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.

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 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.

scholarly journals Hepatitis C Virus Core Protein Is a Dimeric Alpha-Helical Protein Exhibiting Membrane Protein Features

2005 ◽  
Vol 79 (17) ◽  
pp. 11353-11365 ◽  
Author(s):  
Steeve Boulant ◽  
Christophe Vanbelle ◽  
Christine Ebel ◽  
François Penin ◽  
Jean-Pierre Lavergne
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Binding ◽  
Core Protein ◽  
Limited Proteolysis ◽  
Hydrophobic Domain ◽  
Fluorescence Measurements ◽  
Hcv Core Protein ◽  
Core Proteins

ABSTRACT The building block of hepatitis C virus (HCV) nucleocapsid, the core protein, together with viral RNA, is composed of different domains involved in RNA binding and homo-oligomerization. The HCV core protein 1-169 (CHCV169) and its N-terminal region from positions 1 to 117 (CHCV117) were expressed in Escherichia coli and purified to homogeneity suitable for biochemical and biophysical characterizations. The overall conformation and the oligomeric properties of the resulting proteins CHCV169 and CHCV117 were investigated by using analytical centrifugation, circular dichroism, intrinsic fluorescence measurements, and limited proteolysis. Altogether, our results show that core protein (CHCV169) behaves as a membranous protein and forms heterogeneous soluble micelle-like aggregates of high molecular weight in the absence of detergent. In contrast, it behaves, in the presence of mild detergent, as a soluble, well-folded, noncovalent dimer. Similar to findings observed for core proteins of HCV-related flaviviruses, the HCV core protein is essentially composed of α-helices (50%). In contrast, CHCV117 is soluble and monodispersed in the absence of detergent but is unfolded. It appears that the folding of the highly basic domain from positions 2 to 117 (2-117 domain) depends on the presence of the 117-169 hydrophobic domain, which contains the structural determinants ensuring the binding of core with cellular membranes. Finally, our findings provide valuable information for further investigations on isolated core protein, as well as for attempts to reconstitute nucleocapsid particles in vitro.

scholarly journals Characterization of Hepatitis C Virus Core Protein Multimerization and Membrane Envelopment: Revelation of a Cascade of Core-Membrane Interactions

2009 ◽  
Vol 83 (19) ◽  
pp. 9923-9939 ◽  
Author(s):  
Li-Shuang Ai ◽  
Yu-Wen Lee ◽  
Steve S.-L. Chen
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Complex Formation ◽  
Signal Peptide ◽  
Core Protein ◽  
The Core ◽  
Hcv Core Protein ◽  
Core Proteins ◽  
Multimeric Complex ◽  
Protein Multimerization

ABSTRACT The molecular basis underlying hepatitis C virus (HCV) core protein maturation and morphogenesis remains elusive. We characterized the concerted events associated with core protein multimerization and interaction with membranes. Analyses of core proteins expressed from a subgenomic system showed that the signal sequence located between the core and envelope glycoprotein E1 is critical for core association with endoplasmic reticula (ER)/late endosomes and the core's envelopment by membranes, which was judged by the core's acquisition of resistance to proteinase K digestion. Despite exerting an inhibitory effect on the core's association with membranes, (Z-LL)2-ketone, a specific inhibitor of signal peptide peptidase (SPP), did not affect core multimeric complex formation, suggesting that oligomeric core complex formation proceeds prior to or upon core attachment to membranes. Protease-resistant core complexes that contained both innate and processed proteins were detected in the presence of (Z-LL)2-ketone, implying that core envelopment occurs after intramembrane cleavage. Mutations of the core that prevent signal peptide cleavage or coexpression with an SPP loss-of-function D219A mutant decreased the core's envelopment, demonstrating that SPP-mediated cleavage is required for core envelopment. Analyses of core mutants with a deletion in domain I revealed that this domain contains sequences crucial for core envelopment. The core proteins expressed by infectious JFH1 and Jc1 RNAs in Huh7 cells also assembled into a multimeric complex, associated with ER/late-endosomal membranes, and were enveloped by membranes. Treatment with (Z-LL)2-ketone or coexpression with D219A mutant SPP interfered with both core envelopment and infectious HCV production, indicating a critical role of core envelopment in HCV morphogenesis. The results provide mechanistic insights into the sequential and coordinated processes during the association of the HCV core protein with membranes in the early phase of virus maturation and morphogenesis.

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 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 Proteasome Activator PA28γ-Dependent Nuclear Retention and Degradation of Hepatitis C Virus Core Protein

2003 ◽  
Vol 77 (19) ◽  
pp. 10237-10249 ◽  
Author(s):  
Kohji Moriishi ◽  
Tamaki Okabayashi ◽  
Kousuke Nakai ◽  
Kyoji Moriya ◽  
Kazuhiko Koike ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Regulatory Protein ◽  
Hcv Infection ◽  
Nuclear Retention ◽  
Proteasome Activator ◽  
Hcv Core Protein ◽  
Core Proteins ◽  
Chronic Hcv

ABSTRACT Hepatitis C virus (HCV) core protein plays an important role in the formation of the viral nucleocapsid and a regulatory protein involved in hepatocarcinogenesis. In this study, we have identified proteasome activator PA28γ (11S regulator γ) as an HCV core binding protein by using yeast two-hybrid system. This interaction was demonstrated not only in cell culture but also in the livers of HCV core transgenic mice. These findings are extended to human HCV infection by the observation of this interaction in liver specimens from a patient with chronic HCV infection. Neither the interaction of HCV core protein with other PA28 subtypes nor that of PA28γ with other Flavivirus core proteins was detected. Deletion of the PA28γ-binding region from the HCV core protein or knockout of the PA28γ gene led to the export of the HCV core protein from the nucleus to the cytoplasm. Overexpression of PA28γ enhanced the proteolysis of the HCV core protein. Thus, the nuclear retention and stability of the HCV core protein is regulated via a PA28γ-dependent pathway through which HCV pathogenesis may be exerted.

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