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A novel combination of three codon inserts in the pol coding region of HIV-1 RNA was identified in a highly antiretroviral experienced study subject with HIV-1 infection. A one codon insert was observed in the protease region between codon 40 and 41 simultaneously with a two codon insert present in the reverse transcriptase region at codon 69.
The objective of HIV treatment is to suppress viral replication, and currently more than 20 different drugs have been approved to treat HIV.1 Shortly after antiretroviral drugs were in clinical trial, drug resistance-associated mutations were first described.2 HIV drug resistance has since been described for every active drug3 and drug resistance testing has been incorporated as part of standard clinical management and clinical trial design.4 In the course of a clinical study to assess the treatment of protease-resistant virus we discovered a previously unreported three base insert in HIV-1 clade B protease (PR) simultaneously with a six base insert in reverse transcriptase (RT) (GenBank accession number FJ159426).
HIV RNA was extracted, reverse transcribed, and the polymerase (pol) gene was amplified according to the manufacturer's instructions using the ViroSeq HIV-1 Genotyping System Version 2.0 (Celera Diagnostics, Foster City, CA). Sequence data were analyzed using ViroSeq Version 2.6 Sequence Analysis Software (Celera Diagnostics).
The six nucleotide insert (Fig. 1A) has been described previously and is known as a T69S+XX insertion.5 These inserts usually have a “T”-to-“S” point mutation at codon 69 and then a two amino acid insertion added to the functional protein. The proposed mechanism of decreasing susceptibility to ART by this insertion is to stall or to cause the slippage of RT during reverse transcription.6 Virus isolates containing these insertions have reduced susceptibility to all nucleoside and nucleotide RT inhibitors.7 Stalling or slippage has also been hypothesized to be the mechanism behind the generation of PR inserts. Several PR insert strains have been identified both with and without major PR resistance mutations, although these PR inserts have not been shown to directly contribute to decreased susceptibility to protease inhibitors (PI).8 The insert we describe (Fig.1B) has not yet been evaluated with site-directed mutants with and without the associated PI resistance mutations for impact on PI susceptibility in vitro. This insert, however, was identified in the setting of extensive, prolonged, and intermittent ART pressure (Fig. 2). The antiretroviral susceptibility profiles of the viruses with these inserts using the Monogram Phenosense assay are shown in Table 1.
To model the function of the described PR insert, we generated superimposed computer models of PR with or without our specific insert (Fig. 3). The close proximity of the one codon insert to the functional binding cleft of the PR homodimer could impact drug susceptibility, since the morphology and nature of the binding cleft may be altered by the addition of amino acids extending near or into the binding site leading to decreased PI binding.9 Further in vitro characterization of this novel PR insert with and without corresponding PR mutations associated with decreased susceptibility to PI still need to be evaluated.
This work was supported by Grants AI69432 (ACTG), AI043638 (AIEDRP), MH62512 (HNRC), MH083552 (Clade), AI077304 (Dual Infection), AI36214 (the Viral Pathogenesis Core of the UCSD Center for AIDS Research), AI047745 (Dynamics), AI74621 (Transmission), AI069464 (Quintiles Clinical Trial), and AI69423 and AI50410 from the National Institutes of Health and the California HIV/AIDS Research Program RN07-SD-702. We would particularly like to thank all of the participants and investigators involved in the ACTG 5126 trial. Written informed consent was obtained from all patients and the human experimentation guidelines of the U.S. Department of Health and Human Services and the individual institutions were followed in conducting this research. GenBank accession number: FJ159426.
No competing financial interests exist.