AN IN-VIVO MUTATION FROM LEUCINE TO TRYPTOPHAN AT POSITION-210 IN HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE CONTRIBUTES TO HIGH-LEVEL RESISTANCE TO 3'-AZIDO-3'-DEOXYTHYMIDINE

Sequencing of the reverse transcription (RT) region of 26 human immuno deficiency virus type (HIV-1) isolates from eight patients treated wit h 3'-azido-3'-deoxythymidine (AZT) revealed a mutation at codon 210 fr om TTG (leucine) to TGG (tryptophan) exclusively in association with r esistance to AZT. The mutation Trp-210 was observed in 15 of the 20 is olates phenotypically resistant to AZT, being more commonly observed t han resistance-associated mutations at codons 67, 70, and 219. Trp-210 was never observed before the emergence of resistance-associated muta tions Leu-41 and Tyr-215, and in a sequential series of five isolates from one patient the order of emergence of mutations was found to be T yr-215, Leu-41, and then Trp-210. Trp-210 was also found in associatio n with the Leu-41, Asn-67, Arg-70, and Tyr-215 resistance genotype. To define the role of Trp-210 in AZT resistance, molecular HIV-1 clones were constructed with various combinations of RT mutations at codons 4 1, 67, 70, 210, and 215 and tested for susceptibility to AZT. In clone s with polymerase genes derived either from HXB2-D or clinical isolate s, Trp-210 alone did not increase AZT resistance, whereas in conjuncti on with Leu-41 and Tyr-215, Trp-210 into the genetic context of mutati ons at codons 41, 67, 70, and 215 further enhanced resistance from a 5 0% inhibitory concentration of 1.44 mu M to 8.41 mu M. Molecular model ing of the tertiary structure of HIV-1 RT revealed that the distance b etween the side chains of Trp-210 (in helix alpha F) and Tyr-215 (in s trand beta 11a) approximated 4 Angstrom (1 Angstrom = 0.1 nm), suffici ently close to result in significant energetic interaction between the se two aromatic side chains. In conclusion, Trp-210 contributes signif icantly to phenotypic AZT resistance of HIV-1 by augmenting resistance at least three- to sixfold in the context of two resistant genotypes, and its effect may require an interaction with an aromatic amino acid at position 215.