The ORF2 Protein of Hepatitis E Virus Binds the 5′ Region of Viral RNA

ABSTRACT Hepatitis E virus (HEV) is a major human pathogen in much of the developing world. It is a plus-strand RNA virus with a 7.2-kb polyadenylated genome consisting of three open reading frames, ORF1, ORF2, and ORF3. Of these, ORF2 encodes the major capsid protein of the virus and ORF3 encodes a small protein of unknown function. Using the yeast three-hybrid system and traditional biochemical techniques, we have studied the RNA binding activities of ORF2 and ORF3, two proteins encoded in the 3′ structural part of the genome. Since the genomic RNA from HEV has been postulated to contain secondary structures at the 5′ and 3′ ends, we used these two terminal regions, besides other regions within the genome, in this study. Experiments were designed to test for interactions between the genomic RNA fusion constructs with ORF2 and ORF3 hybrid proteins in a yeast cellular environment. We show here that the ORF2 protein contains RNA binding activity. The ORF2 protein specifically bound the 5′ end of the HEV genome. Deletion analysis of this protein showed that its RNA binding activity was lost when deletions were made beyond the N-terminal 111 amino acids. Finer mapping of the interacting RNA revealed that a 76-nucleotide (nt) region at the 5′ end of the HEV genome was responsible for binding the ORF2 protein. This 76-nt region included the 51-nt HEV sequence, conserved across alphaviruses. Our results support the requirement of this conserved sequence for interaction with ORF2 and also indicate an increase in the strength of the RNA-protein interaction when an additional 44 bases downstream of this 76-nt region were included. Secondary-structure predictions and the location of the ORF2 binding region within the HEV genome indicate that this interaction may play a role in viral encapsidation.

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Cytoplasmic Localization of the ORF2 Protein of Hepatitis E Virus Is Dependent on Its Ability To Undergo Retrotranslocation from the Endoplasmic Reticulum

Journal of Virology ◽

10.1128/jvi.02039-06 ◽

2007 ◽

Vol 81 (7) ◽

pp. 3339-3345 ◽

Cited By ~ 55

Author(s):

Milan Surjit ◽

Shahid Jameel ◽

Sunil K. Lal

Keyword(s):

Endoplasmic Reticulum ◽

Hepatitis E Virus ◽

Major Capsid Protein ◽

Rna Virus ◽

Hepatitis E ◽

26S Proteasome ◽

Orf2 Protein ◽

Surface Localization ◽

Positive Strand Rna Virus ◽

Positive Strand Rna

ABSTRACT Hepatitis E virus (HEV) is a positive-strand RNA virus that is prevalent in much of the developing world. ORF2 is the major capsid protein of HEV. Although ORF2 is an N-linked glycoprotein, it is abundantly located in the cytoplasm in addition to having membrane and surface localization. The mechanism by which ORF2 protein obtains access to the cytoplasm is unknown. In this report, we prove that initially all ORF2 protein is present in the endoplasmic reticulum and a fraction of it becomes retrotranslocated to the cytoplasm. The ability of ORF2 to be retrotranslocated is dependent on its glycosylation status and follows the canonical dislocation pathway. However, in contrast to general substrates of the dislocation pathway, retrotranslocated ORF2 protein is not a substrate of the 26S proteasome complex and is readily detectable in the cytoplasm in the absence of any protease inhibitor, suggesting that the retrotranslocated protein is stable in the cytoplasm. This study thus defines the pathway by which ORF2 obtains access to the cytoplasm.

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Activation of CXCL-8 Transcription by Hepatitis E Virus ORF-1 via AP-1

Mediators of Inflammation ◽

10.1155/2015/495370 ◽

2015 ◽

Vol 2015 ◽

pp. 1-6 ◽

Cited By ~ 1

Author(s):

Zhubing Li ◽

Lu Chen ◽

Qiang Liu

Keyword(s):

Transcriptional Activation ◽

Promoter Activity ◽

Hepatitis E Virus ◽

Rna Virus ◽

Ectopic Expression ◽

Hepatitis E ◽

Genomic Rna ◽

Host Interactions ◽

Positive Sense ◽

Binding Sequence

Hepatitis E virus (HEV) is a small nonenveloped single-stranded positive-sense RNA virus and is one of the major causes for acute hepatitis worldwide. CXCL-8 is a small multifunctional proinflammatory chemokine. It was reported recently that HEV infection significantly upregulates CXCL-8 gene expression. In this study, we investigated the mechanism of HEV-induced CXCL-8 transcriptional activation. Using CXCL-8 promoter reporters of different lengths ranging from −1400 to −173, we showed that −173 promoter has the highest promoter activity in the presence of HEV genomic RNA, indicating that the −173 promoter contains sequences responsible for CXCL-8 activation by HEV. Ectopic expression of the ORF-1 protein can upregulate the −173 CXCL-8 promoter activity. In contrast, expression of the ORF-2 protein suppresses the CXCL-8 promoter activity and expression of the ORF-3 protein has no effect on the CXCL-8 promoter activity. We further showed that AP-1 is required for CXCL-8 activation because neither HEV genomic RNA nor the ORF-1 protein can upregulate the −173 CXCL-8 promoter in the absence of the AP-1 binding sequence. Taken together, our results showed that HEV and HEV ORF-1 protein activate the CXCL-8 promoter via AP-1. This novel function of HEV ORF-1 protein should contribute to our understanding of HEV-host interactions and HEV-associated pathogenesis.

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A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses

Journal of Virology ◽

10.1128/jvi.01897-08 ◽

2008 ◽

Vol 83 (4) ◽

pp. 1981-1991 ◽

Cited By ~ 76

Author(s):

Huiquan Liu ◽

Yanping Fu ◽

Daohong Jiang ◽

Guoqing Li ◽

Jun Xie ◽

...

Keyword(s):

Sclerotinia Sclerotiorum ◽

Hepatitis E Virus ◽

Rna Viruses ◽

Rna Virus ◽

Hepatitis E ◽

Genomic Rna ◽

Human Virus ◽

Positive Strand ◽

Positive Strand Rna Virus ◽

Positive Strand Rna

ABSTRACT Previously, we reported that three double-stranded RNA (dsRNA) segments, designated L-, M-, and S-dsRNAs, were detected in Sclerotinia sclerotiorum strain Ep-1PN. Of these, the M-dsRNA segment was derived from the genomic RNA of a potexvirus-like positive-strand RNA virus, Sclerotinia sclerotiorum debilitation-associated RNA virus. Here, we present the complete nucleotide sequence of the L-dsRNA, which is 6,043 nucleotides in length, excluding the poly(A) tail. Sequence analysis revealed the presence of a single open reading frame (nucleotide positions 42 to 5936) that encodes a protein with significant similarity to the replicases of the “alphavirus-like” supergroup of positive-strand RNA viruses. A sequence comparison of the L-dsRNA-encoded putative replicase protein containing conserved methyltransferase, helicase, and RNA-dependent RNA polymerase motifs showed that it has significant sequence similarity to the replicase of Hepatitis E virus, a virus infecting humans. Furthermore, we present convincing evidence that the virus-like L-dsRNA could replicate independently with only a slight impact on growth and virulence of its host. Our results suggest that the L-dsRNA from strain Ep-1PN is derived from the genomic RNA of a positive-strand RNA virus, which we named Sclerotinia sclerotiorum RNA virus L (SsRV-L). As far as we know, this is the first report of a positive-strand RNA mycovirus that is related to a human virus. Phylogenetic and sequence analyses of the conserved motifs of the RNA replicase of SsRV-L showed that it clustered with the rubi-like viruses and that it is related to the plant clostero-, beny- and tobamoviruses and to the insect omegatetraviruses. Considering the fact that these related alphavirus-like positive-strand RNA viruses infect a wide variety of organisms, these findings suggest that the ancestral positive-strand RNA viruses might be of ancient origin and/or they might have radiated horizontally among vertebrates, insects, plants, and fungi.

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

Journal of Virology ◽

10.1128/jvi.00754-17 ◽

2017 ◽

Vol 91 (21) ◽

Cited By ~ 36

Author(s):

Nidhi Kaushik ◽

Chandru Subramani ◽

Saumya Anang ◽

Rajagopalan Muthumohan ◽

Shalimar ◽

...

Keyword(s):

Rna Polymerase ◽

Hepatitis E Virus ◽

Zinc Supplementation ◽

Hepatitis E ◽

Viral Rna ◽

Health Concern ◽

Healthy Individuals ◽

Genomic Rna ◽

Rna Dependent Rna Polymerase ◽

Zinc Salts

ABSTRACT 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.

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The Capsid (ORF2) Protein of Hepatitis E Virus in Feces Is C-Terminally Truncated

Pathogens ◽

10.3390/pathogens11010024 ◽

2021 ◽

Vol 11 (1) ◽

pp. 24

Author(s):

Takashi Nishiyama ◽

Koji Umezawa ◽

Kentaro Yamada ◽

Masaharu Takahashi ◽

Satoshi Kunita ◽

...

Keyword(s):

Amino Acids ◽

Cell Culture ◽

Structure Prediction ◽

Hepatitis E Virus ◽

Culture Media ◽

Molecular Size ◽

Hepatitis E ◽

Terminal Region ◽

Translation Product ◽

Orf2 Protein

The hepatitis E virus (HEV) is a causative agent of hepatitis E. HEV virions in circulating blood and culture media are quasi-enveloped, while those in feces are nonenveloped. The capsid (ORF2) protein associated with an enveloped HEV virion is reported to comprise the translation product of leucine 14/methionine 16 to 660 (C-terminal end). However, the nature of the ORF2 protein associated with fecal HEV remains unclear. In the present study, we compared the molecular size of the ORF2 protein among fecal HEV, cell-culture-generated HEV (HEVcc), and detergent-treated protease-digested HEVcc. The ORF2 proteins associated with fecal HEV were C-terminally truncated and showed the same size as those of the detergent-treated protease-digested HEVcc virions (60 kDa), in contrast to those of the HEVcc (68 kDa). The structure prediction of the ORF2 protein (in line with previous studies) demonstrated that the C-terminal region (54 amino acids) of an ORF2 protein is in flux, suggesting that proteases target this region. The nonenveloped nondigested HEV structure prediction indicates that the C-terminal region of the ORF2 protein moves to the surface of the virion and is unnecessary for HEV infection. Our findings clarify the maturation of nonenveloped HEV and will be useful for studies on the HEV lifecycle.

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Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription

mBio ◽

10.1128/mbio.00769-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 13

Author(s):

Qiang Ding ◽

Ila Nimgaonkar ◽

Nicholas F. Archer ◽

Yaron Bram ◽

Brigitte Heller ◽

...

Keyword(s):

Hepatitis E Virus ◽

Regulatory Element ◽

Hepatitis E ◽

Regulatory Elements ◽

Genome Replication ◽

Genomic Rna ◽

Chronic Infections ◽

Particle Assembly ◽

Subgenomic Rna ◽

General Importance

ABSTRACT Approximately 20 million hepatitis E virus (HEV) infections occur annually in both developing and industrialized countries. Most infections are self-limiting, but they can lead to chronic infections and cirrhosis in immunocompromised patients, and death in pregnant women. The mechanisms of HEV replication remain incompletely understood due to scarcity of adequate experimental platforms. HEV undergoes asymmetric genome replication, but it produces an additional subgenomic (SG) RNA encoding the viral capsid and a viroporin in partially overlapping open reading frames. Using a novel transcomplementation system, we mapped the intragenomic subgenomic promoter regulating SG RNA synthesis. This cis -acting element is highly conserved across all eight HEV genotypes, and when the element is mutated, it abrogates particle assembly and release. Our work defines previously unappreciated viral regulatory elements and provides the first in-depth view of the intracellular genome dynamics of this emerging human pathogen. IMPORTANCE HEV is an emerging pathogen causing severe liver disease. The genetic information of HEV is encoded in RNA. The genomic RNA is initially copied into a complementary, antigenomic RNA that is a template for synthesis of more genomic RNA and for so-called subgenomic RNA. In this study, we identified the precise region within the HEV genome at which the synthesis of the subgenomic RNA is initiated. The nucleotides within this region are conserved across genetically distinct variants of HEV, highlighting the general importance of this segment for the virus. To identify this regulatory element, we developed a new experimental system that is a powerful tool with broad utility to mechanistically dissect many other poorly understood functional elements of HEV.

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Synthetic Peptides Containing Three Neutralizing Epitopes of Genotype 4 Swine Hepatitis E Virus ORF2 induced Protection against Swine HEV Infection in Rabbit

Vaccines ◽

10.3390/vaccines8020178 ◽

2020 ◽

Vol 8 (2) ◽

pp. 178

Author(s):

Yiyang Chen ◽

Tianxiang Chen ◽

Yuhang Luo ◽

Jie Fan ◽

Meimei Zhang ◽

...

Keyword(s):

Hepatitis E Virus ◽

Keyhole Limpet Hemocyanin ◽

Hepatitis E ◽

Genotype 4 ◽

Virus Shedding ◽

Fecal Shedding ◽

Orf2 Protein ◽

Swine Hepatitis ◽

Phosphate Buffered Saline ◽

Neutralizing Epitopes

Genotype 4 hepatitis E virus (HEV) is a zoonotic pathogen transmitted to humans through food and water. Previously, three genotype 4 swine HEV ORF2 peptides (407EPTV410, 410VKLYTS415, and 458PSRPF462) were identified as epitopes of virus-neutralizing monoclonal antibodies that partially blocked rabbit infection with swine HEV. Here, individual and tandem fused peptides were synthesized, conjugated to keyhole limpet hemocyanin (KLH), then evaluated for immunoprotection of rabbits against swine HEV infection. Forty New Zealand White rabbits were randomly assigned to eight groups; groups 1 thru 5 received three immunizations with EPTV-KLH, VKLYTS-KLH, PSRPF-KLH, EPTVKLYTS-KLH, or EPTVKLYTSPSRPF-KLH, respectively; group 6 received truncated swine HEV ORF2 protein (sp239), and group 7 received phosphate-buffered saline. After an intravenous swine HEV challenge, all group 7 rabbits exhibited viremia and fecal virus shedding by 2–4 weeks post challenge (wpc), seroconversion by 4–9 wpc, elevated alanine aminotransferase (ALT) at 2 wpc, and severe liver lymphocytic venous periphlebitis. Only 1–2 rabbits/group in groups 1–4 exhibited delayed viremia, fecal shedding, seroconversion, increased ALT levels, and slight liver lymphocytic venous periphlebitis; groups 5–6 showed no pathogenic effects. Collectively, these results demonstrate that immunization with a polypeptide containing three genotype 4 HEV ORF2 neutralizing epitopes completely protected rabbits against swine HEV infection.

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