Human immunodeficiency virus type 1 reverse transcriptase genotype and drug susceptibility changes in infected individuals receiving dideoxyinosine monotherapy for 1 to 2 years

Abstract

The genetic mechanisms of human immunodeficiency virus type 1 (HIV-1) resistance to dideoxyinosine (ddI) in vivo have been described based on data from primary HIV-1 isolates. To better define the spectrum of HIV-1 reverse transcriptase (RT) changes occurring during ddI therapy, we determined the genotype and ddI susceptibility of the RT gene of HIV RNA isolated from the plasma of 23 patients who had received 1 to 2 years (mean, 87 +/- 16 weeks) of ddI monotherapy. Population-based sequencing of plasma virus showed that 12 of 23 (52%) patients developed known ddI resistance mutations: L74V (7 patients), K65R (2 patients), L74V with M184V (3 patients), and L74V with K65R (1 patient). Five patients developed one or more known zidovudine resistance mutations (at codons 41, 67, 70, 215, and/or 219) during the study. Other amino acid substitutions were found, but only S68G and L210W occurred in more than one patient. Studies of sensitivity to ddI were performed on population-based recombinant-virus stocks generated by homologous recombination between a plasmid containing an HXB2 clone with the RT gene deleted and RT-PCR products of the RT genes from patients' plasma RNA. The sequences of the virus stocks produced by this procedure were typically identical to the sequence of the input PCR product from plasma RNA. Both an MT-2 cell-based culture assay and a cell-free virion-associated RT inhibition assay showed that viruses possessing an L74V and/or M184V mutation or a K65R mutation had reduced sensitivity to ddI. Viruses without these specific mutations had no change in sensitivity to ddI. The results presented here show that the spectrum of RT mutations in a population of patients on ddI monotherapy is more complex than previously described. The development of multiple mutational patterns, including those that confer resistance to other nucleoside analogs, highlights the complexity of using the currently available nucleoside analogs for antiretroviral therapy.

Nucleoside and nucleotide inhibitors of HIV-1 replication

“…2B). Mutation at position 74 (mostly L74V) is frequently found in patients receiving ddI mono-therapy [48,68,75,76] and also occurs during ABC mono-therapy [56,74]. In the former case, L74V is associated with other mutations (mainly M184V) [77].…”

“…Hence, the reduced fitness of the double-mutant virus is explained by the general reduction in incorporation of the natural dNTPs by the corresponding mutated RT as well as reduced processivity. 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].…”

“…This set of six mutations providing resistance towards AZT and d4T was therefore named the thymidine-associated mutations (TAMs). TAMs also occur in about 10% of individuals receiving ddI mono-therapy [47,48]. The TAMs are located both in the fingers and in the palm subdomains of RT, at the front of the nucleotide-binding pocket ( fig.…”

“…2B). Mutation K65R emerges after mono-therapy with ddI [48,80], ddC [80,81] or ABC [74], and during tenofovir intensification [60]. K65R is also frequently selected in cell culture by ABC [77], tenofovir [60] and d4T [82], and its incidence increased recently, mainly in patients receiving tri-therapies excluding AZT [83].…”

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

“…Thus, L74V may mediate disruption of this hydrogen bond network in a secondary fashion. K65R is an important mutation found in clinical isolates that confers resistance to several NRTIs including didanosine, zalcitibine, stavudine, abacavir, tenofovir, lamivudine, and emtricitabine [1, 39,40]. As mentioned above, K65 is located in the fingers domain, and conformational changes initiated by nucleotide binding bring K65 into the proximity of the dNTP-binding site, where it serves to orient the triphosphate moiety of the nucleotide [23,31].…”