Immune responses of blood donors to peptides of various lengths and those with genotypic sequence variations corresponding to the N-terminal portion of the core protein of hepatitis C virus

ABSTRACT We have previously reported on the ubiquitylation and degradation of hepatitis C virus core protein. Here we demonstrate that proteasomal degradation of the core protein is mediated by two distinct mechanisms. One leads to polyubiquitylation, in which lysine residues in the N-terminal region are preferential ubiquitylation sites. The other is independent of the presence of ubiquitin. Gain- and loss-of-function analyses using lysineless mutants substantiate the hypothesis that the proteasome activator PA28γ, a binding partner of the core, is involved in the ubiquitin-independent degradation of the core protein. Our results suggest that turnover of this multifunctional viral protein can be tightly controlled via dual ubiquitin-dependent and -independent proteasomal pathways.

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A Disulfide-Bonded Dimer of the Core Protein of Hepatitis C Virus Is Important for Virus-Like Particle Production

Journal of Virology ◽

10.1128/jvi.00402-10 ◽

2010 ◽

Vol 84 (18) ◽

pp. 9118-9127 ◽

Cited By ~ 23

Author(s):

Yukihiro Kushima ◽

Takaji Wakita ◽

Makoto Hijikata

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Particle Production ◽

Rna Binding ◽

Core Protein ◽

Dominant Negative ◽

Proteinase K ◽

Dominant Negative Effect ◽

The Core ◽

Virus Like Particle

ABSTRACT Hepatitis C virus (HCV) core protein forms the nucleocapsid of the HCV particle. Although many functions of core protein have been reported, how the HCV particle is assembled is not well understood. Here we show that the nucleocapsid-like particle of HCV is composed of a disulfide-bonded core protein complex (dbc-complex). We also found that the disulfide-bonded dimer of the core protein (dbd-core) is formed at the endoplasmic reticulum (ER), where the core protein is initially produced and processed. Mutational analysis revealed that the cysteine residue at amino acid position 128 (Cys128) of the core protein, a highly conserved residue among almost all reported isolates, is responsible for dbd-core formation and virus-like particle production but has no effect on the replication of the HCV RNA genome or the several known functions of the core protein, including RNA binding ability and localization to the lipid droplet. The Cys128 mutant core protein showed a dominant negative effect in terms of HCV-like particle production. These results suggest that this disulfide bond is critical for the HCV virion. We also obtained the results that the dbc-complex in the nucleocapsid-like structure was sensitive to proteinase K but not trypsin digestion, suggesting that the capsid is built up of a tightly packed structure of the core protein, with its amino (N)-terminal arginine-rich region being concealed inside.

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The N-terminal half of the core protein of hepatitis C virus is sufficient for nucleocapsid formation

Journal of General Virology ◽

10.1099/vir.0.79775-0 ◽

2004 ◽

Vol 85 (4) ◽

pp. 971-981 ◽

Cited By ~ 54

Author(s):

Nathalie Majeau ◽

Valérie Gagné ◽

Annie Boivin ◽

Marilène Bolduc ◽

Josée-Anne Majeau ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Core Protein ◽

Host Cells ◽

Yeast Cells ◽

Main Function ◽

C Protein ◽

Multifunctional Protein ◽

The Core

The core (C) protein of hepatitis C virus (HCV) appears to be a multifunctional protein that is involved in many viral and cellular processes. Although its effects on host cells have been extensively discussed in the literature, little is known about its main function, the assembly and packaging of the viral genome. We have studied the in vitro assembly of several deleted versions of recombinant HCV C protein expressed in E. coli. We demonstrated that the 75 N-terminal residues of the C protein were sufficient to assemble and generate nucleocapsid-like particles (NLPs) in vitro. However, homogeneous particles of regular size and shape were observed only when NLPs were produced from at least the first 79 N-terminal amino acids of the C protein. This small protein unit fused to the endoplasmic reticulum-anchoring domain also generated NLPs in yeast cells. These data suggest that the N-terminal half of the C protein is important for formation of NLPs. Similarities between the HCV C protein and C proteins of other members of the Flaviviridae are discussed.

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Interaction of Hepatitis C Virus Core Protein with Viral Sense RNA and Suppression of Its Translation

Journal of Virology ◽

10.1128/jvi.73.12.9718-9725.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 9718-9725 ◽

Cited By ~ 117

Author(s):

Takashi Shimoike ◽

Shigetaka Mimori ◽

Hideki Tani ◽

Yoshiharu Matsuura ◽

Tatsuo Miyamura

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Core Protein ◽

Firefly Luciferase ◽

Encephalomyocarditis Virus ◽

Hcv Rna ◽

Dependent Manner ◽

Genomic Rna ◽

The Core ◽

Hcv Core Protein

ABSTRACT To clarify the binding properties of hepatitis C virus (HCV) core protein and its viral RNA for the encapsidation, morphogenesis, and replication of HCV, the specific interaction of HCV core protein with its genomic RNA synthesized in vitro was examined in an in vivo system. The positive-sense RNA from the 5′ end to nucleotide (nt) 2327, which covers the 5′ untranslated region (5′UTR) and a part of the coding region of HCV structural proteins, interacted with HCV core protein, while no interaction was observed in the same region of negative-sense RNA and in other regions of viral and antiviral sense RNAs. The internal ribosome entry site (IRES) exists around the 5′UTR of HCV; therefore, the interaction of the core protein with this region of HCV RNA suggests that there is some effect on its cap-independent translation. Cells expressing HCV core protein were transfected with reporter RNAs consisting of nt 1 to 709 of HCV RNA (the 5′UTR of HCV and about two-thirds of the core protein coding regions) followed by a firefly luciferase gene (HCV07Luc RNA). The translation of HCV07Luc RNA was suppressed in cells expressing the core protein, whereas no significant suppression was observed in the case of a reporter RNA possessing the IRES of encephalomyocarditis virus followed by a firefly luciferase. This suppression by the core protein occurred in a dose-dependent manner. The expression of the E1 envelope protein of HCV or β-galactosidase did not suppress the translation of both HCV and EMCV reporter RNAs. We then examined the regions that are important for suppression of translation by the core protein and found that the region from nt 1 to 344 was enough to exert this suppression. These results suggest that the HCV core protein interacts with viral genomic RNA at a specific region to form nucleocapsids and regulates the expression of HCV by interacting with the 5′UTR.

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The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice

Nature Medicine ◽

10.1038/2053 ◽

1998 ◽

Vol 4 (9) ◽

pp. 1065-1067 ◽

Cited By ~ 860

Author(s):

Kyoji Moriya ◽

Hajime Fujie ◽

Yoshizumi Shintani ◽

Hiroshi Yotsuyanagi ◽

Takeya Tsutsumi ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Transgenic Mice ◽

Core Protein ◽

The Core

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Ubiquitin-Independent Degradation of Hepatitis C Virus F Protein

Journal of Virology ◽

10.1128/jvi.00832-08 ◽

2008 ◽

Vol 83 (2) ◽

pp. 612-621 ◽

Cited By ~ 43

Author(s):

Kamile Yuksek ◽

Wen-ling Chen ◽

David Chien ◽

Jing-hsiung James Ou

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Core Protein ◽

Binding Domain ◽

Full Length ◽

Dependent Manner ◽

F Protein ◽

Proteasome Subunit ◽

The Core ◽

In Cells

ABSTRACT Hepatitis C virus (HCV) F protein is encoded by the +1 reading frame of the viral genome. It overlaps with the core protein coding sequence, and multiple mechanisms for its expression have been proposed. The full-length F protein that is synthesized by translational ribosomal frameshift at codons 9 to 11 of the core protein sequence is a labile protein. By using a combination of genetic, biochemical, and cell biological approaches, we demonstrate that this HCV F protein can bind to the proteasome subunit protein α3, which reduces the F-protein level in cells in a dose-dependent manner. Deletion-mapping analysis identified amino acids 40 to 60 of the F protein as the α3-binding domain. This α3-binding domain of the F protein together with its upstream sequence could significantly destabilize the green fluorescent protein, an otherwise stable protein. Further analyses using an F-protein mutant lacking lysine and a cell line that contained a temperature-sensitive E1 ubiquitin-activating enzyme indicated that the degradation of the F protein was ubiquitin independent. Based on these observations as well as the observation that the F protein could be degraded directly by the 20S proteasome in vitro, we propose that the full-length HCV F protein as well as the F protein initiating from codon 26 is degraded by an ubiquitin-independent pathway that is mediated by the proteasome subunit α3. The ability of the F protein to bind to α3 raises the possibility that the HCV F protein may regulate protein degradation in cells.

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