Four Amino Acid Changes in HIV-2 Protease Confer Class-Wide Sensitivity to Protease Inhibitors

Abstract

Protease is essential for retroviral replication, and protease inhibitors (PI) are important for treating HIV infection. HIV-2 exhibits intrinsic resistance to most FDA-approved HIV-1 PI, retaining clinically useful susceptibility only to lopinavir, darunavir, and saquinavir. The mechanisms for this resistance are unclear; although HIV-1 and HIV-2 proteases share just 38 to 49% sequence identity, all critical structural features of proteases are conserved. Structural studies have implicated four amino acids in the ligand-binding pocket (positions 32, 47, 76, and 82). We constructed HIV-2 ROD9 molecular clones encoding the corresponding wild-type HIV-1 amino acids (I32V, V47I, M76L, and I82V) either individually or together (clone PR⌬4) and compared the phenotypic sensitivities (50% effective concentration [EC 50 ]) of mutant and wild-type viruses to nine FDA-approved PI. Single amino acid replacements I32V, V47I, and M76L increased the susceptibility of HIV-2 to multiple PI, but no single change conferred class-wide sensitivity. In contrast, clone PR⌬4 showed PI susceptibility equivalent to or greater than that of HIV-1 for all PI. We also compared crystallographic structures of wild-type HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir to models of the PR⌬4 enzyme. These models suggest that the amprenavir sensitivity of PR⌬4 is attributable to stabilizing enzyme-inhibitor interactions in the P2 and P2= pockets of the protease dimer. Together, our results show that the combination of four amino acid changes in HIV-2 protease confer a pattern of PI susceptibility comparable to that of HIV-1, providing a structural rationale for intrinsic HIV-2 PI resistance and resolving long-standing questions regarding the determinants of differential PI susceptibility in HIV-1 and HIV-2. IMPORTANCEProteases are essential for retroviral replication, and HIV-1 and HIV-2 proteases share a great deal of structural similarity. However, only three of nine FDA-approved HIV-1 protease inhibitors (PI) are active against HIV-2. The underlying reasons for intrinsic PI resistance in HIV-2 are not known. We examined the contributions of four amino acids in the ligand-binding pocket of the enzyme that differ between HIV-1 and HIV-2 by constructing HIV-2 clones encoding the corresponding HIV-1 amino acids and testing the PI susceptibilities of the resulting viruses. We found that the HIV-2 clone containing all four changes (PR⌬4) was as susceptible as HIV-1 to all nine PI. We also modeled the PR⌬4 enzyme structure and compared it to existing crystallographic structures of HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir. Our findings demonstrate that four positions in the ligand-binding cleft of protease are the primary cause of HIV-2 PI resistance.

Analysis of the HIV-2 protease’s adaptation to various ligands: characterization of backbone asymmetry using a structural alphabet

“…(1999) detected a structural asymmetry adjacent to the nucleotide-binding sites of aspartate transcarbamoylase that would appear to account for their separation into high- and low-affinity binding sites 33 . Despite their strong chemical similarity (Tanimoto coefficient = 0.95), DRV and APV exhibit completely different susceptibility against PR2 2 , 7 . By comparing the asymmetric profiles of PR2 dimers complexed with APV and DRV, we observed some differences that may be involved in the better recognition or higher affinity between DRV and PR2, particularly in the binding pockets at positions 23, 31, 46, and 75.…”

Inhibitors for Novel Coronavirus Protease Identified by Virtual Screening of 687 Million Compounds

“…12 However, these compounds remained in the preclinical or early clinical stage not leading to an approved medicine 18 and their effectiveness for the novel virus might suffer due differences in single amino acids. 19 Accordingly, the development of specific inhibitors for SARS-CoV-2 main protease remains an urgent necessity in the scientific community 10-12 which is reflected by the multiple projects focusing on this protein. For example, three studies investigating the repurposing of marketed drugs proposed several candidates for SARS-CoV-2 treatment.…”

Exploration of the effect of sequence variations located inside the binding pocket of HIV-1 and HIV-2 proteases

“…(2016), who reported the opposite observation by incorporating four HIV-1-like mutations, I32V, V47I, L76M, and I82V, into a wild-type PR2. This PR2 mutant exhibited susceptibility to all PIs perfectly equivalent to PR1 3 . In this last study, the authors also showed that mutations in the binding site induced only small rearrangements, modifying the internal interactions between PR2 and DRV compared to the PR1-DRV complex.…”

“…Currently, nine FDA (Food and Drug Administration)-approved PIs are available for HIV-1 therapy, including saquinavir (SQV), ritonavir, indinavir, nelfinavir, amprenavir (APV), lopinavir (LPV), atazanavir, tipranavir, and darunavir (DRV). Only three of these are commonly recommended for the treatment of HIV-2 infection: SQV, LPV, and DRV 1 , 3 , 4 . Greater understanding of the structural mechanisms underlying HIV-2 resistance to PIs is important for the development of new efficacious anti-HIV-2 drugs.…”