title: Hepatitis E virus (HEV) protease: a chymotrypsin-like enzyme that processes both non-structural (pORF1) and capsid (pORF2) protein.
abstract: Hepatitis E virus (HEV), a major cause of acute viral hepatitis across the world, is a non-enveloped, plus-strand RNA virus. Its genome codes three proteins, pORF1 (multifunctional polyprotein), pORF2 (capsid protein) and pORF3 (multi-regulatory protein). pORF1 encodes methyltransferase, putative papain-like cysteine protease, helicase and replicase enzymes. Of these, the protease domain has not been characterized. On the basis of sequence analysis, we cloned and expressed a protein covering aa 440-610 of pORF1, expression of which led to cell death in Escherichia coli BL-21 and Huh7 hepatoma cells. Finally, we expressed and purified this protein from E. coli C43 cells (resistant to toxic proteins). The refolded form of this protein showed protease activity in gelatin zymography. Digestion assays showed cleavage of both pORF1 and pORF2 as observed previously. MS revealed digestion of capsid protein at both the N and C termini. N-terminal sequencing of the ~35 kDa methyltransferase, ~35 kDa replicase and ~56 kDa pORF2 proteins released by protease digestion revealed that the cleavage sites were alanine15/isoleucine16, alanine1364/valine1365 in pORF1 and leucine197/valine198 in pORF2. Specificity of these cleavage sites was validated by site-directed mutagenesis. Further characterization of the HEV protease, carried out using twelve inhibitors, showed chymostatin and PMSF to be the most efficient inhibitors, indicating this protein as a chymotrypsin-like protease. The specificity was further confirmed by cleavage of the chymotrypsin-specific fluorogenic peptide N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. Mutational analysis of the conserved serine/cysteine/histidine residues suggested that H443 and C472/C481/C483 are possibly the active site residues. To our knowledge, this is the first direct demonstration of HEV protease and its function.
authors.affiliation: Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.
authors.forename: Daizy
authors.initials: D
authors.lastname: Paliwal
authors.affiliation: Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.
authors.forename: Subrat Kumar
authors.initials: SK
authors.lastname: Panda
authors.affiliation: Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.
authors.forename: Neeraj
authors.initials: N
authors.lastname: Kapur
authors.affiliation: Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.
authors.forename: Satya Pavan Kumar
authors.initials: SPK
authors.lastname: Varma
authors.affiliation: Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.
authors.forename: Hemlata
authors.initials: H
authors.lastname: Durgapal
mesh_terms: D036022:Capsid Proteins / Q000378:metabolism*; D020134:Catalytic Domain; D002460:Cell Line; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D016752:Hepatitis E virus / Q000201:enzymology* / Q000235:genetics; D022781:Hepatocytes; D006801:Humans; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
chemical_list: D036022:Capsid Proteins; D017361:Viral Nonstructural Proteins; C011545:semen liquefaction factor; D010447:Peptide Hydrolases