Expression of processed envelope protein of hepatitis C virus in mammalian and insect cells

Matsuura, Yoshiharu; Harada, Shizuko; Suzuki, Ritsuro; Watanabe, Yoji; Inoue, Yasushi; Saito, Izumu; Miyamura, Tatsuo

doi:10.1128/jvi.66.3.1425-1431.1992

Cited by 94 publications

(33 citation statements)

References 25 publications

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“…One possible explanation for this difference is that E l from HCV-Bac 4 is more heavily glycosylated than is the E l from HCV-Bac 5. This is supported by the data of Matsuura et al, (14) who found also that the E l protein ranged from 22 to 35 kD, depending on the extent of glycosylation.…”

Section: Production and Processing Of Hcv Structural Pro-supporting

confidence: 78%

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Characterization of hepatitis C virus structural proteins with a recombinant baculovirus expression system,

Hsu¹

1993

Hepatology

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We cloned and expressed the sequences encoding the structural proteins of the hepatitis C virus in a baculovirus eukaryotic expression system. Four recombinant constructs expressed sufficient hepatitis C virus-specific proteins in insect cell culture to allow analysis of protein cleavage, glycosylation and immunoreactivity. Using immunoblot analysis, we detected a 22-kD protein corresponding to the hepatitis C virus capsid protein cleaved from a larger precursor. Recombinant constructs encoding the presumptive envelope (E1) protein produced products ranging from 30 to 35 kD, whereas constructs encoding the presumptive E2/NS1 protein expressed products ranging in size from 68 to 73 kD. The recombinant envelope proteins were glycosylated, as shown by sensitivity to endoglycosidase F digestion, whereas the capsid was not. We examined the immunoreactivity of these recombinant proteins using sera from 50 patients chronically infected with HCV. Forty-seven of 50 of these sera contained antibodies against the capsid, 14 (28%) also had antibodies against E1 and at least 5 (10%) had antibody against E2/NS1. Forty-seven of 50 sera (94%) were viremic, as determined on hepatitis C virus polymerase chain reaction. The three sera that were hepatitis C virus polymerase chain reaction negative did not have envelope antibodies, whereas all sera that had envelope antibodies were also hepatitis C virus polymerase chain reaction positive. Thus antibodies to baculovirus-expressed hepatitis C virus structural proteins, including E1 and E2/NS1, are found in the presence of viremia.

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Section: Production and Processing Of Hcv Structural Pro-supporting

confidence: 78%

“…Matsuura et al (14) have reported the expression of the E l glycoprotein of HCV in baculovirus. Our results are largely in accord with theirs with respect to cleavage and glycosylation.…”

Section: Discussionmentioning

confidence: 99%

Characterization of hepatitis C virus structural proteins with a recombinant baculovirus expression system,

Hsu¹

1993

Hepatology

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“…The HCV plasmids used in this report were obtained from Tatsuo Miyamura, Yoshiharu Matsuura, Makoto Hijikata, or generated using HCV genotype 1b as templates (31)(32)(33). The plasmids E1 and E2 encode the respective envelope proteins with their endogenous signal peptides.…”

Section: Plasmidsmentioning

confidence: 99%

Hepatitis C virus envelope proteins regulate CHOP via induction of the unfolded protein response

2005

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Unfolded protein response (UPR) is a cellular adaptive response that functions to reduce stress caused by malfolded proteins in the endoplasmic reticulum (ER). UPR can be induced under physiological or pathological conditions and is responsible for the pathogenesis of many human diseases. Hepatitis C virus (HCV) is a single-stranded, positive-sense RNA virus causing chronic diseases. Its genome encodes two envelope proteins E1 and E2, which mature in the ER to form a noncovalently bound, native complex and disulfide aggregates and have previously been shown to induce expression of the molecular chaperone immunoglobulin heavy chain binding protein. In this study, we show that HCV envelope protein expression regulates another stress indicator CCAAT/enhancer-binding protein-homologous protein (CHOP). The ER-stress element and the activating transcription factor 4 element in the CHOP promoter were activated to a similar extent by HCV envelope protein expression. Using mouse embryonic fibroblasts deficient in the ER stress kinase RNA-activated protein kinase-like ER-resident kinase (PERK), we showed that PERK was necessary and sufficient for activating the CHOP promoter. Expression of HCV E1 and/or E2 also induced splicing of X-box binding protein 1 and transactivation of the unfolded protein response element, leading to the speculation that HCV E1 and E2 not only regulate the UPR but also ER-associated degradation.

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“…5 Other studies have also shown that HCV can coexist with antienvelope antibodies, suggesting that the antibody is not neutralizing in all patients. 6 Studies showing that chimpanzees can be reinfected with the same or a different viral strain, 7 and that humans can develop multiple episodes of transfusion-associated HCV, suggest that the host immune responses do not provide protective immunity. 8 The finding of high rates of amino acid variation in the HVR of HCV during chronic infection, both in human and chimpanzee infections, led to the suggestion that changes in the HVR could result from humoral immune pressure leading to the selection of escape mutants.…”

mentioning

confidence: 99%

Comparison of the rate of sequence variation in the hypervariable region of E2/NS1 region of hepatitis C virus in normal and hypogammaglobulinemic patients

et al. 1998

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The hypervariable region (HVR) of the E2/NS1 region of hepatitis C virus (HCV) varies greatly between viral isolates with high rates of genomic change reported during the course of chronic infection. The HVR is thought to encode a structurally unconstrained envelope protein containing several linear B cell epitopes recognized by neutralizing antibody. It has been postulated that amino acid changes in the HVR could result from humoral immune pressure leading to the selection of escape mutants. The aim of this study was to compare the rates of nucleotide and amino acid variation in the HVR of control patients to patients with common variable immunodeficiency (CVID) where the effect of the humoral immune system is reduced. Five controls and four patients with CVID were studied. Serum samples were taken over periods of between 1 and 6 years. HCV was detected by polymerase chain reaction (PCR) with primers derived from conserved flanking regions of the HVR. PCR products were cloned into a plasmid vector and recombinant clones identified by restriction enzyme digestion. Purified DNA from at least three individual clones from each time point was sequenced by the dideoxynucleotide chain-termination method. Consensus sequences were extracted from the three clones, and the DNA and deduced protein sequences were compared. Control patients had a mean rate of nucleotide change of 6.954 nucleotide substitutions per year, compared with patients with CVID with a rate of 0.415 nucleotide substitutions per year (P F .02). The corresponding rates for amino acid variation were 3.868 amino acid substitutions per year for the control patients compared with 0.185 amino acid substitutions per year for the patients with CVID. These findings suggest that in the absence of humoral immune selective pressure, the frequency of occurrence of genetic variation in the major viral species is reduced. The mutations occur, but in the absence of immune selection remain as minor species. The evolution of viral mutants capable of evading the host's immune system may contribute to the ability of HCV to establish chronic infection. (HEPATOLOGY 1998;26:223-227.)Genomic analysis has shown hepatitis C virus (HCV) to be distantly related to both flaviviruses and pestiviruses with structural genes at the 5Ј end preceding those encoding the nonstructural proteins. Analysis of HCV isolates from around the world has revealed extensive heterogeneity between isolates, although the sequence variation is not distributed evenly throughout the genome. In particular, a 27-amino acid region at the amino terminus of E2/NS1 has been shown to vary greatly between isolates and has become known as hypervariable region 1 (HVR). 1-3 The HVR appears to be structurally unconstrained and tolerates extensive amino acid variation; it probably encodes part of an envelope peptide that is exposed to the host' s immune response. 4 Studies have indicated the presence within this region of linear B-cell epitopes recognized by neutralizing antibody, and amino acid variation occurring du...

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Expression of processed envelope protein of hepatitis C virus in mammalian and insect cells

Cited by 94 publications

References 25 publications

Characterization of hepatitis C virus structural proteins with a recombinant baculovirus expression system,

Characterization of hepatitis C virus structural proteins with a recombinant baculovirus expression system,

Hepatitis C virus envelope proteins regulate CHOP via induction of the unfolded protein response

Comparison of the rate of sequence variation in the hypervariable region of E2/NS1 region of hepatitis C virus in normal and hypogammaglobulinemic patients

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