ABSTRACT
Alphaviruses require conserved cysteine residues for proper folding and assembly of the E1 and E2 envelope glycoproteins, and likely depend on host protein disulfide isomerase-family enzymes (PDI) to aid in facilitating disulfide bond formation and isomerization in these proteins. Here, we show that in human HEK293 cells, commercially available inhibitors of PDI or modulators thereof (thioredoxin reductase, TRX-R; endoplasmic reticulum oxidoreductin-1, ERO-1) inhibit the replication of CHIKV chikungunya virus (CHIKV) in vitro in a dose-dependent manner. Further, the TRX-R inhibitor auranofin inhibited Venezuelan equine encephalitis virus and the flavivirus Zika virus replication in vitro, while PDI inhibitor 16F16 reduced replication but demonstrated notable toxicity. 16F16 significantly altered the viral genome: plaque-forming unit (PFU) ratio of CHIKV in vitro without affecting relative intracellular viral RNA quantities and inhibited CHIKV E1-induced cell-cell fusion, suggesting that PDI inhibitors alter progeny virion infectivity through altered envelope function. Auranofin also increased the extracellular genome:PFU ratio but decreased the amount of intracellular CHIKV RNA, suggesting an alternative mechanism of action. Finally, auranofin reduced footpad swelling and viremia in the C57BL/6 murine model of CHIKV infection. Our results suggest that targeting oxidative folding pathways represents a potential new anti-alphavirus therapeutic strategy.
Subject(s)
Antiviral Agents/pharmacology , Chikungunya Fever/virology , Chikungunya virus/drug effects , Chikungunya virus/physiology , Host-Pathogen Interactions/physiology , Alphavirus Infections/virology , Animals , Auranofin/antagonists & inhibitors , Chikungunya Fever/mortality , Chikungunya virus/pathogenicity , Disease Models, Animal , Encephalitis Virus, Venezuelan Equine/drug effects , Flavivirus/drug effects , HEK293 Cells , Humans , Membrane Glycoproteins , Mice , Mice, Inbred C57BL , Protein Disulfide-Isomerases/pharmacology , Protein Folding , Thioredoxin-Disulfide Reductase/pharmacology , Viral Envelope Proteins/metabolism , Virus Replication/drug effects , Zika Virus/drug effects , Zika Virus Infection/virologyABSTRACT
1. Acute promyelocytic leukaemia (APL) is characterized by a block in differentiation at the promyelocyte stage. Here, we describe the effects of auranofin (AF), a coordinated gold compound, on apoptosis and differentiation of APL cells. 2. Nucleosomal DNA fragmentation assay and Hoechst 33342 staining indicated that AF induced apoptosis in APL-derived NB4 cells at low concentrations (0.5-1.0 microm). The AF-induced apoptosis involved caspase-3 activation and specific cleavage of poly-ADP-ribose polymerase. 3. The AF-treated NB4 cells also produced reactive oxygen species (ROS) and cotreatment with N-acetyl-l-cysteine protected the NB4 cells from AF-induced apoptosis. 4. Expression of the CD11b cell surface marker and C/EBPepsilon was increased when the cells were treated for 4 days with 0.3 microm AF and a physiological concentration of all-trans retinoic acid (ATRA, 5 nm). Treatment with AF in combination with ATRA markedly increased the number of cells with differentiated features, such as lobed or multiple nuclei and numerous granules and vacuoles. At these low concentrations, neither AF nor ATRA alone induced significant cell differentiation. 5. These findings suggest not only that AF induces caspase-3-dependent apoptosis via a mechanism involving ROS, but also that the combined treatment with AF and ATRA induces differentiation of NB4 cells. Our results demonstrate a novel characteristic of AF from which an effective drug treatment of APL might be developed.
Subject(s)
Apoptosis/drug effects , Auranofin/adverse effects , Drug Therapy, Combination , Leukemia, Promyelocytic, Acute/drug therapy , Tretinoin/therapeutic use , Acetylcysteine/pharmacology , Antigens, Surface/drug effects , Antigens, Surface/genetics , Antirheumatic Agents/adverse effects , Antirheumatic Agents/antagonists & inhibitors , Antirheumatic Agents/chemistry , Apoptosis/physiology , Auranofin/antagonists & inhibitors , Auranofin/chemistry , Benzimidazoles , CD11b Antigen/genetics , CD11b Antigen/metabolism , Caspase 3 , Caspases/metabolism , Cell Differentiation/drug effects , DNA/chemistry , DNA/drug effects , Fluorescent Dyes , HL-60 Cells , Humans , Korea , Leukemia, Promyelocytic, Acute/pathology , Leukemia, Promyelocytic, Acute/physiopathology , Nucleosomes/chemistry , Nucleosomes/drug effects , Reactive Oxygen Species/metabolism , Signal Transduction/physiology , Tretinoin/pharmacology , Tumor Cells, CulturedABSTRACT
Metallothioneins (MTs) are low molecular weight, thiol-rich, metal-binding proteins. Auranofin (AF) is a gold compound active in the treatment of rheumatoid arthritis. The effects of AF on regulation of MT gene expression in Chinese hamster ovary cells were studied. AF-resistant cells accumulated substantial amounts of MT mRNA and protein, whereas no induction was observed in AF-sensitive cells. Cells capable of inducing MT in the presence of AF were much less sensitive to AF-mediated cytotoxicity. Induction of MT by low concentrations of Cd protected cells from subsequently administered doses of AF. The level of protection correlated with the level of induced MT. These findings indicate that MT plays a central role in the mechanisms underlying cellular resistance to gold compounds.
