HIV-1 protease is one of the major antiviral targets in the treatment of patients infected with HIV-1. The nine FDA approved HIV-1 protease inhibitors were developed with extensive use of structure-based drug design, thus the atomic details of how the inhibitors bind are well characterized. From this structural understanding the molecular basis for drug resistance in HIV-1 protease can be elucidated. Selected mutations in response to therapy and diversity between clades in HIV-1 protease have altered the shape of the active site, potentially altered the dynamics and even altered the sequence of the cleavage sites in the Gag polyprotein. All of these interdependent changes act in synergy to confer drug resistance while simultaneously maintaining the fitness of the virus. New strategies, such as incorporation of the substrate envelope constraint to design robust inhibitors that incorporate details of HIV-1 protease’s function and decrease the probability of drug resistance, are necessary to continue to effectively target this key protein in HIV-1 life cycle.

Drug resistance is an important cause of antiretroviral therapy failure in human immunodeficiency virus (HIV)-infected patients. Mutations in the protease render the virus resistant to protease inhibitors (PIs). Gag cleavage sites also mutate, sometimes correlating with resistance mutations in the protease, but their contribution to resistance has not been systematically analyzed. The present study examines mutations in Gag cleavage sites that associate with protease mutations and the impact of these associations on drug susceptibilities. Significant associations were observed between mutations in the nucleocapsid-p1 (NC-p1) and p1-p6 cleavage sites and various PI resistance-associated mutations in the protease. Several patterns were frequently observed, including mutations in the NC-p1 cleavage site in combination with I50L, V82A, and I84V within the protease and mutations within the p1-p6 cleavage site in combination with D30N, I50V, and I84V within the protease. For most patterns, viruses with mutations both in the protease and in either cleavage site were significantly less susceptible to specific PIs than viruses with mutations in the protease alone. Altered PI resistance in HIV-1 was found to be associated with the presence of Gag cleavage site mutations. These studies suggest that associated cleavage site mutations may contribute to PI susceptibility in highly specific ways depending on the particular combinations of mutations and inhibitors. Thus, cleavage site mutations should be considered when assessing the level of PI resistance.

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The selective pressure of the competitive protease inhibitors causes both HIV-1 protease and occasionally its substrates to evolve drug resistance. We hypothesize that this occurs particularly in substrates that protrude beyond the substrate envelope and contact residues that mutate in response to a particular protease inhibitor. To validate this hypothesis, we analyzed substrate and protease sequences for covariation. Using the chi2 test, we show a positive correlation between the nelfinavir-resistant D30N/N88D protease mutations and mutations at the p1-p6 cleavage site as compared to the other cleavage sites. Both nelfinavir and the substrate p1-p6 protrude beyond the substrate envelope and contact residue 30, thus possibly making the p1-p6 cleavage site more vulnerable to co-evolution.

Other than cleavage site mutations, there is little data on specific positions within Gag that impact on HIV protease inhibitor susceptibility. We have recently shown that non-cleavage site mutations in gag, particularly within matrix protein can restore replication capacity and further reduce protease inhibitor drug susceptibility when coexpressed with a drug-resistant (mutant) protease. The matrix protein of this patient-derived virus was studied in order to identify specific changes responsible for this phenotype. Three amino acid changes in matrix (R76K, Y79F, and T81A) had an impact on replication capacity as well as drug susceptibility. Introduction of these three changes into wild-type (WT) matrix resulted in an increase in the replication capacity of the protease mutant virus to a level similar to that achieved by all the changes within the mutant matrix and part of the capsid protein. Pairs of changes to wild-type matrix led to an increased replication capacity of the protease mutant (although less than with all three changes). Having only these three changes to matrix in a wild-type virus (with wild-type protease) resulted in a 5-to 7-fold change in protease inhibitor 50% effective concentration (EC 50 ). Individual changes did not have as great an effect on replication capacity or drug susceptibility, demonstrating an interaction between these positions, also confirmed by sequence covariation analysis. Molecular modeling predicts that each of the three mutations would result in a loss of hydrogen bonds within ␣-helix-4 of matrix, leading to the hypothesis that more flexibility within this region or altered matrix structure would account for our findings.Current British HIV Association (BHIVA) and other guidelines for highly active antiretroviral therapy (HAART) in the treatment of HIV and AIDS recommend first-line therapy with three active drugs: two nucleoside reverse transcriptase (RT) inhibitors and a nonnucleoside RT inhibitor. Protease inhibitors (PIs) are used with two active RT inhibitors in second-line therapy after the failure of first-line therapy (11). PIs are some of the most potent of the antiretroviral drugs in HIV clinical practice. Resistance to PIs develops by the accumulation of mutations within the protease gene that change amino acids within the enzymatic active site, reducing the binding of the inhibitor. Many of these primary PI resistance mutations have a negative effect on virus fitness or replication capacity, resulting in further secondary mutations that do not cause resistance themselves but instead increase the replication capacity of the resistant virus (2-5, 20, 22, 26). HIV protease cleaves Gag and Gag-Pol polyproteins, resulting in viral maturation after cellular release. Mutations within the Gag protein, particularly at the cleavage sites (cleavage site mutations [CSMs]) have also been associated with the recovery of replication capacity (9,10,24,31,35) as well as with PI resistance without protease mutations (27). Structural analysis showed that the A431V CSM has incr...