scholarly journals NTPase and 5′ to 3′ RNA Duplex-Unwinding Activities of the Hepatitis E Virus Helicase Domain

2010 ◽  
Vol 84 (7) ◽  
pp. 3595-3602 ◽  
Author(s):  
Yogesh A. Karpe ◽  
Kavita S. Lole
Keyword(s):  
Atpase Activity ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Rna Helicase ◽  
Binding Motif ◽  
Helicase Domain ◽  
Helicase Activity ◽  
Reading Frame ◽  
Rna Duplexes ◽  
Multiple Domains

ABSTRACT Hepatitis E virus (HEV) is a causative agent of acute hepatitis, and it is the sole member of the genus Hepevirus in the family Hepeviridae. The open reading frame 1 (ORF1) protein of HEV encodes nonstructural polyprotein with putative domains for methyltransferase, cysteine protease, helicase and RNA-dependent RNA polymerase. It is not yet known whether ORF1 functions as a single protein with multiple domains or is processed to form separate functional units. On the basis of amino acid conserved motifs, HEV helicase has been grouped into helicase superfamily 1 (SF-1). In order to examine the RNA helicase activity of the NTPase/helicase domain of HEV, the region (amino acids 960 to 1204) was cloned and expressed as histidine-tagged protein in Escherichia coli (HEV Hel) and purified. HEV Hel exhibited NTPase and RNA unwinding activities. Enzyme hydrolyzed all rNTPs efficiently, dATP and dCTP with moderate efficiency, while it showed less hydrolysis of dGTP and dTTP. Enzyme showed unwinding of only RNA duplexes with 5′ overhangs showing 5′-to-3′ polarity. We also expressed and purified two HEV Hel mutants. Helicase mutant I, with substitution in the nucleotide-binding motif I (GKS to GAS), showed 30% ATPase activity. Helicase mutant II, with substitutions in the Mg2+ binding motif II (DEAP to AAAP), showed 50% ATPase activity. Both mutants completely lost ability to unwind RNA duplexes with 5′ overhangs. These findings represent the first report demonstrating NTPase/RNA helicase activity of the helicase domain of HEV ORF1.

scholarly journals RNA 5′-Triphosphatase Activity of the Hepatitis E Virus Helicase Domain

2010 ◽  
Vol 84 (18) ◽  
pp. 9637-9641 ◽  
Author(s):  
Yogesh A. Karpe ◽  
Kavita S. Lole
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Open Reading Frame ◽  
Helicase Domain ◽  
Reading Frame ◽  
Essential Step ◽  
Positive Sense ◽  
Rna Triphosphatase ◽  
Rna Duplexes ◽  
Open Reading Frame 1

ABSTRACT Hepatitis E virus (HEV) has a positive-sense RNA genome with a 5′-m7G cap. HEV open reading frame 1 (ORF1) encodes a polyprotein with multiple enzyme domains required for replication. HEV helicase is a nucleoside triphosphatase (NTPase) with the ability to unwind RNA duplexes in the 5′-to-3′ direction. When incubated with 5′-[γ-32P]RNA and 5′-[α-32P]RNA, HEV helicase released 32P only from 5′-[γ-32P]RNA, showing specificity for the γ-β-triphosphate bond. Removal of γ-phosphate from the 5′ end of the primary transcripts (pppRNA to ppRNA) by RNA triphosphatase is an essential step during cap formation. It is suggested that HEV employs the helicase to mediate the first step of 5′ cap synthesis.

scholarly journals Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase

2001 ◽  
Vol 75 (17) ◽  
pp. 8289-8297 ◽  
Author(s):  
Chun-Ling Tai ◽  
Wen-Ching Pan ◽  
Shwu-Huey Liaw ◽  
Ueng-Cheng Yang ◽  
Lih-Hwa Hwang ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Atpase Activity ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Rna Helicase ◽  
Helicase Domain ◽  
Helicase Activity ◽  
Conserved Sequence

ABSTRACT The carboxyl terminus of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole microenvironment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3.

scholarly journals Hepatitis E and Pregnancy: An Unholy Alliance Unmasked from Kashmir, India

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1329
Author(s):  
Mohammad Sultan Khuroo
Keyword(s):  
Viral Replication ◽  
Pregnant Women ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Polymerase Activity ◽  
Severe Liver ◽  
Reading Frame ◽  
Multiple Factors ◽  
Severe Liver Disease ◽  
Development And Management

The adverse relationship between viral hepatitis and pregnancy in developing countries had been interpreted as a reflection of retrospectively biased hospital-based data collection by the West. However, the discovery of hepatitis E virus (HEV) as the etiological agent of an epidemic of non-A, non-B hepatitis in Kashmir, and the documenting of the increased incidence and severity of hepatitis E in pregnancy via a house-to-house survey, unmasked this unholy alliance. In the Hepeviridae family, HEV-genotype (gt)1 from genus Orthohepevirus A has a unique open reading frame (ORF)4-encoded protein which enhances viral polymerase activity and viral replication. The epidemics caused by HEV-gt1, but not any other Orthohepevirus A genotype, show an adverse relationship with pregnancy in humans. The pathogenesis of the association is complex and at present not well understood. Possibly multiple factors play a role in causing severe liver disease in the pregnant women including infection and damage to the maternal-fetal interface by HEV-gt1; vertical transmission of HEV to fetus causing severe fetal/neonatal hepatitis; and combined viral and hormone related immune dysfunction of diverse nature in the pregnant women, promoting viral replication. Management is multidisciplinary and needs a close watch for the development and management of acute liver failure. (ALF). Preliminary data suggest beneficial maternal outcomes by early termination of pregnancy in patients with lower grades of encephalopathy.

scholarly journals Antigenic Characterization of ORF2 and ORF3 Proteins of Hepatitis E Virus (HEV)

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1385
Author(s):  
Giulia Pezzoni ◽  
Lidia Stercoli ◽  
Eleonora Pegoiani ◽  
Emiliana Brocchi
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Recombinant Antigen ◽  
Open Reading Frame ◽  
Reading Frame ◽  
E Coli ◽  
Antigenic Characterization ◽  
Antigenic Properties ◽  
Open Reading Frame 2

To evaluate the antigenic properties of Hepatitis E Virus (HEV) Open Reading Frame 2 and 3 (ORF2 and ORF3) codified proteins, we expressed different portions of ORF2 and the entire ORF3 in E. coli, a truncated ORF2, was also expressed in baculovirus. A panel of 37 monoclonal antibodies (MAbs) was raised against ORF2 (1–660 amino acids) and MAbs were mapped and characterized using the ORF2 expressed portions. Selected HEV positive and negative swine sera were used to evaluate ORF2 and ORF3 antigens’ immunogenicity. The MAbs were clustered in six groups identifying six antigenic regions along the ORF2. Only MAbs binding to the sixth ORF2 antigenic region (394–608 aa) were found to compete with HEV positive sera and efficiently catch the recombinant antigen expressed in baculovirus. The ORF2 portion from 394–608 aa demonstrated to include most immunogenic epitopes with 85% of HEV positive swine sera reacting against the region from 461–544 aa. Only 5% of the selected HEV sera reacted against the ORF3 antigen.

Hepatitis E virus open reading frame 3 protein interacts with porcine liver-specific plasminogen and α2-antiplasmin

2013 ◽  
Vol 86 (3) ◽  
pp. 487-495 ◽  
Author(s):  
Yan Zhou ◽  
Yansheng Geng ◽  
Jun Yang ◽  
Chenyan Zhao ◽  
Tim J. Harrison ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Open Reading Frame ◽  
Porcine Liver ◽  
Reading Frame

scholarly journals Hepatitis E Virus (HEV) Open Reading Frame 2 Antigen Kinetics in Human-Liver Chimeric Mice and Its Impact on HEV Diagnosis

2019 ◽  
Vol 220 (5) ◽  
pp. 811-819 ◽  
Author(s):  
Ibrahim M Sayed ◽  
Lieven Verhoye ◽  
Claire Montpellier ◽  
Florence Abravanel ◽  
Jacques Izopet ◽  
...  
Keyword(s):  
Density Gradient ◽  
Hepatitis E Virus ◽  
Gradient Analysis ◽  
Hepatitis E ◽  
Open Reading Frame ◽  
Viral Rna ◽  
Humanized Mice ◽  
Molecular Diagnostic ◽  
Reading Frame ◽  
Open Reading Frame 2

Abstract Background Hepatitis E virus infection (HEV) is an emerging problem in developed countries. Diagnosis of HEV infection is based on the detection of HEV-specific antibodies, viral RNA, and/or antigen (Ag). Humanized mice were previously reported as a model for the study of HEV infection, but published data were focused on the quantification of viral RNA. However, the kinetics of HEV Ag expression during infection remains poorly understood. Methods Plasma specimens and suspensions of fecal specimens from HEV-infected and ribavirin-treated humanized mice were analyzed using HEV antigen–specific enzyme-linked immunosorbent assay, reverse transcription–quantitative polymerase chain reaction analysis, density gradient analysis, and Western blotting. Result Open reading frame 2 (ORF2) Ag was detected in both plasma and stool from HEV-infected mice, and levels increased over time. Contrary to HEV RNA, ORF2 Ag levels were higher in mouse plasma than in stool. Interestingly, ORF2 was detected in plasma from mice that tested negative for HEV RNA in plasma but positive for HEV RNA in stool and was detected after viral clearance in mice that were treated with ribavirin. Plasma density gradient analysis revealed the presence of the noninfectious glycosylated form of ORF2. Conclusion ORF2 Ag can be used as a marker of active HEV infection and for assessment of the effect of antiviral therapy, especially when fecal samples are not available or molecular diagnostic tests are not accessible.

Primary structure of open reading frame 2 and 3 of the hepatitis E virus isolated from Morocco

1999 ◽  
Vol 57 (2) ◽  
pp. 126-133 ◽  
Author(s):  
Jihong Meng ◽  
Mian-er Cong ◽  
Xing Dai ◽  
Jacques Pillot ◽  
Michael A. Purdy ◽  
...  
Keyword(s):  
Primary Structure ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Open Reading Frame 2

scholarly journals Effect of NS3 and NS5B proteins on classical swine fever virus internal ribosome entry site-mediated translation and its host cellular translation

2008 ◽  
Vol 89 (4) ◽  
pp. 994-999 ◽  
Author(s):  
Ming Xiao ◽  
Yan Bai ◽  
Hui Xu ◽  
Xiaolu Geng ◽  
Jun Chen ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Classical Swine Fever ◽  
Rna Helicase ◽  
Helicase Domain ◽  
Dependent Manner ◽  
Helicase Activity ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Cellular Translation

A full-length NS3 (NS3F) and a truncated NS3 protein (NS3H) with an RNA helicase domain possess RNA helicase activity. Using an in vitro system with a monocistronic reporter RNA or DNA, containing the CSFV 5′-UTR, we observed that both NS3F and NS3H enhanced internal ribosome entry site (IRES)-mediated and cellular translation in a dose-dependent manner, but NS3 protease (NS3P) that lacks a helicase domain did not. NS3F was stronger than NS3H in promoting both translations. These results showed that viral RNA helicase could promote viral and cellular translation, and higher RNA helicase activity might be more efficient. The NS5B protein, the viral replicase, did not significantly affect the IRES-directed or cellular translation alone. NS5B significantly enhanced the stimulative effect of NS3F on both IRES-mediated and cellular translation, but did not affect that of NS3H or NS3P. This suggests that NS5B and NS3 interact via the protease domain during the enhancement of translation.

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