ABSTRACT
An infectious chimeric feline immunodeficiency virus (FIV)/HIV strain carrying six HIV-like protease (PR) mutations (I37V/N55M/V59I/I98S/Q99V/P100N) was subjected to selection in culture against the PR inhibitor lopinavir (LPV), darunavir (DRV), or TL-3. LPV selection resulted in the sequential emergence of V99A (strain S-1X), I59V (strain S-2X), and I108V (strain S-3X) mutations, followed by V37I (strain S-4X). Mutant PRs were analyzed in vitro, and an isogenic virus producing each mutant PR was analyzed in culture for LPV sensitivity, yielding results consistent with the original selection. The 50% inhibitory concentrations (IC50s) for S-1X, S-2X, S-3X, and S-4X were 95, 643, 627, and 1,543 nM, respectively. The primary resistance mutations, V99(82)A, I59(50)V, and V37(32)I, are consistent with the resistance pattern developed by HIV-1 under similar selection conditions. While resistance to LPV emerged readily, similar PR mutations causing resistance to either DRV or TL-3 failed to emerge after passage for more than a year. However, a G37D mutation in the nucleocapsid (NC) was observed in both selections and an isogenic G37D mutant replicated in the presence of 100 nM DRV or TL-3, whereas parental chimeric FIV could not. An additional mutation, L92V, near the PR active site in the folded structure recently emerged during TL-3 selection. The L92V mutant PR exhibited an IC50 of 50 nM, compared to 35 nM for 6s-98S PR, and processed the NC-p2 junction more efficiently, consistent with increased viral fitness. These findings emphasize the role of mutations outside the active site of PR in increasing viral resistance to active-site inhibitors and suggest additional targets for inhibitor development.
Subject(s)
Drug Resistance, Viral , HIV Protease Inhibitors/pharmacology , HIV Protease/metabolism , HIV-1/enzymology , Immunodeficiency Virus, Feline/drug effects , Mutation, Missense , Selection, Genetic , DNA Mutational Analysis , HIV Protease/genetics , HIV-1/genetics , Immunodeficiency Virus, Feline/genetics , Immunodeficiency Virus, Feline/growth & development , Immunodeficiency Virus, Feline/isolation & purification , Inhibitory Concentration 50 , Microbial Sensitivity Tests , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Serial PassageABSTRACT
Heparan sulfate proteoglycans (HSPGs) act as binding receptors or attachment factors for the viral envelope of many viruses, including strains of HIV and feline immunodeficiency virus (FIV). The FIV gp95 glycoprotein (SU) from laboratory-adapted strains (tissue culture adapted [TCA]) such as FIV-34TF10 can bind to HSPG, whereas SU from field strains (FS) such as FIV-PPR cannot. Previous studies indicate that SU-HSPG interactions occur within the V3 loop. We utilized a series of nested V3 peptides to further map the HSPG binding sites and found that both sides of the predicted V3 loop stem were critical for the binding but not the CXCR4 binding domain near the predicted tip of the V3 loop. Neutralization assays for TCA strain entry using the same set of V3 peptides showed that peptides targeting CXCR4 or HSPG binding sites can block infection, supporting the V3 loop as a critical neutralization target. Site-directed mutagenesis identified two highly conserved arginines, R379 and R389, on the N-terminal side of the V3 stem as critical for the contact between SU and HSPG. Residues K407, K409, K410, and K412 on the C-terminal side of the V3 stem form a second nonconserved domain necessary for HSPG binding, consistent with the observed specificity distinctions with FS FIV. Our findings discriminate structural determinants important for HSPG and CXCR4 binding by FIV SU and thus further define the importance of the V3 loop for virus entry and infection.
Subject(s)
Amino Acids/metabolism , Glycoproteins/metabolism , Heparan Sulfate Proteoglycans/metabolism , Immunodeficiency Virus, Feline/metabolism , Viral Envelope Proteins/metabolism , Amino Acid Sequence , Amino Acids/chemistry , Animals , Base Sequence , Cats , Cell Line , DNA Primers , Flow Cytometry , Glycoproteins/chemistry , Glycoproteins/genetics , Immunodeficiency Virus, Feline/physiology , Membrane Fusion , Molecular Sequence Data , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Protein Binding , Receptors, CXCR4/metabolism , Sequence Homology, Amino Acid , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/geneticsABSTRACT
A library of 68 brominated fragments was screened against a new crystal form of inhibited HIV-1 protease in order to probe surface sites in soaking experiments. Often, fragments are weak binders with partial occupancy, resulting in weak, difficult-to-fit electron density. The use of a brominated fragment library addresses this challenge, as bromine can be located unequivocally via anomalous scattering. Data collection was carried out in an automated fashion using AutoDrug at SSRL. Novel hits were identified in the known surface sites: 3-bromo-2,6-dimethoxybenzoic acid (Br6) in the flap site and 1-bromo-2-naphthoic acid (Br27) in the exosite, expanding the chemistry of known fragments for development of higher affinity potential allosteric inhibitors. At the same time, mapping the binding sites of a number of weaker binding Br-fragments provides further insight into the nature of these surface pockets.
Subject(s)
Bromine/chemistry , HIV Protease/chemistry , HIV-1/enzymology , Protease Inhibitors/chemistry , Small Molecule Libraries/chemistry , Allosteric Regulation , Binding Sites , Crystallography, X-Ray , Drug Evaluation, Preclinical , HIV Protease/genetics , HIV Protease/metabolism , Humans , Hydroxybenzoate Ethers/chemistry , Molecular Docking Simulation , Naphthalenes/chemistry , Protease Inhibitors/metabolism , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Small Molecule Libraries/metabolismABSTRACT
The fragment indole-6-carboxylic acid (1F1), previously identified as a flap site binder in a fragment-based screen against HIV protease (PR), has been cocrystallized with pepstatin-inhibited PR and with apo-PR. Another fragment, 3-indolepropionic acid (1F1-N), predicted by AutoDock calculations and confirmed in a novel inhibition of nucleation crystallization assay, exploits the same interactions in the flap site in two crystal structures. Both 1F1 and 1F1-N bind to the closed form of apo-PR and to pepstatin:PR. In solution, 1F1 and 1F1-N raise the Tm of apo-PR by 3.5-5 °C as assayed by differential scanning fluorimetry (DSF) and show equivalent low-micromolar binding constants to both apo-PR and pepstatin:PR, assayed by backscattering interferometry (BSI). The observed signal intensities in BSI are greater for each fragment upon binding to apo-PR than to pepstatin-bound PR, consistent with greater conformational change in the former binding event. Together, these data indicate that fragment binding in the flap site favors a closed conformation of HIV PR.