A Family of Insertion Mutations between Codons 67 and 70 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confer Multinucleoside Analog Resistance

Abstract

To investigate the occurrence of multinucleoside analog resistance during therapy failure, we surveyed the drug susceptibilities and genotypes of nearly 900 human immunodeficiency virus type 1 (HIV-1) samples. For 302 of these, the 50% inhibitory concentrations of at least four of the approved nucleoside analogs had fourfold-or-greater increases. Genotypic analysis of the reverse transcriptase (RT)-coding regions from these samples revealed complex mutational patterns, including the previously recognized codon 151 multidrug resistance cluster. Surprisingly, high-level multinucleoside resistance 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). Treatment history information showed that a common factor for the development of these variants was AZT (3′-azido-3′-deoxythymidine, zidovudine) therapy in combination with 2′,3′-dideoxyinosine or 2′,3′-dideoxycytidine, although treatment patterns varied considerably. Site-directed mutagenesis studies confirmed that 69Ser-(Ser-Ser) in an AZT resistance mutational background conferred simultaneous resistance to multiple nucleoside analogs. The insertions are located in the “fingers” domain of RT. Modelling the 69Ser-(Ser-Ser) insertion into the RT structure demonstrated the profound direct effect that this change is likely to have in the nucleoside triphosphate binding site of the enzyme. Our data highlight the increasing problem of HIV-1 multidrug resistance and underline the importance of continued resistance surveillance with appropriate, sufficiently versatile genotyping technology and phenotypic drug susceptibility analysis.

Molecular mechanisms of HIV-1 resistance to nucleoside reverse transcriptase inhibitors (NRTIs)

“…We think the answer is more complex. A recent study showed that the insertion mutations provide multidrug resistance mainly when they are present in a background of AZT resistance mutations [71]. Our experiments show that the enhanced phosphorolysis associated with the D67N/ K70R/T215F/K219Q RT is relatively specific for AZT-terminated DNA [46].…”

Nucleoside and nucleotide inhibitors of HIV-1 replication

“…In the case of mutations K65R and L74V, both selected by ddI, an 84-fold loss in the catalytic rate constant of the double-mutated RT compared to the WT also accounts for the poor ability to use natural dNTPs and the resulting poor viral fitness [129] and explains why K65R and L74V are never selected together in the clinic [48]. Finally, clinical data revealed that K65R hypersensitizes HIV-1 to AZT [55,95,130] and does not develop in patients receiving AZT-containing regimens [131]. Accordingly, in a more recent clinical study, a low frequency of K65R mutation emerged, in treatment-experienced patients, but only for patients without detectable TAMs at baseline, suggesting a functional and/or structural incompatibility for the two substitutions within the same RT.…”

“…ddI [89], and also contributes to resistance to each of the NRTIs in the context of TAMs [60,[90][91][92][93]. In about 2% of heavily treated HIV-1-infected patients [94] having received prolonged AZT treatment followed by administration of other NRTIs, RT has a dipeptide insertion (SA, SG or mainly SS) between position 69 and 70, associated with other amino acid substitutions such as T69S or T215Y and other TAMs [94][95][96]. Individually, the dipeptide insertions confer only a low level of resistance to each of the NRTIs, but in the context of T215Y and other TAMs, high levels of resistance to AZT and moderate levels to d4T, ddC, ddI and tenofovir are achieved [95][96][97][98][99] ( fig.…”

Overcoming HIV drug resistance through rational drug design based on molecular, biochemical, and structural profiles of HIV resistance

“…Insertion mutations between positions 69 and 70 of the fingers domain (69-insertions) have been described that result in multidrug-resistant HIV [50]. These mutations are associated with NAMs in clinical isolates [51].…”

“…5a). Each monomer of the protease also contributes amino acids (positions [45][46][47][48][49][50][51][52][53][54][55][56] to form a flap that extends over the substrate-binding cleft. The flap must be flexible enough to allow entry and exit of the polypeptide substrates.…”