Characterization of the hepatitis C virus E2/NS1 gene product expressed in mammalian cells

Spaete, Richard R.; Alexander, D. L.; Rugroden, Mary E.; Choo, Qui‐Lim; Berger, Kimberly; Crawford, Kevin; Kuo, C.; Leng, Song; Lee, Cindy; Ralston, Robert; Thudium, Kent; Tung, James W.; Kuo, George; Houghton, Michael

doi:10.1016/0042-6822(92)90537-y

Cited by 118 publications

(93 citation statements)

References 53 publications

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“…The presence of ER retention signals within the C-terminal regions of both E1 and E2 (5,6,13) could explain these observations. Consistent with these data, truncation of E2 at its C terminus leads to its secretion from expressing cells (20,22,32,34). These observations suggest that HCV particle morphogenesis occurs by budding into the ER and subsequent transport of viral particles through the host cell secretory pathway before release into the extracellular space.…”

supporting

confidence: 69%

“…Both glycoproteins are heavily modified by N-linked glycosylation and are believed to be type I integral transmembrane proteins, with C-terminal hydrophobic anchor domains. Expression of the E1 and E2 glycoproteins in mammalian cell lines demonstrates their ER retention with no cell surface glycoprotein expression detectable (9,10,27,32,34). Immunoelectron microscopic studies localized the glycoproteins to the ER (7,9).…”

mentioning

confidence: 99%

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Functional Characterization of Intracellular and Secreted Forms of a Truncated Hepatitis C Virus E2 Glycoprotein

Flint

Dubuisson

Maidens

et al. 2000

J Virol

106

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The E2 protein of hepatitis C virus (HCV) is believed to be a virion surface glycoprotein that is a candidate for inclusion in an antiviral vaccine. A truncated soluble version of E2 has recently been shown to interact with CD81, suggesting that this protein may be a component of the receptor for HCV. When expressed in eukaryotic cells, a significant proportion of E2 forms misfolded aggregates. To analyze the specificity of interaction between E2 and CD81, the aggregated and monomeric forms of a truncated E2 glycoprotein (E2 661 ) were separated by high-pressure liquid chromatography and analyzed for CD81 binding. Nonaggregated forms of E2 preferentially bound CD81 and a number of conformation-dependent monoclonal antibodies (MAbs). Furthermore, intracellular forms of E2 661 were found to bind CD81 with greater affinity than the extracellular forms. Intracellular and secreted forms of E2 661 were also found to differ in reactivity with MAbs and human sera, consistent with differences in antigenicity. Together, these data indicate that proper folding of E2 is important for its interaction with CD81 and that modifications of glycans can modulate this interaction. Identification of the biologically active forms of E2 will assist in the future design of vaccines to protect against HCV infection.

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supporting

confidence: 69%

mentioning

confidence: 99%

Functional Characterization of Intracellular and Secreted Forms of a Truncated Hepatitis C Virus E2 Glycoprotein

Flint

Dubuisson

Maidens

et al. 2000

J Virol

106

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“…It was previously reported that the full-length E2 protein remained membrane-associated, presumably due to the putative transmembrane domain, and that deletion of the C-terminal hydrophobic region appeared to facilitate the secretion of the truncated E2 protein (10,27,31,32). The signal peptide of other proteins such as tissue plasminogen activator and rabies virus glycoprotein was shown to further facilitate the secretion of the C-terminal-truncated E2 protein, but not the full-length E2 protein (11,32). In contrast, our data showed that the fusion of hGH to the E2 protein enables full-length E2 protein as well as the C-terminal-truncated form to efficiently secrete into culture medium, suggesting that HCV E2 protein could be secreted, depending upon the signal peptide and/or secretory protein fused to the E2 protein.…”

Section: Discussionmentioning

confidence: 99%

Hepatitis C Virus E2 Protein Purified from Mammalian Cells Is Frequently Recognized by E2-specific Antibodies in Patient Sera

Lee

Suh

Cho

et al. 1997

Journal of Biological Chemistry

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The envelope protein of hepatitis C virus (HCV) is composed of two membrane-associated glycoproteins, E1 and E2. To obtain HCV E2 protein as a secretory form at a high level, we constructed a recombinant chinese hamster ovary (CHO) cell line expressing a C-terminal truncated E2 (E2t) fused to human growth hormone (hGH), CHO/hGHE2t. The hGHE2t fusion protein was purified from the culture supernatant using anti-hGH mAb affinity chromatography at approximately 80% purity. The purified hGHE2t protein appeared to be assembled into oligomers linked by intermolecular disulfide bond(s) when density gradient centrifugation and SDSpolyacrylamide gel electrophoresis were employed. When the purified fusion protein was used for testing its ability to bind to antibodies specific for HCV by enzymelinked immunosorbent assay, the protein was recognized by antibodies in sera from 90% of HCV-positive patients. Treatment of hGHE2t protein by ␤-mercaptoethanol, but not by heat and SDS, significantly reduced its reactivity to the antibodies of patient sera, suggesting that intermolecular and/or intramolecular disulfide bonds are important for its ability to recognize its specific antibody and that the E2 protein contains discontinuous antigenic epitope(s). Hepatitis C virus (HCV)1 is a major causative agent of posttransfusion and sporadic non-A, non-B hepatitis throughout the world (1, 2). In most cases, the virus appears to cause a persistent infection. Previous studies indicate that the development of chronic liver diseases, cirrhosis, and hepatocellular carcinoma is associated with chronic HCV infection (3).Comparative analyses of the genomes from several HCV strains indicate that HCV is a member of the family Flaviviridae, which includes flaviviruses and pestiviruses (4). The HCV genome is a 9.5-kilobase positive-strand RNA from which a single polypeptide is expressed and processed by cellular and viral proteinases to produce the putative viral structural and nonstructural proteins (4 -6). It was previously shown that structural proteins were composed of the core protein of 18 -22 kDa and two glycosylated envelope proteins, E1 of 31-35 kDa and E2 of 58 -74 kDa (5, 7-11). Although some lymphocyte cell lines have shown to support the limited replication of HCV, there has not been in vitro cell culture system efficiently enough to be used for viral propagation and for detailed virological studies (12). Expression studies using recombinant cDNA templates are the only means for identifying individual HCV proteins and to study their roles in the pathogenesis of HCV infection.The hydrophobicity profile of HCV polyprotein suggested that the HCV E2 protein corresponds to the flavivirus NS1 glycoprotein and the major pestivirus envelope protein gp53/ gp55 (E2; gp53 in bovine viral diarrhea virus and gp55 in hog cholera virus), which were reported to induce protective immunity in experimental animals (13,14). HCV envelope proteins are of considerable interest, because experimentally challenged chimpanzees were either protected or shown to a...

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“…These animals express the full length lines has generated conflicting data in terms of protein size, core protein in cytoplasm of their hepatocytes at levels posttranslational modification, and subcellular localizacomparable to those detected in naturally infected pation. 12,15,21,22 [25][26][27][28][29] They are both believed to be that it inhibits HBV replication in HuH-7 cells after trantransmembrane glycoproteins, with C-terminal hydrophobic sient transfection in vitro. We have also produced anchors.…”

mentioning

confidence: 99%

“…24 HCV E1 and E2 are heavily glycosylated, HBV gene expression or replication, contrary to reports putative envelope proteins. [25][26][27][28][29] They are both believed to be that it inhibits HBV replication in HuH-7 cells after trantransmembrane glycoproteins, with C-terminal hydrophobic sient transfection in vitro. We have also produced anchors.…”

mentioning

confidence: 99%

Hepatitis C Virus Core and E2 Protein Expression in Transgenic Mice

et al. 1997

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to other flaviviruses. [12][13][14][15] Recently it was demonstrated that Transgenic mice have been produced that express the HCV core can form homodimers and probably multimers 16 hepatitis C virus (HCV) core protein in the liver under similar to the nucleocapsid proteins of retroviruses 17,18 and the transcriptional control of the mouse major urinary HBV. 19,20 In vitro expression of HCV core in different cell protein promoter. These animals express the full length lines has generated conflicting data in terms of protein size, core protein in cytoplasm of their hepatocytes at levels posttranslational modification, and subcellular localizacomparable to those detected in naturally infected pation. 12,15,21,22 [25][26][27][28][29] They are both believed to be that it inhibits HBV replication in HuH-7 cells after trantransmembrane glycoproteins, with C-terminal hydrophobic sient transfection in vitro. We have also produced anchors. After synthesis and glycosylation, E1 and E2 fold transgenic mice in which a C-terminally truncated and associate primarily through noncovalent interactions (aa384-715) glycosylated HCV E2 protein is expressed in with a slow kinetics as shown in vitro by pulse-chase experithe liver under the transcriptional control of the mouse ments. 30 The HCV glycoproteins are predominantly localized albumin promoter. Despite the high level expression of in the endoplasmic reticulum (ER) and are not translocated HCV E2 protein, no evidence of liver disease was debeyond the cis Golgi. 28,30 Purified E1E2 oligomers have been tected in these animals. These results suggest that the used to successfully immunize chimpanzees against chal-HCV core and E2 proteins are not cytopathic for the lenge with low doses of homologous HCV-1, 31 but it is not hepatocyte in vivo, and they represent an initial step in known if the protection is mediated by neutralizing antibodthe development of a small animal model of HCV immuies or other immune mechanisms. nopathology. (HEPATOLOGY 1997;25:719-727.)In the absence of an efficient cell culture system and convenient animal models to study HCV pathogenesis, we have Hepatitis C virus (HCV) is a parenterally transmitted hep-produced transgenic mice in which HCV core and HCV E2 atotropic virus that, like hepatitis B virus (HBV), causes are expressed in the liver under the control of the developacute and chronic hepatitis and hepatocellular carcinoma. [1][2][3][4] mentally regulated, liver-specific mouse major urinary pro-HCV is a positive stranded RNA virus whose genome (about tein (MUP) and constitutively active mouse albumin (ALB) 9.6 kb) contains a single uninterrupted open reading frame promoters. The characteristics of these animals form the ba-(ORF) that encodes a polyprotein of 3010-3011 amino sis for the current report. acids. 1,[5][6][7][8] In vitro studies have demonstrated that the polyprotein is processed by cellular and viral proteases to produce MATERIALS AND METHODSat least ten different viral structural and nonstructural proteins, whose order is C-E1-E2-E2/p7...

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Characterization of the hepatitis C virus E2/NS1 gene product expressed in mammalian cells

Cited by 118 publications

References 53 publications

Functional Characterization of Intracellular and Secreted Forms of a Truncated Hepatitis C Virus E2 Glycoprotein

Functional Characterization of Intracellular and Secreted Forms of a Truncated Hepatitis C Virus E2 Glycoprotein

Hepatitis C Virus E2 Protein Purified from Mammalian Cells Is Frequently Recognized by E2-specific Antibodies in Patient Sera

Hepatitis C Virus Core and E2 Protein Expression in Transgenic Mice

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