Selection of HIV-1 for Resistance to Fourth Generation Protease Inhibitors Reveals Two Independent Pathways to High-Level Resistance

Well-designed viral protease inhibitors (PIs) potently inhibit replication as well as create a high genetic barrier for resistance. Through in vivo selective pressure, we have generated high-level resistance against ten HIV-1 PIs and their precursor, the FDA-approved drug darunavir (DRV), achieving 1,000-fold resistance over the starting EC50. The accumulation of mutations revealed two pathways to high-level resistance, resulting in protease variants with up to 14 mutations in and outside of the active site. The two pathways demonstrate the interplay between drug resistance and viral fitness. Replicate selections showed that one inhibitor could select for resistance through either pathway, although subtle changes in chemical structure of the inhibitors led to preferential use of one pathway over the other. Viral variants from the two pathways showed differential selection of compensatory mutations in Gag cleavage sites. These results reveal the high-level of selective pressure that is attainable with these fourth-generation protease inhibitors, and the interplay between selection of mutations to confer resistance while maintaining viral fitness.