A family of insertion mutations between codons 67 and 70 of human immunodeficiency virus type 1 reverse transcriptase confer multinucleoside analog resistance

To investigate the occurrence of multinucleoside analog resistance during t herapy failure, we surveyed the drug susceptibilities and genotypes of near ly 900 human immunodeficiency virus type 1 (HIV-1) samples. For 302 of thes e, the 50% inhibitory concentrations of at least four of the approved nucle oside analogs had fourfold-or-greater increases. Genotypic analysis of the reverse transcriptase (RT)-coding regions from these samples revealed compl ex mutational patterns, including the previously recognized codon 151 multi drug resistance cluster. Surprisingly, high-level multinucleoside resistanc e was associated with a diverse family of amino acid insertions in addition to "conventional" point mutations. These insertions were found between RT codons 67 and 70 and were commonly 69Ser-(Ser-Ser) or 69Ser-(Ser-Gly). Trea tment history information showed that a common factor for the development o f these variants was AZT (3'-azido-3'-deoxythymidine, zidovudine) therapy i n combination with 2',3'-dideoxyinosine or 2',3'-dideoxycytidine, although treatment patterns varied considerably. Site-directed mutagenesis studies c onfirmed that 69Ser-(Ser-Ser) in an AZT resistance mutational background co nferred simultaneous resistance to multiple nucleoside analogs. The inserti ons are located in the "fingers" domain of RT. Modelling the 69Ser-(Ser-Ser ) insertion into the RT structure demonstrated the profound direct effect t hat this change is likely to have in the nucleoside triphosphate binding si te of the enzyme. Our data highlight the increasing problem of HIV-1 multid rug resistance and underline the importance of continued resistance surveil lance with appropriate, sufficiently versatile genotyping technology and ph enotypic drug susceptibility analysis.