Structural basis for drug and substrate specificity exhibited by FIV encoding a chimeric FIV/HIV protease

Abstract

A chimeric feline immunodeficiency virus (FIV) protease (PR) has been engineered that supports infectivity but confers sensitivity to the human immunodeficiency virus (HIV) PR inhibitors darunavir (DRV) and lopinavir (LPV). The 6s-98S PR has five replacements mimicking homologous residues in HIV PR and a sixth which mutated from Pro to Ser during selection. Crystal structures of the 6s-98S FIV PR chimera with DRV and LPV bound have been determined at 1.7 and 1.8 Å resolution, respectively. The structures reveal the role of a flexible 90s loop and residue 98 in supporting Gag processing and infectivity and the roles of residue 37 in the active site and residues 55, 57 and 59 in the flap in conferring the ability to specifically recognize HIV PR drugs. Specifically, Ile37Val preserves tertiary structure but prevents steric clashes with DRV and LPV. Asn55Met and Val59Ile induce a distinct kink in the flap and a new hydrogen bond to DRV. Ile98Pro→Ser and Pro100Asn increase 90s loop flexibility, Gln99Val contributes hydrophobic contacts to DRV and LPV, and Pro100Asn forms compensatory hydrogen bonds. The chimeric PR exhibits a comparable number of hydrogen bonds, electrostatic interactions and hydrophobic contacts with DRV and LPV as in the corresponding HIV PR complexes, consistent with IC(50) values in the nanomolar range.

Selection of Drug-Resistant Feline Immunodeficiency Virus (FIV) Encoding FIV/HIV Chimeric Protease in the Presence of HIV-Specific Protease Inhibitors

“…The 6s-98S FIV employed in this study produces a chimeric FIV/ HIV PR with six mutations (I37 32 V, N55 46 M, V59 50 I, I98 81 S, Q99 82 V, and P100 83 N) and is sensitive to the HIV-1 PR inhibitors LPV, DRV, and TL-3 both in in vitro assays of PR activity and in tissue culture infectivity studies. This chimera was previously shown to be sensitive to 200 nM LPV (25), so selection of drugresistant mutants employed an initial concentration of 100 nM LPV.…”

“…Infectious 6s-98S contains five substitutions for structurally equivalent residues of HIV-1 PR, including I37 32 V, N55 46 M, V59 50 I, Q99 82 V, and P100 83 N, and an additional I98 81 P-ϾS mutation (FIV numbering with equivalent HIV-1 numbering in superscript) with a replication capacity much better than that of the original chimeric FIV 6s mutant (I37 32 V, N55 46 M, V59 50 I, Q99 82 V, P100 83 N, and I98 81 P) emerged from a long-term culture. The structural location of I37 32 V is in the active core, whereas N55 46 M and V59 50 I are in the flap region. I98 81 S, Q99 82 V, and P100 83 N are in the 90s loop region (Fig.…”

“…The FIV and HIV-1 PRs have 27 identical residues and display distinct substrate and inhibitor specificities, and none of the current FDA-approved drugs against HIV-1 PR inhibit FIV PR. Of importance, nine of the aforementioned HIV-1 resistance mutations in HIV-1 PR, namely, V11I, K20I, V32I, I50V, I62V, I64L, A71I, N88D, and L90M, are already present in the equivalent positions of FIV PR (16 11 I, 25 20 I, 37 32 I, 59 50 (8,9), in essence making the feline enzyme the ultimate drug-resistant "mutant" enzyme. Rational design based on a comparison of the structures of the FIV and HIV-1 PRs led to the development of TL-3, a broad-based PR inhibitor capable of inhibiting FIV, SIV (simian immunodeficiency virus), HIV-1, and resistant HIV-1 mutants in cell culture (10)(11)(12)(13)(14)(15).…”

“…We designed and expressed several chimeric FIV/HIV PRs that demonstrated HIV-like specificity by substituting as few as four amino acids (I37 32 V/N55 46 M/M56 47 I/V59 50 I) into FIV PR, causing the K i values for many HIV-1 PR inhibitors to drop from the high micromolar to the nanomolar range (21,22). The use of a cell-based FIV Gag-Pol expression system (23) further allowed assessment of the temporal processing of Gag polyprotein by chimeric PRs in the context of the natural substrate (24).…”

“…However, the generation of infectious mutant viruses producing chimeric PR was problematic because of deleterious effects on the cleavage of the Gag polyprotein caused by certain substitutions. Once these problem residues were identified, we were able to generate an infectious mutant FIV producing FIV/ HIV chimeric PR with six mutations, i.e., I37 32 V in the active core, N55 46 M/V59 50 I in the flaps, and I98 81 H(S)/Q99 82 V/P100 83 N in the "90s loop" (25). The chimeric PR was sensitive to the potent HIV-1 PR inhibitors lopinavir (LPV) and darunavir (DRV) and the broad-based inhibitor TL-3, with 50% inhibitory concentrations (IC 50 s) of about 30 nM in vitro.…”