Cloning, sequencing, and expression of the hepatitis E virus (HEV) nonstructural open reading frame 1 (ORF1)

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117

Hepatitis E virus (HEV) is an important etiological agent of epidemic and sporadic hepatitis, which is endemic to the Indian subcontinent and prevalent in most of the developing parts of the world. The infection is often associated with acute liver failure and high mortality, particularly in pregnant women. In order to develop methods of intervention, it is essential to understand the biology of the virus. This is particularly important as no reliable in vitro culture system is available. We have constructed a cDNA clone encompassing the complete HEV genome from independently characterized subgenomic fragments of an Indian epidemic isolate. Transfection studies were carried out with HepG2 cells using in vitro-transcribed RNA from this full-length HEV cDNA clone. The presence of negative-sense RNA, indicative of viral replication, was demonstrated in the transfected cells by strand-specific reverse transcription-PCR and slot blot hybridization. The viral proteins pORF2 and pORF3 and processed components of the pORF1 polyprotein (putative methyltransferase, helicase, and RNA-dependent RNA polymerase) were identified in the transfected cells by metabolic pulse-labeling with Hepatitis E virus (HEV) is established as an etiological agent of the epidemic and sporadic forms of waterborne hepatitis (6, 25). The first well-characterized HEV epidemic was reported in Delhi, India, in 1955 (35). Several other epidemics have since been described in developing countries on nearly every continent (19,21). HEV has a positive-strand polyadenylated RNA genome ϳ7.2 kb in length (27) containing three open reading frames (ORFs). Nonstructural ORF1 (5Ј end) codes for a polyprotein of 1,693 amino acids (pORF1) and contain domains homologous to a viral methyltransferase, a papainlike cysteine protease, an RNA helicase, and an RNAdependent RNA polymerase (RdRp). The second ORF (3Ј end) codes for the major viral capsid protein of 660 amino acid (pORF2), while the third and smallest ORF (ORF3) codes for a 123-amino-acid-long polypeptide (pORF3) whose function is unknown (32). Apart from its coding region, the viral genome has 27-and 68-nucleotide (nt)-long noncoding regions at its 5Ј and 3Ј ends, respectively (32). The genome sequences of HEV have been reported from different geographical isolates and show a high degree of homology at both the nucleotide and amino acid levels (3,4,12,32,33). Expression of structural proteins pORF2 and pORF3 in prokaryotic and eukaryotic systems has been reported by different investigators (7,11,14,23,29). Earlier, we have expressed and characterized pORF2 and pORF3 in animal cells. pORF2 is an 88-kDa glycoprotein which is expressed intracellularly, as well as on the cell surface (14, 37). The ORF3 protein (pORF3) is a 13.5-kDa phosphoprotein which is phosphorylated by the cellular mitogen-activated protein kinase and associates with the cytoskeleton (36). We have also recently expressed the ORF1 polyprotein in both prokaryotic and eukaryotic systems (2).In the absence of a reliable in vitro culture system...

Section: Methodsmentioning

confidence: 99%

“…The ORF3 protein (pORF3) is a 13.5-kDa phosphoprotein which is phosphorylated by the cellular mitogen-activated protein kinase and associates with the cytoskeleton (36). We have also recently expressed the ORF1 polyprotein in both prokaryotic and eukaryotic systems (2).…”

mentioning

confidence: 99%

The In Vitro-Synthesized RNA from a cDNA Clone of Hepatitis E Virus Is Infectious

Panda

Ansari

Durgapal

et al. 2000

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117

“…HEV is a plusstranded RNA virus with a 7.2-kb genome containing three open reading frames (ORF), ORF1, ORF2, and ORF3, encoding three different proteins (20,32,35). ORF1 (5,079 bp) is at the 5Ј end of the genome and is predicted to code for the putative nonstructural proteins with sequences homologous to those encoding viral methyltransferases, proteases, helicases, and RNA-dependent RNA polymerases (1,20,27,35). In the absence of a reliable in vitro culture system for HEV, fundamental studies on its replication and expression strategy have not been undertaken.…”

mentioning

confidence: 99%

The 41-Amino-Acid C-Terminal Region of the Hepatitis E Virus ORF3 Protein Interacts with Bikunin, a Kunitz-Type Serine Protease Inhibitor

Tyagi

Surjit

Lal

2005

Hepatitis E virus (HEV), a human plus-stranded RNA virus, contains three open reading frames (ORF).Of these, ORF1 encodes the viral nonstructural polyprotein, ORF2 encodes the major capsid protein, and ORF3 codes for a phosphoprotein of undefined function. Recently, using the yeast two-hybrid system to screen a human cDNA liver library, we have isolated and characterized AMBP (␣ 1 -microglobulin/bikunin precursor), which specifically interacts with the ORF3 protein of HEV. The ORF3 protein expedites the processing and secretion of ␣ 1 -microglobulin. When checked individually for interaction, the second processed protein from AMBP, bikunin, strongly interacted with the full-length ORF3 protein. This protein-protein interaction has been validated by immunoprecipitation in both COS-1 and Huh7 cells and by His 6 pull-down assays. In dual-labeling immunofluorescent staining, followed by fluorescence microscopy of transfected human liver cells, ORF3 colocalized with endogenously expressed bikunin. Finally, a 41-amino-acid C-terminal region of ORF3 has been found to be responsible for interacting with bikunin. The importance of this virus-host protein-protein interaction, with reference to the viral life cycle, has been discussed.Hepatitis E is an acute disease endemic in many countries throughout developing parts of the world, in particular on the continents of Africa and Asia, where it causes epidemics and sporadic infections. The causative agent, hepatitis E virus (HEV), is transmitted via the fecal-oral route, predominantly through contaminated water (7,10,25,26). HEV is a plusstranded RNA virus with a 7.2-kb genome containing three open reading frames (ORF), ORF1, ORF2, and ORF3, encoding three different proteins (20,32,35). ORF1 (5,079 bp) is at the 5Ј end of the genome and is predicted to code for the putative nonstructural proteins with sequences homologous to those encoding viral methyltransferases, proteases, helicases, and RNA-dependent RNA polymerases (1,20,27,35). In the absence of a reliable in vitro culture system for HEV, fundamental studies on its replication and expression strategy have not been undertaken. ORF2 and ORF3 have been expressed in Escherichia coli, animal cells, baculovirus, and yeast and in vitro in a coupled transcription-translation system (12,15,17,22,23). ORF2, the major capsid protein for HEV, is synthesized as a precursor and is processed through signal sequence cleavage into the mature protein, which is capable of selfassociation (18, 36). ORF3 encodes a small 13.5-kDa phosphoprotein that is expressed intracellularly, associates with the cytoskeleton, and shows no major processing (3, 42). The ORF3 protein dimerizes using a 43-amino-acid region, interacts with SH3 domains, and activates mitogen-activated protein kinase (21, 37). The phosphorylated form of the ORF3 protein has also been shown to interact with the nonglycosylated form of the major capsid protein, ORF2 (38). These properties of ORF3 clearly indicate that this protein may have multiple roles in HEV pathogenesis. Rece...

“…It has a single-stranded positive-sense RNA genome of about 7.5 kb with three forward open reading frames (ORF1, ORF2, and ORF3) encoding three different proteins (19,28,31,32). ORF1 (5,079 bp) is at the 5Ј end of the genome and is predicted to code for putative nonstructural proteins with sequences homologous to those encoding viral methyltransferases, proteases, helicases, and RNA-dependent RNA polymerases (1,15,19). In the absence of a reliable culture system for HEV, fundamental studies on its replication and expression strategy have not been undertaken.…”

mentioning

confidence: 99%

Self-Association and Mapping of the Interaction Domain of Hepatitis E Virus ORF3 Protein

Tyagi

Jameel

Lal

2001

Self Cite

Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information on the basic biology of the virus exists. A small protein (ϳ13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. The N-terminal region of pORF3 is associated with the cytoskeleton using one of its hydrophobic domains. The C-terminal half of pORF3 is rich in proline residues and contains a putative src homology 3 (SH3) binding domain and a mitogen-activated protein kinase phosphorylation site. In this study, we demonstrate that pORF3 can homodimerize in vivo, using the yeast two-hybrid system. We have isolated a 43-amino-acid interaction domain of pORF3 which is capable of self-association in vivo and in vitro. The overlap of the dimerization domain with the SH3 binding and phosphorylation domains suggests that pORF3 may have a dimerization-dependent regulatory role to play in the signal transduction pathway.Hepatitis E virus (HEV), the causative agent of hepatitis E, is a waterborne pathogen which is responsible for sporadic infections as well as large epidemics of acute viral hepatitis in developing countries (4,5,16,17,18,25,26,35). The HEV genome organization resembles that of many alphaviruses, with nonstructural genes at the 5Ј end and structural genes at the 3Ј end (18,27). It has a single-stranded positive-sense RNA genome of about 7.5 kb with three forward open reading frames (ORF1, ORF2, and ORF3) encoding three different proteins (19,28,31,32). ORF1 (5,079 bp) is at the 5Ј end of the genome and is predicted to code for putative nonstructural proteins with sequences homologous to those encoding viral methyltransferases, proteases, helicases, and RNA-dependent RNA polymerases (1,15,19). In the absence of a reliable culture system for HEV, fundamental studies on its replication and expression strategy have not been undertaken. ORF2 and ORF3 have been expressed in Escherichia coli, animal cells, baculovirus and yeast and in vitro in a coupled transcriptiontranslation system (10,12,20,24). ORF2 encodes the major HEV structural protein (pORF2), which has been shown to be an 88-kDa glycoprotein that is expressed intracellularly as well as on the cell surface. It is synthesized as a precursor and is processed through signal sequence cleavage into the mature protein. When expressed through the baculoviral expression system, pORF2 was shown to assemble into virus-like particles which were cell associated as well as secreted in the culture medium (16,22,33).ORF3 encodes a small, 13.5-kDa cytoskeleton-associated phosphoprotein (pORF3) (2, 36), which is expressed intracellularly and shows no major processing. The location of ORF3 on the 3Ј end of the genome classifies it as a structural protein, though its function is yet to be defined.In earlier expression studies, a dimeric form of pORF3 was always observed by using polyacrylamide gel electrophoresis (12). In its primary sequence, pORF3 contains two distinct hydrophobic domains in its ...