ABSTRACT
The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV.
Subject(s)
Apoptosis , Caspases/metabolism , Viral Nonstructural Proteins/physiology , West Nile virus/physiology , Amino Acid Sequence , Animals , Caspase 8 , Cell Line , Cell Membrane/pathology , Cell Membrane/ultrastructure , Cell Nucleus/pathology , Cell Nucleus/ultrastructure , Crows/virology , DNA Helicases/genetics , DNA Helicases/physiology , Enzyme Inhibitors/pharmacology , Gene Silencing , Humans , Molecular Sequence Data , Peptide Hydrolases/genetics , Peptide Hydrolases/physiology , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , RNA, Small Interfering/metabolism , Transfection , Vacuoles/pathology , Vacuoles/ultrastructure , Viral Nonstructural Proteins/genetics , West Nile Fever/veterinary , West Nile Fever/virology , West Nile virus/genetics , West Nile virus/isolation & purificationABSTRACT
West Nile virus (WNV) is a member of the Flaviviridae family of vector-borne pathogens. Clinical signs of WNV infection include neurologic symptoms, limb weakness, and encephalitis, which can result in paralysis or death. We report that the WNV-capsid by itself induces rapid nuclear condensation and cell death in tissue culture. Apoptosis is induced through the mitochondrial pathway resulting in caspase-9 activation and downstream caspase-3 activation. Capsid gene delivery into the striatum of mouse brain or interskeletal muscle resulted in cell death and inflammation, likely through capsid-induced apoptosis in vivo. These studies demonstrate that the capsid protein of WNV may be responsible for aspects of viral pathogenesis through induction of the apoptotic cascade.
