In Vivo Emergence of a Novel Protease Inhibitor Resistance Signature in HIV-1 Matrix

BackgroundProtease Inhibitors (PIs) are the second- and last-line therapy for the majority of HIV-infected patients worldwide. Only around 20% of individuals who fail PI regimens develop major resistance mutations in protease. We sought to explore the role of mutations in gag-protease genotypic and phenotypic changes within six Nigerian patients who failed PI-based regimens without known drug resistance associated protease mutations in order to identify novel determinants of PI resistance. MethodsTarget enrichment and NGS by Illumina Miseq were followed by haplotype reconstruction. Full length gag-protease regions were amplified from baseline (pre-PI) and virologic failure (VF) samples, sequenced and used to construct gag/protease pseudotyped viruses. Phylogenetic analysis was performed using maximum likelihood methods. Susceptibility to lopinavir (LPV) and darunavir (DRV) were measured using a single-cycle replication assay. Western blotting was used to analyse Gag cleavage. ResultsIn one of six participants (subtype CRF02_AG) we found 4-fold lower LPV susceptibility in viral clones during failure of second line treatment. A combination of four mutations (S126del, H127del, T122A and G123E) in p17 matrix of baseline virus generated a similar 4x decrease in susceptibility to LPV but not darunavir. These four amino acid changes were also able to confer LPV resistance to a subtype B gag-protease backbone. Western blotting did not demonstrate significant Gag cleavage differences between sensitive and resistant isolates. Resistant viruses had around 2-fold lower infectivity compared to sensitive clones in the absence of drug. NGS combined with haplotype reconstruction revealed resistant, less fit clones emerged from a minority population at baseline and thereafter persisted alongside sensitive fitter viruses. ConclusionsWe have used a multi-pronged genotypic and phenotypic approach to document emergence and temporal dynamics of a novel protease inhibitor resistance signature in p17 matrix, revealing the interplay between Gag associated resistance and fitness.