Three Residues in HIV-1 Matrix Contribute to Protease Inhibitor Susceptibility and Replication Capacity

Abstract

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...

Genetic Changes in HIV-1 Gag-Protease Associated with Protease Inhibitor-Based Therapy Failure in Pediatric Patients

“…The remaining four positively selected sites (positions 15, 54, 69, and 339) have not previously been associated with PI exposure nor predicted to be HLA/KIR. Two additional sites in Gag have been reported to be associated with PI failure, 17,28 including the HLA/KIR-selected position 79, but were not found to be positively selected in this analysis. In total, 21 of the 53 changes identified in the entire Gag gene following PI failure showed some evidence of selection pressure ( Fig.…”

Inference of epistatic effects leading to entrenchment and drug resistance in HIV-1 protease

“…The results presented here advance recent work in the field, using Potts models to study HIV-1 evolution (Barton et al 2016b;Butler et al 2016), by providing systematic prospective predictions quantifying the influence of specific multi-residue patterns on the tolerance of drug resistance mutations. Recent publications have reported that mutations near or distal to Gag cleavage sites play a role in promoting cleavage by drug-resistant and enzymatically deficient proteases, by selecting for mutations that increase substrate contacts with the protease active site, altering the flexibility of the cleavage site vicinity, or by as of yet unknown mechanisms Kolli et al 2009;Breuer et al 2011;Parry et al 2011;Fun et al 2012;Flynn et al 2015). This suggests that viral coevolution of Gag with selective protease mutations may further stabilize multiple resistance mutations; thus, the analysis of protease mutation patterns can be extended to include amino acid substitutions within Gag and the Gag-Pol polyprotein.…”

Transient HIV-1 Gag–protease interactions revealed by paramagnetic NMR suggest origins of compensatory drug resistance mutations

“…The regions of Gag and protease that undergo compensatory mutations are strikingly similar to those exhibiting significant intermolecular PREs, thereby providing a plausible underlying structural basis behind these mutations. For example, conservative noncleavage site mutations in MA, specifically K30R, R76K, Y79F, and T81A, restore the fitness deficit arising from drug-resistant protease by improving replication capacity and generating a significant reduction in susceptibility to protease inhibitors in vivo (14,29). These residues along with two other drug-resistance mutations, E12K and L75R, reside in regions of MA that give large intermolecular PREs.…”

“…A comparison of regions involved in transient Gag-protease encounter complexes with residues associated with drug-resistance mutations (14)(15)(16)(17)28) in Gag and protease is provided in Figs. 2D and 3C, respectively (also see Table S2 for additional details).…”

“…Synthetic peptides, however, are a poor substitute for the Gag polyprotein in terms of long-range intermolecular interactions and conformational changes that can alter the accessibility of Gag cleavage junctions. Additionally both protease and Gag mutate and coevolve in response to protease inhibitors in current clinical use (10)(11)(12)(13)(14)(15)(16)(17). Primary mutations (i.e., mutations at the catalytic site of protease) and mutations within the Gag cleavage sites are readily amenable to investigation using peptide analogs.…”