Abstract
Hepatitis E virus (HEV) has emerged as a global health concern during the last decade. In spite of a high mortality rate in pregnant women with fulminant hepatitis, no antiviral drugs or licensed vaccine is available in India. HEV-protease is a pivotal enzyme responsible for ORF1 polyprotein processing leading to cleavage of the non-structural enzymes involved in virus replication. HEV-protease region encoding 432–592 amino acids of Genotype-1 was amplified, expressed in Sf21 cells and purified in its native form. The recombinant enzyme was biochemically characterized using SDS-PAGE, Western blotting and Immunofluorescence. The enzyme activity and the inhibition studies were conducted using Zymography, FTC-casein based protease assay and ORF1 polyprotein digestion. To conduct ORF1 digestion assay, the polyprotein, natural substrate of HEV-protease, was expressed in E. coli and purified. Cleavage of 186 kDa ORF1 polyprotein by the recombinant HEV-protease lead to appearance of non-structural proteins viz. Methyltransferase, Protease, Helicase and RNA dependent RNA polymerase which were confirmed through immunoblotting using antibodies generated against specific epitopes of the enzymes. FTC-casein substrate was used for kinetic studies to determine Km and Vmax of the enzyme and also the effect of different metal ions and other protease inhibitors. A 95% inhibition was observed with E-64 which was validated through in silico analysis. The correlation coefficient between inhibition and docking score of Inhibitors was found to have a significant value of r2 = 0.75. The predicted 3D model showed two domain architecture structures similar to Papain like cysteine protease though they differed in arrangements of alpha helices and beta sheets. Hence, we propose that HEV-protease has characteristics of “Papain-like cysteine protease,” as determined through structural homology, active site residues and class-specific inhibition. However, conclusive nature of the enzyme remains to be established.
Highlights
Hepatitis E virus (HEV) is one of the most important viruses responsible for water born epidemics (Kamar et al, 2014)
We developed a robust cell free assay with high sensitivity, selectivity and reproducibility to screen targeted compounds with anti-HEV protease activity
Positive-strand RNA viruses are supposed to encode proteases to process their polyproteins usually required for enzymatic activities, interactions with other proteins, subcellular localization, and the viral assembly (Debing et al, 2016)
Summary
Hepatitis E virus (HEV) is one of the most important viruses responsible for water born epidemics (Kamar et al, 2014). Computational analysis of ORF1 has identified seven putative domains (Koonin et al, 1992) These include an active methyltransferase domain (Met), Y domain (Y) (Parvez, 2017), papain-like cysteine protease (PCP) (Parvez, 2013; Paliwal et al, 2014), a proline -rich region that contains a hypervariable region (H), X -domain (X), helicase (Hel), and an RNA dependent RNA polymerase (RdRP) From N- to C-terminal (Koonin et al, 1992; Parvez, 2017). Mutation in conserved cysteine and histidine residues in the putative protease inhibits its proteolytic activity indicating its role in ORF1 processing and viral replication (Paliwal et al, 2014). This study significantly advances our understanding of the structure and function of HEV protease
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