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

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
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.

scholarly journals Evidence that the Genomic RNA of Hepatitis E Virus Is Capped

1999 ◽  
Vol 73 (10) ◽  
pp. 8848-8850 ◽  
Author(s):  
Yamina Kabrane-Lazizi ◽  
Xiang-Jin Meng ◽  
Robert H. Purcell ◽  
Suzanne U. Emerson
Keyword(s):  
Monoclonal Antibody ◽  
Reverse Transcription ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Genomic Rna ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Replication Strategy ◽  
Positive Sense ◽  
Rna Genome

ABSTRACT Hepatitis E virus (HEV) is an unclassified virus with a positive-sense RNA genome and an undefined replication strategy. In order to determine whether the HEV genome is capped or not, we developed a reverse transcription-PCR assay that is based on the ability of a monoclonal antibody to recognize 7-methylguanosine (m7G). Antibody to m7G bound RNA extracted from virions of two different HEV genotypes. The cap analog competitively inhibited the binding of virion RNAs, demonstrating that HEV has a capped RNA genome.

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

2008 ◽  
Vol 83 (4) ◽  
pp. 1981-1991 ◽  
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.

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

2004 ◽  
Vol 78 (1) ◽  
pp. 320-328 ◽  
Author(s):  
Milan Surjit ◽  
Shahid Jameel ◽  
Sunil K. Lal
Keyword(s):  
Hepatitis E Virus ◽  
Rna Binding ◽  
Rna Virus ◽  
Hepatitis E ◽  
Binding Activity ◽  
Genomic Rna ◽  
Structural Part ◽  
Conserved Sequence ◽  
Cellular Environment ◽  
Orf2 Protein

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.

scholarly journals Hepatitis E virus: a review

2008 ◽  
Vol 52 (No. 9) ◽  
pp. 365-384 ◽  
Author(s):  
P. Vasickova ◽  
I. Psikal ◽  
P. Kralik ◽  
F. Widen ◽  
Z. Hubalek ◽  
...  
Keyword(s):  
Developing Countries ◽  
Hepatitis E Virus ◽  
Rna Virus ◽  
Hepatitis E ◽  
Oral Route ◽  
Human Infection ◽  
Risk Of Infection ◽  
Potential Source ◽  
Increased Risk ◽  
Positive Sense

The hepatitis E virus (HEV), the causative agent of hepatitis E, is a non-enveloped RNA virus. The HEV genome is formed by a non-segmented positive-sense RNA chain. The 3´end of the chain is polyadenylated and the 5´end is structurally characterised by the so called “capping”. According to currently accepted taxonomy, HEV is classified in the genus <i>Hepevirus</i>, the only member of the Hepeviridae family. HE is usually transmitted via the faecal-oral route due to the fact that drinking water or water for industrial purposes is contaminated due to poor sanitation. This spread of HEV has been reported in developing countries of Asia, Africa, South and Central America. However, cases in countries with the sporadic occurrence of HEV have been associated with travelling to countries with an increased risk of infection (developing countries in Asia, Africa and America). HEV infections have subsequently been described in people who have not travelled to endemic countries. Further studies of the HEV suggested other routes of transmission and a zoonotic potential of the virus (pigs and deer as the potential source of human infection).

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

2017 ◽  
Vol 91 (21) ◽  
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.

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

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
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.

Hepatitis E virus

2011 ◽  
Author(s):  
X. J. Meng
Keyword(s):  
Hepatitis E Virus ◽  
Animal Species ◽  
Rna Virus ◽  
Hepatitis E ◽  
Open Reading Frames ◽  
Industrialized Countries ◽  
Efficient Cell Culture System ◽  
The Family ◽  
Avian Hev ◽  
Animal Strains

Hepatitis E virus (HEV) is a small, non-enveloped, single-strand, positive-sense RNA virus of approximately 7.2 kb in size. HEV is classified in the family Hepeviridae consisting of four recognized major genotypes that infect humans and other animals. Genotypes 1 and 2 HEV are restricted to humans and often associated with large outbreaks and epidemics in developing countries with poor sanitation conditions, whereas genotypes 3 and 4 HEV infect humans, pigs and other animal species and are responsible for sporadic cases of hepatitis E in both developing and industrialized countries. The avian HEV associated with Hepatitis-Splenomegaly syndrome in chickens is genetically and antigenically related to mammalian HEV, and likely represents a new genus in the family. There exist three open reading frames in HEV genome: ORF1 encodes non-structural proteins, ORF2 encodes the capsid protein, and the ORF3 encodes a small phosphoprotein. ORF2 and ORF3 are translated from a single bicistronic mRNA, and overlap each other but neither overlaps ORF1. Due to the lack of an efficient cell culture system and a practical animal model for HEV, the mechanisms of HEV replication and pathogenesis are poorly understood. The recent identification and characterization of animal strains of HEV from pigs and chickens and the demonstrated ability of cross-species infection by these animal strains raise potential public health concerns for zoonotic HEV transmission. It has been shown that the genotypes 3 and 4 HEV strains from pigs can infect humans, and vice versa. Accumulating evidence indicated that hepatitis E is a zoonotic disease, and swine and perhaps other animal species are reservoirs for HEV. A vaccine against HEV is not yet available.

Mechanisms for Transcriptional Activation of the Human Activated Leukocyte Cell Adhesion Molecule Gene and Its Silencing by Immunosuppressive Toxins.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1637-1637 ◽  
Author(s):  
Fang Tan ◽  
Flaubert Mbeunkui ◽  
Crystal Harris ◽  
Solomon F. Ofori-Acquah
Keyword(s):  
Dendritic Cells ◽  
Cell Adhesion ◽  
Inflammatory Response ◽  
Transcriptional Activation ◽  
Promoter Activity ◽  
Cell Adhesion Molecule ◽  
Start Site ◽  
Translational Start Site ◽  
Translational Start ◽  
Binding Sequence

Abstract Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a member of the immunoglobulin super-family. It is expressed on the surfaces of activated monocytes, dendritic cells and macrophages. These immune cells use ALCAM through homotypic and heterotypic adhesions to control multiple stages in the inflammatory response. Indeed, anti-ALCAM antibodies and recombinant soluble ALCAM significantly inhibit monocyte transendothelial migration, stabilization of the immunological synapse and dendritic cell-mediated T-lymphocyte proliferation. Despite this significance, there is currently no understanding of how the human ALCAM gene is regulated. In this study, we identified the mechanisms for transcription, basal transcriptional activation and immunosuppressive silencing of the ALCAM gene. A common site for transcription of the ALCAM gene was identified 350 base pairs (bp) upstream from the translational start site. Multiple truncated fragments of the ALCAM promoter was cloned from genomic DNA and sub-cloned upstream of a promoterless luciferase vector. A proximal 650-bp promoter sequence conferred tissue-independent activation in hematopoietic, epithelial and endothelial cells. A canonical Sp1 binding sequence at −550 upstream of the translational start site was mapped within this proximal positive regulatory promoter region. Site-directed mutagenesis revealed this sequence was essential for optimum ALCAM promoter activity. Importantly, Sp1 occupied the cognate sequence in vivo as determined by chromatin immunoprecipitation assays. Over-expression of Sp1 significantly increased ALCAM promoter activity whereas a control expression vector had no impact. DNA sequences in the interval −600 to −800 negatively influenced promoter activity in a tissue-specific manner. This region contained a putative binding sequence for the aryl hydrocarbon receptor (Ahr), which highlighted ALCAM as a potential target of the immunosuppressing ligand dioxin. This hypothesis was tested by examination of whether ALCAM activation is blocked by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) in monocytes differentiating into macrophages and dendritic cells. Expression of ALCAM was increased 3–5-fold in HL-60 and THP-1 monocytes treated with the differentiating agent phorbol 12-myristate 13-acetate. TCDD dose dependently blocked this activation, indeed, the highest concentration of TCDD (25 nM) used in this study completely blocked ALCAM activation in both monocytic cells. In conclusion, we have unveiled for the first time, the molecular basis for transcription and basal trans-activation of the human ALCAM gene, and identified the Ahr-pathway as a powerful silencer of ALCAM gene activation. Further studies of the ALCAM promoter, may clarify how this gene is up-regulated as part of the inflammatory response, and how it is silenced by immunotoxins. Heterologous expression of ALCAM may be a potential strategy to mitigate the immunosuppressive effects of dioxins and polycyclic aromatic hydrocarbons.

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

2007 ◽  
Vol 81 (7) ◽  
pp. 3339-3345 ◽  
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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