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

2004 ◽  
Vol 85 (4) ◽  
pp. 971-981 ◽  
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.

Interfering with hepatitis C virus assembly in vitro using affinity peptides directed towards core protein

2012 ◽  
Vol 58 (4) ◽  
pp. 475-482
Author(s):  
Jean-Baptiste Duvignaud ◽  
Nathalie Majeau ◽  
Priscilla Delisle ◽  
Normand Voyer ◽  
Stéphane M. Gagné ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Self Assembly ◽  
Core Protein ◽  
Virus Assembly ◽  
Viral Assembly ◽  
Structural Protein ◽  
Ns5a Protein ◽  
The Core

Viral assembly is a crucial key step in the life cycle of every virus. In the case of Hepatitis C virus (HCV), the core protein is the only structural protein to interact directly with the viral genomic RNA. Purified recombinant core protein is able to self-assemble in vitro into nucleocapsid-like particles upon addition of a structured RNA, providing a robust assay with which to study HCV assembly. Inhibition of self-assembly of the C170 core protein (first 170 amino acids) was tested using short peptides derived from the HCV core, from HCV NS5A protein, and from diverse proteins (p21 and p73) known to interact with HCV core protein. Interestingly, peptides derived from the core were the best inhibitors. These peptides are derived from regions of the core predicted to be involved in the interaction between core subunits during viral assembly. We also demonstrated that a peptide derived from the C-terminal end of NS5A protein moderately inhibits the assembly process.

scholarly journals Self-Assembly of Nucleocapsid-Like Particles from Recombinant Hepatitis C Virus Core Protein

2001 ◽  
Vol 75 (5) ◽  
pp. 2119-2129 ◽  
Author(s):  
Meghan Kunkel ◽  
Marta Lorinczi ◽  
René Rijnbrand ◽  
Stanley M. Lemon ◽  
Stanley J. Watowich
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Self Assembly ◽  
High Throughput Screening ◽  
Core Protein ◽  
Rna Molecules ◽  
The Core ◽  
Assembly Pathway ◽  
Core Proteins

ABSTRACT Little is known about the assembly pathway and structure of hepatitis C virus (HCV) since insufficient quantities of purified virus are available for detailed biophysical and structural studies. Here, we show that bacterially expressed HCV core proteins can efficiently self-assemble in vitro into nucleocapsid-like particles. These particles have a regular, spherical morphology with a modal distribution of diameters of approximately 60 nm. Self-assembly of nucleocapsid-like particles requires structured RNA molecules. The 124 N-terminal residues of the core protein are sufficient for self-assembly into nucleocapsid-like particles. Inclusion of the carboxy-terminal domain of the core protein modifies the core assembly pathway such that the resultant particles have an irregular outline. However, these particles are similar in size and shape to those assembled from the 124 N-terminal residues of the core protein. These results provide novel opportunities to delineate protein-protein and protein-RNA interactions critical for HCV assembly, to study the molecular details of HCV assembly, and for performing high-throughput screening of assembly inhibitors.

scholarly journals Hepatitis C Virus Core Protein Binds to the Cytoplasmic Domain of Tumor Necrosis Factor (TNF) Receptor 1 and Enhances TNF-Induced Apoptosis

1998 ◽  
Vol 72 (5) ◽  
pp. 3691-3697 ◽  
Author(s):  
Nongliao Zhu ◽  
Ali Khoshnan ◽  
Robert Schneider ◽  
Masayuki Matsumoto ◽  
Gunther Dennert ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Tumor Necrosis ◽  
Core Protein ◽  
Cytoplasmic Domain ◽  
The Core ◽  
Induced Apoptosis ◽  
Necrosis Factor

ABSTRACT The hepatitis C virus (HCV) core protein is known to be a multifunctional protein, besides being a component of viral nucleocapsids. Previously, we have shown that the core protein binds to the cytoplasmic domain of lymphotoxin β receptor, which is a member of tumor necrosis factor receptor (TNFR) family. In this study, we demonstrated that the core protein also binds to the cytoplasmic domain of TNFR 1. The interaction was demonstrated both by glutathioneS-transferase fusion protein pull-down assay in vitro and membrane flotation method in vivo. Both the in vivo and in vitro binding required amino acid residues 345 to 407 of TNFR 1, which corresponds to the “death domain” of this receptor. We have further shown that stable expression of the core protein in a mouse cell line (BC10ME) or human cell lines (HepG2 and HeLa cells) sensitized them to TNF-induced apoptosis, as determined by the TNF cytotoxicity or annexin V apoptosis assay. The presence of the core protein did not alter the level of TNFR 1 mRNA in the cells or expression of TNFR 1 on the cell surface, suggesting that the sensitization of cells to TNF by the viral core protein was not due to up-regulation of TNFR 1. Furthermore, we observed that the core protein blocked the TNF-induced activation of RelA/NF-κB in murine BC10ME cells, thus at least partially accounting for the increased sensitivity of BC10ME cells to TNF. However, NF-κB activation was not blocked in core protein-expressing HeLa or HepG2 cells, implying another mechanism of TNF sensitization by core protein. These results together suggest that the core protein can promote cell death during HCV infection via TNF signaling pathways possibly as a result of its interaction with the cytoplasmic tail of TNFR 1. Therefore, TNF may play a role in HCV pathogenesis.

scholarly journals In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

2003 ◽  
Vol 77 (6) ◽  
pp. 3669-3679 ◽  
Author(s):  
Caterina Trozzi ◽  
Linda Bartholomew ◽  
Alessandra Ceccacci ◽  
Gabriella Biasiol ◽  
Laura Pacini ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
In Vitro Selection ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
In Vitro Systems

ABSTRACT The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

scholarly journals The genotype 3-specific hepatitis C virus core protein residue phenylalanine 164 increases steatosis in an in vitro cellular model

Gut ◽  
2007 ◽  
Vol 56 (9) ◽  
pp. 1302-1308 ◽  
Author(s):  
C Hourioux ◽  
R Patient ◽  
A Morin ◽  
E Blanchard ◽  
A Moreau ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Cellular Model ◽  
Genotype 3 ◽  
Virus Core

scholarly journals Cytokine-Modulated Natural Killer Cells Differentially Regulate the Activity of the Hepatitis C Virus

2018 ◽  
Vol 19 (9) ◽  
pp. 2771 ◽  
Author(s):  
Yoo Cho ◽  
Hwan Lee ◽  
Hyojeung Kang ◽  
Hyosun Cho
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nk Cells ◽  
Natural Killer ◽  
Core Protein ◽  
Lymphoid Cells ◽  
Hcv Rna ◽  
Cell Infection ◽  
Hcv Core Protein

HCV genotype 2a strain JFH-1 replicates and produces viral particles efficiently in human hepatocellular carcinoma (huh) 7.5 cells, which provide a stable in vitro cell infection system for the hepatitis C virus (HCVcc system). Natural killer (NK) cells are large lymphoid cells that recognize and kill virus-infected cells. In this study, we investigated the interaction between NK cells and the HCVcc system. IL-10 is a typical immune regulatory cytokine that is produced mostly by NK cells and macrophages. IL-21 is one of the main cytokines that stimulate the activation of NK cells. First, we used anti-IL-10 to neutralize IL-10 in a coculture of NK cells and HCVcc. Anti-IL-10 treatment increased the maturation of NK cells by enhancing the frequency of the CD56+dim population in NK-92 cells. However, with anti-IL-10 treatment of NK cells in coculture with J6/JFH-1-huh 7.5 cells, there was a significant decrease in the expression of STAT1 and STAT5 proteins in NK-92 cells and an increase in the HCV Core and NS3 proteins. In addition, rIL-21 treatment increased the frequency of the CD56+dim population in NK-92 cells, Also, there was a dramatic increase in the expression of STAT1 and STAT5 proteins in rIL-21 pre-stimulated NK cells and a decrease in the expression of HCV Core protein in coculture with J6/JFH-1-huh 7.5 cells. In summary, we found that the functional activation of NK cells can be modulated by anti-IL-10 or rIL-21, which controls the expression of HCV proteins as well as HCV RNA replication.

scholarly journals Proteasomal Turnover of Hepatitis C Virus Core Protein Is Regulated by Two Distinct Mechanisms: a Ubiquitin-Dependent Mechanism and a Ubiquitin-Independent but PA28γ-Dependent Mechanism

2008 ◽  
Vol 83 (5) ◽  
pp. 2389-2392 ◽  
Author(s):  
Ryosuke Suzuki ◽  
Kohji Moriishi ◽  
Kouichirou Fukuda ◽  
Masayuki Shirakura ◽  
Koji Ishii ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Loss Of Function ◽  
Proteasome Activator ◽  
Binding Partner ◽  
The Core ◽  
Virus Core ◽  
Lysine Residues ◽  
Dependent Mechanism

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.

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

2010 ◽  
Vol 84 (18) ◽  
pp. 9118-9127 ◽  
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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