title: 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-2ROD9 molecular clones encoding the corresponding wild-type HIV-1 amino acids (I32V, V47I, M76L, and I82V) either individually or together (clone PRΔ4) and compared the phenotypic sensitivities (50% effective concentration [EC50]) 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 PRΔ4 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 PRΔ4 enzyme. These models suggest that the amprenavir sensitivity of PRΔ4 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.
  
  
  OBJECTIVE
  Proteases 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 (PRΔ4) was as susceptible as HIV-1 to all nine PI. We also modeled the PRΔ4 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.
authors.affiliation: Department of Medicine, Division of Allergy & Infectious Diseases and Center for Emerging & Re-emerging Infectious Diseases, School of Medicine, University of Washington, Seattle, Washington, USA raugid@uw.edu.
authors.forename: Dana N
authors.initials: DN
authors.lastname: Raugi
authors.affiliation: Department of Medicine, Division of Allergy & Infectious Diseases and Center for Emerging & Re-emerging Infectious Diseases, School of Medicine, University of Washington, Seattle, Washington, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Smith
authors.affiliation: Department of Medicine, Division of Allergy & Infectious Diseases and Center for Emerging & Re-emerging Infectious Diseases, School of Medicine, University of Washington, Seattle, Washington, USA Department of Global Health, School of Public Health, University of Washington, Seattle, Washington, USA.
authors.forename: Geoffrey S
authors.initials: GS
authors.lastname: Gottlieb
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000235:genetics*; D001665:Binding Sites; D002460:Cell Line; D024882:Drug Resistance, Viral; D016333:HIV Protease / Q000235:genetics* / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D006801:Humans; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D020125:Mutation, Missense; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D000596:Amino Acids; D017320:HIV Protease Inhibitors; D016333:HIV Protease; C065184:p16 protease, Human immunodeficiency virus 2