n) or r(pU)(n) at the 3' end. In all cases, deter mination of kinetic parameters was done in the presence or absence of compo unds known to modify protein conformation, such as dimethyl sulfoxide (DMSO ), urea, and Triton X-100. Although we found similar K-m values for all RTs , there was generally an increase in the affinity when enzymes were tested in the presence of DMSO, urea, and Triton X-100. Then, we analyzed the nucl eation and elongation steps of the polymerization process. The efficiency o f formation of the first base pair was determined by measuring K-m1, the af finity between RT and the 3' terminal nucleotide of the primer. An importan t difference was found: in the presence of DMSO, urea, and Triton X-100, th e K-m1 values for mutated enzymes were higher than those of wild type RTs. Thus, the presence of compounds able to change protein conformation led to a marked destabilization of the interaction of mutated RTs with the 3' term inal nucleotide of the primer. From these results, it can be hypothesized t hat resistance to AZT is not due to the direct influence of mutations on RT , but rather to conformational changes of the mutated RT in complex with th e template-primer altering the ability of the enzyme to select or reject an incoming dNTP. (C) 1998 Elsevier Science Ltd. All rights reserved.