Calcium-dependent calpain proteases are implicated in processing of the hepatitis C virus NS5A protein.
The nonstructural 5A (NS5A) protein of the hepatitis C virus (HCV) is a multifunctional phosphoprotein that is implicated in viral replication and HCV-mediated pathogenesis. We report here that the NS5A protein from the HCV genotype 1a is processed into shorter distinct forms when expressed in mammalian cells (Vero, HepG2, HuH-7, and WRL68) infected with an NS5A-expressing HSV-1-based amplicon vector or when transiently transfected with NS5A-expressing plasmids in the absence of exogenous apoptotic stimuli. Inhibitor studies combined with cell-free cleavage assays suggest that calcium-dependent calpain proteases, in addition to caspase-like proteases, are involved in NS5A processing. Interestingly, His-tagging experiments indicated that all the detectable NS5A-cleaved products are N-terminal forms of the protein. Additionally, immunofluorescence studies showed that, despite proteolytic cleavage, the NS5A protein exhibits a cytoplasm-perinuclear localization similar to that of the full-length protein. Thus, our results are consistent with recent data that demonstrated that NS5A is capable of perturbing intracellular calcium homeostasis and suggest that NS5A is both an inducer and a substrate of the calcium-dependent calpain protease(s). This may imply that cleavage of NS5A by calpain(s) could play a role in the modulation of NS5A function.
Hellenic Pasteur Institute, Laboratory of Molecular Virology, 127 Vas. Sofias Ave., Athens, Greece 115 21.
M
M
Kalamvoki
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P
Mavromara
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000502:physiology*; D020169:Caspases / Q000502:physiology; D045744:Cell Line, Tumor; D003546:Cysteine Endopeptidases / Q000502:physiology; D018259:Herpesvirus 1, Human / Q000235:genetics; D006801:Humans; D009097:Multienzyme Complexes / Q000502:physiology; D046988:Proteasome Endopeptidase Complex; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
D002118:Calcium; D002154:Calpain; D020169:Caspases; D003546:Cysteine Endopeptidases; D009097:Multienzyme Complexes; D046988:Proteasome Endopeptidase Complex; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase