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Journal of Virology, November 2004, p. 11865-11878, Vol. 78, No. 21
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.21.11865-11878.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Calcium-Dependent Calpain Proteases Are Implicated in Processing of the Hepatitis C Virus NS5A Protein

M. Kalamvoki and P. Mavromara^*

Molecular Virology Laboratory, Hellenic Pasteur Institute, Athens, Greece

Received 7 April 2004/ Accepted 17 July 2004

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.

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* Corresponding author. Mailing address: Hellenic Pasteur Institute, Laboratory of Molecular Virology, 127 Vas. Sofias Ave., Athens, Greece 115 21. Phone: 32 10 6478875. Fax: 32 10 6478877. E-mail: penelopm{at}hol.gr.

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Journal of Virology, November 2004, p. 11865-11878, Vol. 78, No. 21
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.21.11865-11878.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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