Dataset Information

Molecular characterization of hepatitis E virus ORF1 gene supports a papain-like cysteine protease (PCP)-domain activity.

ABSTRACT: Hepatitis E Virus (HEV) ORF1 encodes the nonstructural polyprotein wherein a role of PCP-domain in ORF1 proteolysis and/or RNA replication still remains contested. A series of ORF1 mutants of HEV-SAR55 replicon were constructed and tested for viability in S10-3 cells. Six of PCP-'cysteine' (C457A, C459A, C471A, C472A, C481A and C483A) and three 'histidine' (H443L, H497L and H590L) mutants were lethal. Further, a highly conserved 'glycine-triad' (G815-G816-G817) in downstream X-domain, homologous to rubella virus protease-substrate (G1299-G1300-G1301) was identified where two of X-mutants (G816V and G817V) turned lethal. However, all ORF1 sequential nucleotide-mutants conserving the amino acids were viable, which clearly showed post-translational regulation of HEV replication by PCP- and X-domains. Moreover, while vector-expressed ORF1-fusion polyprotein yielded a ~191 kDa band in vitro, it produced ~78 and ~35 kDa fragments ex vivo. Collectively, the indispensability and functional effects of 'PCP-catalytic' and 'X-substrate' residues on HEV replication strongly supported a viral protease.

SUBMITTER: Parvez MK

PROVIDER: S-EPMC7125709 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Molecular characterization of hepatitis E virus ORF1 gene supports a papain-like cysteine protease (PCP)-domain activity.

Parvez Mohammad Khalid MK

Virus research 20130823 2

Hepatitis E Virus (HEV) ORF1 encodes the nonstructural polyprotein wherein a role of PCP-domain in ORF1 proteolysis and/or RNA replication still remains contested. A series of ORF1 mutants of HEV-SAR55 replicon were constructed and tested for viability in S10-3 cells. Six of PCP-'cysteine' (C457A, C459A, C471A, C472A, C481A and C483A) and three 'histidine' (H443L, H497L and H590L) mutants were lethal. Further, a highly conserved 'glycine-triad' (G815-G816-G817) in downstream X-domain, homologous ...[more]

PMID: 23978667

Similar Datasets

Project description: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 r 2 = 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.

Dataset Information

Molecular characterization of hepatitis E virus ORF1 gene supports a papain-like cysteine protease (PCP)-domain activity.

Publications

Molecular characterization of hepatitis E virus ORF1 gene supports a papain-like cysteine protease (PCP)-domain activity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets