Hepatitis E Virus Protease Inhibits the Activity of Eukaryotic Initiation Factor 2-Alpha Kinase 4 and Promotes Virus Survival.

Multiple mechanisms exist in a cell to cope with stress. Four independent stress-sensing kinases constitute the integrated stress response machinery of the mammalian cell, and they sense the stress signals and act by phosphorylating the eukaryotic initiation factor 2α (eIF2α) to arrest cellular translation. Eukaryotic initiation factor 2 alpha kinase 4 (eIF2AK4) is one of the four kinases and is activated under conditions of amino acid starvation, UV radiation, or RNA virus infection, resulting in shutdown of global translation. An earlier study in our laboratory constructed the protein interaction network of the hepatitis E virus (HEV) and identified eIF2AK4 as a host interaction partner of the genotype 1 (g1) HEV protease (PCP). Here, we report that PCP's association with the eIF2AK4 results in inhibition of self-association and concomitant loss of kinase activity of eIF2AK4. Site-directed mutagenesis of the 53rd phenylalanine residue of PCP abolishes its interaction with the eIF2AK4. Further, a genetically engineered HEV-expressing F53A mutant PCP shows poor replication efficiency. Collectively, these data identify an additional property of the g1-HEV PCP protein, through which it helps the virus in antagonizing eIF2AK4-mediated phosphorylation of the eIF2α, thus contributing to uninterrupted synthesis of viral proteins in the infected cells. IMPORTANCE Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in humans. It causes chronic infection in organ transplant patients. Although the disease is self-limiting in normal individuals, it is associated with high mortality (~30%) in pregnant women. In an earlier study, we identified the interaction between the genotype 1 HEV protease (PCP) and cellular eukaryotic initiation factor 2 alpha kinase 4 (eIF2AK4). Since eIF2AK4 is a sensor of the cellular integrated stress response machinery, we evaluated the significance of the interaction between PCP and eIF2AK4. Here, we show that PCP competitively associates with and interferes with self-association of the eIF2AK4, thereby inhibiting its kinase activity. Lack of eIF2AK4 activity prevents phosphorylation-mediated inactivation of the cellular eIF2α, which is essential for initiation of cap-dependent translation. Thus, PCP behaves as a proviral factor, promoting uninterrupted synthesis of viral proteins in infected cells, which is crucial for survival and proliferation of the virus.

Molecular docking and dynamic simulation analysis of Hepatitis E virus protease in complexing with the E64 inhibitor.

The unavailability of a suitable treatment for human Hepatitis E virus (HEV) infection necessitate the development of anti HEV drugs. The HEV papain-like cysteine proteases (HEV PCP) is a crucial target to prevent viral replication and progression. E64 is a known HEV PCP inhibitor; however, its molecular mechanism of inhibition is not yet known. Since the crystal structure of HEV PCP is not available, the primary focuses of the present study was to refine the predicted HEV PCP structural model by molecular dynamics (MD) simulation. Further, we performed a 200 ns MD simulation to understand the structural complexity of HEV PCP and the effect of E64 binding with HEV PCP. The E64 binding with active site residues Gln48, Thr51, Gln55, Cys52, Ser81, Gln 98, Cys 132, Arg158, His159, Asn 160 and Ala96 leads to reduced fluctuations in the residue at N-terminal (18-41) that include the CHC motif (26-28). However, most of the other non interacting residues, including the inter-domain linker region (46-87), showed increased fluctuations in the HEV PCP-E64 complex. The residue Asp21 and Ala96 are involved in the formation of interdomain interactions in the HEV PCP apo enzyme. While in the PCP-E64 complex, E64 binds to Ala96 and creates a steric hindrance to prevent interdomain interactions. Thus, the E64 binding reduces interdomain interactions and restrict domain movements in the HEV PCP-E64 complex. This information will be important for the chemically designing more effective derivatives of E64 developing HEV PCP specific inhibitors.Communicated by Ramaswamy H. Sarma.

Methyltransferase of a cell culture-adapted hepatitis E inhibits the MDA5 receptor signaling pathway.

Hepatitis E virus (HEV) is a causative agent of acute hepatitis and jaundice. The number of human infections is approximated to be over 20 million cases per year. The transmission is mainly via the fecal-oral route and contaminated water and food are considered to be a major source of infection. As a mouse model is not available, a recent development of a cell culture-adapted HEV strain (47832c) is considered as a very important tools for molecular analysis of HEV pathogenesis in cells. Previously, we demonstrated that HEV-encoded methyltransferase (MeT) encoded by the 47832c strain inhibits MDA5- and RIG-I-mediated activation of interferon ß (IFN-ß) promoter. Here, we report that MeT impairs the phosphorylation and activation of interferon regulatory factor 3 and the p65 subunit of NF-κB in a dose-dependent manner. In addition, the MeT encoded by the 47832c, but not that of HEV clinical or field isolates (SAR-55, Mex-14, KC-1, and ZJ-1), displays the inhibitory effect. A deeper understanding of MeTmediated suppression of IFN-ß expression would provide basis of the cell culture adaptation of HEV.

Hepatitis E virus polymerase binds to IFIT1 to protect the viral RNA from IFIT1-mediated translation inhibition.

Hepatitis E virus (HEV) induces interferons and regulates the induction of interferon-stimulated genes (ISGs) in the host cell. HEV infection has been shown to promote the expression of different ISGs, such as ISG15, IFIT1, MX1, RSAD2/Viperin and CxCL10, in cell culture and animal models. Interferon-induced protein with tetratricopeptide repeat 1 (IFIT1) is an ISG-encoded protein that inhibits the translation of viral RNA, having 5'-triphosphate or the mRNA lacking 2'-O-methylation on the 5'cap. In this study, we found that IFIT1 binds to HEV RNA to inhibit its translation. HEV replication is also restricted in hepatoma cells with overexpressed IFIT1. However, despite this binding of IFIT1 to HEV RNA, HEV successfully replicates in hepatoma cells in the infection scenario. In an effort to identify the underlying mechanism, we found that HEV RNA-dependent RNA polymerase (RdRp) binds to IFIT1, thereby protecting the viral RNA from IFIT1-mediated translation inhibition. RdRp sequesters IFIT1, resulting in the successful progression of viral replication in the infected cells. Thus, we discovered a distinct pro-viral role of HEV RdRp that is crucial for successful infection in the host, and propose a unique mechanism developed by HEV to overcome IFIT1-mediated host immune response.

Hepatitis E Virus Cysteine Protease Has Papain Like Properties Validated by in silico Modeling and Cell-Free Inhibition Assays.

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.

Hepatitis E Virus Papain-Like Cysteine Protease Inhibits Type I Interferon Induction by Down-Regulating Melanoma Differentiation-Associated Gene 5.

Upon viral infection, the host cell recognizes the invasion through a number of pattern recognition receptors. Melanoma differentiation associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I) recognize RNA molecules derived from invading viruses, activating down-stream signaling cascades, culminating in the induction of the type I interferon. On the other hand, viruses have evolved to evade type I interferon-mediated inhibition. Hepatitis E virus has been shown to encode a few antagonists of type I interferon and it is not surprising that viruses encode multiple mechanisms of viral evasion. In the present study, we demonstrated that HEV PCP strongly down-regulates MDA5-mediated activation of interferon ß induction in a dose-dependent manner. Interestingly, MDA5 protein expression was almost completely abolished. In addition, polyinosinic polycytidylic acid (poly(I:C))- and Sendai virus-mediated activation of type I interferon responses were similarly abrogated in the presence of HEV PCP. Furthermore, HEV PCP down-regulates several molecules that play critical roles in the induction of type I IFN expression. Taken together, these data collectively suggest that HEV-encoded PCP is a strong antagonist of type I interferon.

Molecular Analysis and Modeling of Hepatitis E Virus Helicase and Identification of Novel Inhibitors by Virtual Screening.

The hepatitis E virus- (HEV-) helicase as a novel drug-target was evaluated. While cell culture model was used for mutational characterization of helicase, in silico protein modeling and virtual screening were employed to identify helicase inhibitors. None of the saturation mutant replicons significantly affected RNA replication. Notably, mutants encompassing the Walker motifs replicated as wild-type, showing indispensability of nucleotides conservation in viability compared to known criticality of amino acids. A 3D modeling of HEV-helicase and screening of a compound dataset identified ten most promising inhibitors with drug likeness, notably, JFD02650, RDR03130, and HTS11136 that interacted with Walker A residues Gly975, Gly978, Ser979, and Gly980. Our model building and virtual identification of novel helicase inhibitors warrant further studies towards developing anti-HEV drugs.

Hepatitis E Virus Methyltransferase Inhibits Type I Interferon Induction by Targeting RIG-I.

The type I interferons (IFNs) play a vital role in activation of innate immunity in response to viral infection. Accordingly, viruses have evolved to employ various survival strategies to evade innate immune responses induced by type I IFNs. For example, HEV encoded papainlike cysteine protease (PCP) has been shown to inhibit IFN activation signaling by suppressing K63-linked de-ubiquitination of retinoic acid-inducible gene I (RIG-I) and TANK-binding kinase 1 (TBK1), thus effectively inhibiting down-stream activation of IFN signaling. In present study, we demonstrated that hepatitis E virus (HEV) inhibits poly inosinicpolycytidylic acid (poly(I:C))-induced IFN-ß transcriptional induction. Moreover, by using reporter assay with individual HEV-encoded gene, we showed that HEV methyltransferase (MeT), a non-structural protein, significantly decreases RIG-I-induced IFN-ß induction and NF-κB signaling activities in a dose-dependent manner. Taken together, we report here that MeT, along with PCP, is responsible for the inhibition of RIG-I-induced activation of type I IFNs, expanding the list of HEV-encoded antagonists of the host innate immunity.

Zinc Salts Block Hepatitis E Virus Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase.

Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going up to 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource-starved countries. No specific antiviral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In a human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-Huh7 cell line-based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveal that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection.IMPORTANCE Hepatitis E virus (HEV) is a public health concern in resource-starved countries due to frequent outbreaks. It is also emerging as a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as ribavirin have been used to treat HEV cases, there are known side effects and limitations of such therapy. Our discovery of the ability of zinc salts to block HEV replication by virtue of their ability to inhibit the activity of viral RdRp is important because these findings pave the way to test the efficacy of zinc supplementation therapy in HEV-infected patients. Since zinc supplementation therapy is known to be safe in healthy individuals and since high-dose zinc is used in the treatment of Wilson's disease, it may be possible to control HEV-associated health problems following a similar treatment regimen.

Report of novel H105R, D29N, V27A mutations in the methyltransferase region of the HEV genome in patients with acute liver failure.

BACKGROUND: The Hepatitis E virus (HEV) has been responsible for major outbreaks in the developing countries affecting millions of people and acute sporadic hepatitis worldwide. The HEV methyltransferase is important for capping the 5'-end of the viral pregenomic RNA which is critical for viral infection. OBJECTIVES: We aimed to assess the substitutional profile in the HEV methyltransferase region in patients with acute liver failure (ALF) and acute viral hepatitis (AVH) from North Indian population and associate the substitutions with the poor outcome of the disease. STUDY DESIGN: HEV RNA was detected and partial region encoding the Methyltransferase domain in the HEV genome was amplified by Reverse Transcriptase(RT-PCR). Viral load of HEV was quantified utilizing Real time PCR.32 representative samples consisting of 16 AVH and 16 ALF were directly sequenced and amino acid changes were compared using Fischer's exact (two-tailed) test. RESULTS: Novel mutations Valine27Alanine (V27A), Aspartate29Asparagine (D29N) and Histidine105Arginine (H105R) mutation corresponding to 107T>C, 115G>A and 341 A>G substitutions respectively were significantly (p<0.0001) obtained in 16/16(100%) ALF patients compared to none (0/16) of the AVH patients. HEV viral load and disease severity parameters corresponding to the samples with D29N and V27A mutations were significantly higher compared to the isolates lacking these mutations while the H105R mutation was associated with decreased viremia. CONCLUSION: The D29N and V27A mutations had significant association with the poor outcome in ALF patients suggesting key role in enhancing HEV replication while the association of H105R mutation with decreased viremia creates interest on its antiviral aspects.

Hepatitis E virus RNA-dependent RNA polymerase: RNA template specificities, recruitment and synthesis.

Hepatitis E virus (HEV) is a positive-sense RNA virus and member of the genus Orthohepevirus in the family Hepeviridae. Although HEV RNA-dependent RNA polymerase (HEV-RdRp) plays an important role in the HEV life cycle, its template specificities are not completely understood. We expressed HEV-RdRp protein with His-tag in a bacterial system and analysed template specificities using different putative cis-regulatory elements in the HEV genome. The enzyme showed highest affinity for the 3' non-coding region (NCR), then for the 5'NCR and least for the putative subgenomic promoter (SgP). The enzyme could co-bind to 3'NCR and putative SgP templates together, as evident from the supershift in binding assay, indicating presence of different binding sites for these elements. Proteomic analysis revealed that the RNA elements share two common peptides for binding, while a third peptide, which is highly conserved across different HEV genotypes, is specific for 3'NCR. We propose that, during the early phases of replication, as negative sense antigenome copies accumulate at the replication site, they probably initiate promoter swapping from 3'NCR to SgP, to favour synthesis of subgenomic RNA and to prevent synthesis of genomic RNA. The conserved site for 3'NCR binding could be potential antiviral target and needs further evaluation.

Analysis of complete genome sequences and a V239A substitution in the helicase domain of swine hepatitis E virus strains isolated in Canada.

Among Canadian swine HEV strains, only one complete genome sequence has been published so far, and there are no data on the virulence of these strains. A collection of 28 Canadian swine HEV strains was used in this study. After RNA extraction, a portion of ORF2, the 3' end of the helicase domain, and two complete genomes were amplified and sequenced. These two new Canadian complete genomes belonged to two different subtypes and showed 87.5 and 87.7% sequence identity to the Canadian swine HEV strain Arkell. The V239A substitution within the helicase domain, which is associated with increased virulence of genotype 3 HEV, was detected in one Canadian swine HEV strain. However, no human hepatitis E infections have been associated with this strain.

Sequence analysis reveals a conserved extension in the capping enzyme of the alphavirus supergroup, and a homologous domain in nodaviruses.

BACKGROUND: Members of the alphavirus supergroup include human pathogens such as chikungunya virus, hepatitis E virus and rubella virus. They encode a capping enzyme with methyltransferase-guanylyltransferase (MTase-GTase) activity, which is an attractive drug target owing to its unique mechanism. However, its experimental study has proven very difficult. RESULTS: We examined over 50 genera of viruses by sequence analyses. Earlier studies showed that the MTase-GTase contains a "Core" region conserved in sequence. We show that it is followed by a long extension, which we termed "Iceberg" region, whose secondary structure, but not sequence, is strikingly conserved throughout the alphavirus supergroup. Sequence analyses strongly suggest that the minimal capping domain corresponds to the Core and Iceberg regions combined, which is supported by earlier experimental data. The Iceberg region contains all known membrane association sites that contribute to the assembly of viral replication factories. We predict that it may also contain an overlooked, widely conserved membrane-binding amphipathic helix. Unexpectedly, we detected a sequence homolog of the alphavirus MTase-GTase in taxa related to nodaviruses and to chronic bee paralysis virus. The presence of a capping enzyme in nodaviruses is biologically consistent, since they have capped genomes but replicate in the cytoplasm, where no cellular capping enzyme is present. The putative MTase-GTase domain of nodaviruses also contains membrane-binding sites that may drive the assembly of viral replication factories, revealing an unsuspected parallel with the alphavirus supergroup. CONCLUSIONS: Our work will guide the functional analysis of the alphaviral MTase-GTase and the production of domains for structure determination. The identification of a homologous domain in a simple model system, nodaviruses, which replicate in numerous eukaryotic cell systems (yeast, flies, worms, mammals, and plants), can further help crack the function and structure of the enzyme.

The hepatitis E virus ORF1 'X-domain' residues form a putative macrodomain protein/Appr-1″-pase catalytic-site, critical for viral RNA replication.

The hepatitis E virus (HEV) ORF1 gene encodes the non-structural polyprotein wherein the 'X-domain' still remains poorly defined. Cellular X-domain associated macrodomain protein/ADP-ribose-1″-monophosphatase (Appr-1″-pase) activities are also reported in coronaviruses (CoV), including identification of its homologs in alpha and rubella viruses. The present study investigated the role(s) of X-domain residues in HEV replication cycle. In silico analysis showed a high degree of evolutionary conservation of X-domain (a.a. 785-942) a.a. positions wherein the N-terminus residues 'Asn806, Asn809, His812, Gly815, Gly816, and Gly817' formed a potential catalytic-site homolog of CoVAppr-1″-pase. To experimentally test this prediction, X-domain 'active-site' residues were subjected to mutational analysis using the HEV-SAR55 replicon (pSK-GFP). FACS analysis of mutant RNA transfected S10-3 cells showed that Gly816Ala and Gly817Ala constructs completely abrogated HEV replication, similar to their Gly816Val and Gly817Val counterparts. However, 'Gly815Ala' mutant replicated very poorly in contrast to 'Gly815Val' that completely abolished GFP synthesis. Furthermore, while 'Asn806Ala' mutant retained RNA replication, the 'Asn809Ala' and His812Leu mutants showed non-viability. Notably, in a sequential-nucleotide mutation analysis, the dispensability of X-domain in HEV replication at transcriptional level has already been demonstrated (Parvez, 2013b). Taken together, the present data strongly argue for an essential role of X-domain residues (Asn809, His812, Gly816 and Gly817) at post-translational level, indicating its involvement in viral replication. In conclusion, the speculated regulatory role of ORF1 X-domain in HEV replication cycle critically depends on the 'Asn, Asn, His, Gly, Gly, Gly' segment/secondary structure. Nevertheless, further biochemical or biophysical characterizations of HEV X-domain associated Appr-1″-pase activity would only confirm its biological significance in virus or host-pathogenesis.

A mutation in the hepatitis E virus RNA polymerase promotes its replication and associates with ribavirin treatment failure in organ transplant recipients.

We analyzed blood samples collected from 15 patients with chronic hepatitis E who were recipients of solid-organ transplants. All patients cleared the hepatitis E virus (HEV) except for 2 (nonresponders); 1 patient died. A G1634R mutation in viral polymerase was detected in the HEV RNA of the nonresponders; this mutation did not provide the virus with resistance to ribavirin in vitro. However, the mutant form of a subgenomic replicon of genotype 3 HEV replicated more efficiently in vitro than HEV without this mutation, and the same was true for infectious virus, including in competition assays. Similar results were obtained for genotype 1 HEV. The G1634R mutation therefore appears to increase the replicative capacity of HEV in the human liver and hence reduce the efficacy of ribavirin.

Hepatitis E virus (HEV) protease: a chymotrypsin-like enzyme that processes both non-structural (pORF1) and capsid (pORF2) protein.

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.

Analysis of helicase domain mutations in the hepatitis E virus derived from patients with fulminant hepatic failure: effects on enzymatic activities and virus replication.

Fulminant hepatic failure (FHF) is the severe form of hepatitis E virus infection. Virus sequence analyses from severe cases have shown presence of unique and highly conserved mutations in the helicase domain of genotype 1, 3 and 4 viruses. We evaluated role of two amino acid replacements (L1110F) and (V1120I); found to be frequent in genotype 1 FHF-E viruses from India. Three mutant helicase proteins (two with single point mutations and one with dual mutations) were expressed in Escherichia coli and evaluated for their ATPase and RNA unwinding activities. Both L1110F and V1120I helicase mutants showed marginal decrease in ATPase activity, while L1110F/V1120I dual mutant showed normal ATPase activity. All three mutants proteins showed RNA unwinding activities comparable to wild type protein. Corresponding mutations were made in the helicase domain of HEV RLuc replicon and replication efficiencies were tested in the S10-3 (Huh 7) cells. The mutant replicon V1120I showed lower replication as compared to L1110F and L1110F/V1120I mutants. However, all three replicon mutants showed lower replication efficiencies as compared to the wild type replicon. Walker A and Walker B motif mutant HEV replicons were unable to replicate indicating essential role of the virus encoded helicase domain during HEV replication. FHF-E associated helicase mutations resulted in only marginal decrease in the virus replication suggesting alternate function/s of the helicase protein. Mutations in the helicase domain of FHF-E viruses may be responsible for changing virus or host-virus protein-protein interactions, causing alterations in the host responses, eventually leading to more severe disease manifestations.

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

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 ß-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 'ß-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.

Mutagenesis of hepatitis E virus helicase motifs: effects on enzyme activity.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is a non-enveloped RNA virus. The open reading frame 1 encoded non-structural polyprotein has putative domains for methyltransferase, cysteine protease, helicase and RNA-dependent RNA polymerase, however processing of this polyprotein is still uncertain. HEV helicase belongs to superfamily 1 and has all seven conserved motifs typical of the family. NTPase and RNA duplex unwinding activities of HEV helicase domain were recently demonstrated by us. A non-radioactive RNA unwinding assay was developed using biotin and digoxigenin labeled duplex RNA substrate with 5' overhangs for measuring strand displacement activity of the helicase. A series of deletion mutants were constructed to investigate role of individual motifs in the enzymatic activities. Deletion mutants for motif M I and M IV showed increase in ATPase activity. Deletion mutant M VI retained ATPase activity comparable to wild type protein. Mutant M II showed reduced ATPase activity (P=0.003) with no significant decrease in unwinding activity while mutants M Ia and M III showed major reduction of both ATPase and unwinding activities indicating crucial role of these motifs in the helicase function. Overall analysis of deletion mutants showed that Motif I, IV, V and VI have alternative motifs to carry out enzymatic functions of the protein while motifs Ia and III are critical as well as unique motifs in the protein. Knowing the important role of helicase protein during positive sense RNA virus replication, these unique motifs could be good antiviral targets.

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

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.