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Molecular modeling and analysis of hepatitis E virus (HEV) papain-like cysteine protease.

ABSTRACT: The biochemical or biophysical characterization of a papain-like cysteine protease in HEV ORF1-encoded polyprotein still remains elusive. Very recently, we have demonstrated the indispensability of ORF1 protease-domain cysteines and histidines in HEV replication, ex vivo (Parvez, 2013). In this report, the polyprotein partial sequences of HEV strains and genetically-related RNA viruses were analyzed, in silico. Employing the consensus-prediction results of RUBV-p(150) protease as structural-template, a 3D model of HEV-protease was deduced. Similar to RUBV-p(150), a 'papain-like ?-barrel fold' structurally confirmed the classification of HEV-protease. Further, we recognized a catalytic 'Cys434-His443' dyad homologue of RUBV-p(150) (Cys1152-His1273) and FMDV-L(pro) (Cys51-His148) in line with our previous mutational analysis that showed essentiality of 'His443' but not 'His590' in HEV viability. Moreover, a RUBV 'Zn(2+) binding motif' (Cys1167-Cys1175-Cys1178-Cys1225-Cys1227) equivalent of HEV was identified as 'Cys457-His458-Cys459 and Cys481-Cys483' residues within the '?-barrel fold'. Notably, unlike RUBV, 'His458' also clustered therein, that was in conformity with the consensus cysteine protease 'Zn(2+)-binding motif'. By homology, we also proposed an overlapping 'Ca(2+)-binding site' 'D-X-[DNS]-[ILVFYW]-[DEN]-G-[GP]-XX-DE' signature, and a 'proline-rich motif' interacting 'tryptophan (W437-W472)' module in the modeled structure. Our analysis of the predicted model therefore, warrants critical roles of the 'catalytic dyad' and 'divalent metal-binding motifs' in HEV protease structural-integrity, ORF1 self-processing, and RNA replication. This however, needs further experimental validations.

SUBMITTER: Parvez MK

PROVIDER: S-EPMC7114377 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Molecular modeling and analysis of hepatitis E virus (HEV) papain-like cysteine protease.

Parvez Mohammad Khalid MK Khan Azmat Ali AA

Virus research 20131207

The biochemical or biophysical characterization of a papain-like cysteine protease in HEV ORF1-encoded polyprotein still remains elusive. Very recently, we have demonstrated the indispensability of ORF1 protease-domain cysteines and histidines in HEV replication, ex vivo (Parvez, 2013). In this report, the polyprotein partial sequences of HEV strains and genetically-related RNA viruses were analyzed, in silico. Employing the consensus-prediction results of RUBV-p(150) protease as structural-temp ...[more]

PMID: 24321124

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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 modeling and analysis of hepatitis E virus (HEV) papain-like cysteine protease.

Publications

Molecular modeling and analysis of hepatitis E virus (HEV) papain-like cysteine protease.

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