Gag Mutations Strongly Contribute to HIV-1 Resistance to Protease Inhibitors in Highly Drug-Experienced Patients besides Compensating for Fitness Loss

Abstract
Human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors (PI) results from mutations in the viral protease (PR) that reduce PI binding but also decrease viral replicative capacity (RC). Additional mutations compensating for the RC loss subsequently accumulate within PR and in Gag substrate cleavage sites. We examined the respective contribution of mutations in PR and Gag to PI resistance and RC and their interdependence using a panel of HIV-1 molecular clones carrying different sequences from six patients who had failed multiple lines of treatment. Mutations in Gag strongly and directly contributed to PI resistance besides compensating for fitness loss. This effect was essentially carried by the C-terminal region of Gag (containing NC-SP2-p6) with little or no contribution from MA, CA, and SP1. The effect of Gag on resistance depended on the presence of cleavage site mutations A431V or I437V in NC-SP2-p6 and correlated with processing of the NC/SP2 cleavage site. By contrast, reverting the A431V or I437V mutation in these highly evolved sequences had little effect on RC. Mutations in the NC-SP2-p6 region of Gag can be dually selected as compensatory and as direct PI resistance mutations, with cleavage at the NC-SP2 site behaving as a rate-limiting step in PI resistance. Further compensatory mutations render viral RC independent of the A431V or I437V mutations while their effect on resistance persists. Protease inhibitors are among the most active antiviral drugs used in the treatment of Human immunodeficiency virus type 1 (HIV-1) infection. The efficacy of these compounds, however, can be threatened by the emergence of viral resistance, the result of the gradual accumulation of specific mutations in the viral protease. HIV-1 resistance to protease inhibitors often results in impaired protease function and in the loss of the replicative capacity of the virus, an effect that can be partially corrected by selection of compensatory mutations in one of the natural substrates of the protease, the Gag protein. In this study, we have found that Gag mutations not only correct viral replicative capacity but also play a major and direct role in resistance. We observed that this effect is essentially mediated by mutations in the C-terminal region of Gag, and that it correlates with the extent of cleavage downstream of the Gag nucleocapsid protein. Our results establish that mutations in Gag constitute a second and important pathway of HIV-1 resistance to protease inhibitors in patients failing antiretroviral treatment.