scholarly journals Oligomerization of Hepatitis C Virus Core Protein Is Crucial for Interaction with the Cytoplasmic Domain of E1 Envelope Protein

2006 ◽  
Vol 80 (22) ◽  
pp. 11265-11273 ◽  
Author(s):  
Kousuke Nakai ◽  
Toru Okamoto ◽  
Tomomi Kimura-Someya ◽  
Koji Ishii ◽  
Chang Kweng Lim ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Fusion Protein ◽  
Core Protein ◽  
Type I ◽  
Amino Acid Residues ◽  
Cytoplasmic Loop ◽  
The Core ◽  
Er Membrane

ABSTRACT Hepatitis C virus (HCV) contains two membrane-associated envelope glycoproteins, E1 and E2, which assemble as a heterodimer in the endoplasmic reticulum (ER). In this study, predictive algorithms and genetic analyses of deletion mutants and glycosylation site variants of the E1 glycoprotein were used to suggest that the glycoprotein can adopt two topologies in the ER membrane: the conventional type I membrane topology and a polytopic topology in which the protein spans the ER membrane twice with an intervening cytoplasmic loop (amino acid residues 288 to 360). We also demonstrate that the E1 glycoprotein is able to associate with the HCV core protein, but only upon oligomerization of the core protein in the presence of tRNA to form capsid-like structures. Yeast two-hybrid and immunoprecipitation analyses reveal that oligomerization of the core protein is promoted by amino acid residues 72 to 91 in the core. Furthermore, the association between the E1 glycoprotein and the assembled core can be recapitulated using a fusion protein containing the putative cytoplasmic loop of the E1 glycoprotein. This fusion protein is also able to compete with the intact E1 glycoprotein for binding to the core. Mutagenesis of the cytoplasmic loop of E1 was used to define a region of four amino acids (residues 312 to 315) that is important for interaction with the assembled HCV core. Taken together, our studies suggest that interaction between the self-oligomerized HCV core and the E1 glycoprotein is mediated through the cytoplasmic loop present in a polytopic form of the E1 glycoprotein.

scholarly journals Identification of Basic Amino Acids at the N-Terminal End of the Core Protein That Are Crucial for Hepatitis C Virus Infectivity

2010 ◽  
Vol 84 (24) ◽  
pp. 12515-12528 ◽  
Author(s):  
Khaled Alsaleh ◽  
Pierre-Yves Delavalle ◽  
André Pillez ◽  
Gilles Duverlie ◽  
Véronique Descamps ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Basic Amino Acid ◽  
Amino Acid Residues ◽  
The Core ◽  
Hcv Core Protein ◽  
Viral Particles ◽  
A Cell

ABSTRACT A major function of the hepatitis C virus (HCV) core protein is the interaction with genomic RNA to form the nucleocapsid, an essential component of the virus particle. Analyses to identify basic amino acid residues of HCV core protein, important for capsid assembly, were initially performed with a cell-free system, which did not indicate the importance of these residues for HCV infectivity. The development of a cell culture system for HCV (HCVcc) allows a more precise analysis of these core protein amino acids during the HCV life cycle. In the present study, we used a mutational analysis in the context of the HCVcc system to determine the role of the basic amino acid residues of the core protein in HCV infectivity. We focused our analysis on basic residues located in two clusters (cluster 1, amino acids [aa]6 to 23; cluster 2, aa 39 to 62) within the N-terminal 62 amino acids of the HCV core protein. Our data indicate that basic residues of the first cluster have little impact on replication and are dispensable for infectivity. Furthermore, only four basic amino acids residues of the second cluster (R50, K51, R59, and R62) were essential for the production of infectious viral particles. Mutation of these residues did not interfere with core protein subcellular localization, core protein-RNA interaction, or core protein oligomerization. Moreover, these mutations had no effect on core protein envelopment by intracellular membranes. Together, these data indicate that R50, K51, R59, and R62 residues play a major role in the formation of infectious viral particles at a post-nucleocapsid assembly step.

Influence of amino-acid polymorphism in the core protein on progression of liver disease in patients infected with hepatitis C virus genotype 1b

2010 ◽  
Vol 82 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Mariko Kobayashi ◽  
Norio Akuta ◽  
Fumitaka Suzuki ◽  
Tetsuya Hosaka ◽  
Hitomi Sezaki ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Liver Disease ◽  
Hepatitis C ◽  
Amino Acid ◽  
Core Protein ◽  
Amino Acid Polymorphism ◽  
Virus Genotype ◽  
The Core ◽  
Genotype 1B

scholarly journals The Hepatitis C Virus Core Protein Contains a BH3 Domain That Regulates Apoptosis through Specific Interaction with Human Mcl-1

2009 ◽  
Vol 83 (19) ◽  
pp. 9993-10006 ◽  
Author(s):  
Nur Khairiah Mohd-Ismail ◽  
Lin Deng ◽  
Sunil Kumar Sukumaran ◽  
Victor C. Yu ◽  
Hak Hotta ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Core Protein ◽  
Single Amino Acid ◽  
Hydrophobic Residues ◽  
The Core ◽  
Genotype 1B ◽  
Bh3 Domain ◽  
Infected Cells

ABSTRACT The hepatitis C virus (HCV) core protein is known to modulate apoptosis and contribute to viral replication and pathogenesis. In this study, we have identified a Bcl-2 homology 3 (BH3) domain in the core protein that is essential for its proapoptotic property. Coimmunoprecipitation experiments showed that the core protein interacts specifically with the human myeloid cell factor 1 (Mcl-1), a prosurvival member of the Bcl-2 family, but not with other prosurvival members (Bcl-XL and Bcl-w). Moreover, the overexpression of Mcl-1 protects against core-induced apoptosis. By using peptide mimetics, core was found to release cytochrome c from isolated mitochondria when complemented with Bad. Thus, core is a bona fide BH3-only protein having properties similar to those of Noxa, a BH3-only member of the Bcl-2 family that binds preferentially to Mcl-1. There are three critical hydrophobic residues in the BH3 domain of the core protein, and they are essential for the proapoptotic property of the core protein. Furthermore, the genotype 1b core protein is more effective than the genotype 2a core protein in inducing apoptosis due to a single-amino-acid difference at one of these hydrophobic residues (residue 119). Replacing this residue in the J6/JFH-1 infectious clone (genotype 2a) with the corresponding amino acid in the genotype 1b core protein produced a mutant virus, J6/JFH-1(V119L), which induced significantly higher levels of apoptosis in the infected cells than the parental J6/JFH-1 virus. Furthermore, the core protein of J6/JFH-1(V119L), but not that of J6/JFH-1, interacted with Mcl-1 in virus-infected cells. Taken together, the core protein is a novel BH3-only viral homologue that contributes to the induction of apoptosis during HCV infection.

scholarly journals The C-Terminal Hydrophobic Domain of Hepatitis C Virus RNA Polymerase NS5B Can Be Replaced with a Heterologous Domain of Poliovirus Protein 3A

2006 ◽  
Vol 80 (22) ◽  
pp. 11343-11354 ◽  
Author(s):  
Haekyung Lee ◽  
Ying Liu ◽  
Edison Mejia ◽  
Aniko V. Paul ◽  
Eckard Wimmer
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Decisive Role ◽  
Amino Acid Residues ◽  
Subgenomic Replicon ◽  
Hydrophobic Domain ◽  
Virus Rna ◽  
Membrane Bound

ABSTRACT Replication of the plus-stranded RNA genome of hepatitis C virus (HCV) occurs in a membrane-bound replication complex consisting of viral and cellular proteins and viral RNA. NS5B, the RNA polymerase of HCV, is anchored to the membranes via a C-terminal 20-amino-acid-long hydrophobic domain, which is flanked on each side by a highly conserved positively charged arginine. Using a genotype 1b subgenomic replicon (V. Lohmann, F. Korner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartensclager, Science 285:110-113, 1999), we determined the effect of mutations of some highly conserved residues in this domain. The replacement of arginine 570 with alanine completely abolished the colony-forming ability by the replicon, while a R591A change was found to be highly detrimental to replication, viability, and membrane binding by the mutant NS5B protein. Mutations of two other highly conserved amino acids (L588A and P589A) reduced but did not eliminate colony formation. It was of interest, if specific amino acid residues play a role in membrane anchoring of NS5B and replication, to determine whether a complete exchange of the NS5B hydrophobic domain with a domain totally unrelated to NS5B would ablate replication. We selected the 22-amino-acid-long hydrophobic domain of poliovirus polypeptide 3A that is known to adopt a transmembrane configuration, thereby anchoring 3A to membranes. Surprisingly, either partial or full replacement of the NS5B hydrophobic domain with the anchor sequences of poliovirus polypeptide 3A resulted in the replication of replicons whose colony-forming abilities were reduced compared to that of the wild-type replicon. Upon continued passage of the replicon in Huh-7 cells in the presence of neomycin, the replication efficiency of the replicon increased. However, the sequence of the poliovirus polypeptide 3A hydrophobic domain, in the context of the subgenomic HCV replicon, was stably maintained throughout 40 passages. Our results suggest that anchoring NS5B to membranes is necessary but that the amino acid sequence of the anchor per se does not require HCV origin. This suggests that specific interactions between the NS5B hydrophobic domain and other membrane-bound factors may not play a decisive role in HCV replication.

scholarly journals Efficient Virus Assembly, but Not Infectivity, Determines the Magnitude of Hepatitis C Virus-Induced Interferon Alpha Responses of Plasmacytoid Dendritic Cells

2014 ◽  
Vol 89 (6) ◽  
pp. 3200-3208 ◽  
Author(s):  
Elena Grabski ◽  
Ilka Wappler ◽  
Stephanie Pfaender ◽  
Eike Steinmann ◽  
Sibylle Haid ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Virus Assembly ◽  
Plasmacytoid Dendritic Cells ◽  
Type I ◽  
Hepatitis C Virus Infection ◽  
Infected Cells ◽  
Interferon Responses

ABSTRACTWorldwide, approximately 160 million people are chronically infected with hepatitis C virus (HCV), seven distinct genotypes of which are discriminated. The hallmarks of HCV are its genetic variability and the divergent courses of hepatitis C progression in patients. We assessed whether intragenotypic HCV variations would differentially trigger host innate immunity. To this end, we stimulated human primary plasmacytoid dendritic cells (pDC) with crude preparations of different cell culture-derived genotype 2a HCV variants. Parental Japanese fulminant hepatitis C virus (JFH1) did not induce interferon alpha (IFN-α), whereas the intragenotypic chimera Jc1 triggered massive IFN-α responses. Purified Jc1 retained full infectivity but no longer induced IFN-α. Coculture of pDC with HCV-infected hepatoma cells retrieved the capacity to induce IFN-α, whereas Jc1-infected cells triggered stronger responses than JFH1-infected cells. Since the infectivity of virus particles did not seem to affect pDC activation, we next tested Jc1 mutants that were arrested at different stages of particle assembly. These experiments revealed that efficient assembly and core protein envelopment were critically needed to trigger IFN-α. Of note, sequences within domain 2 of the core that vitally affect virus assembly also crucially influenced the IFN-α responses of pDC. These data showed that viral determinants shaped host innate IFN-α responses to HCV.IMPORTANCEAlthough pegylated IFN-α plus ribavirin currently is the standard of care for the treatment of chronic hepatitis C virus infection, not much is known about the relevance of early interferon responses in the pathogenesis of hepatitis C virus infection. Here, we addressed whether intragenotypic variations of hepatitis C virus would account for differential induction of type I interferon responses mounted by primary blood-derived plasmacytoid dendritic cells. Surprisingly, a chimeric genotype 2a virus carrying the nonstructural genes of Japanese fulminant hepatitis C virus (JFH1) induced massive type I interferon responses, whereas the original genotype 2a JFH1 strain did not. Our detailed analyses revealed that, not the virus infectivity, but rather, the efficiency of virus assembly and core protein envelopment critically determined the magnitude of interferon responses. To our knowledge, this is the first example of hepatitis C virus-associated genetic variations that determine the magnitude of innate host responses.

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 Analysis of genotypes and amino acid residues 2209 to 2248 of the NS5A region of hepatitis C virus in relation to the response to interferon-? therapy

Hepatology ◽  
1997 ◽  
Vol 25 (3) ◽  
pp. 750-753 ◽  
Author(s):  
M Kurosaki ◽  
N Enomoto ◽  
T Murakami ◽  
I Sakuma ◽  
Y Asahina ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Interferon Therapy ◽  
Amino Acid Residues ◽  
Ns5a Region

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.

scholarly journals Amino Acid Polymorphisms in Hepatitis C Virus Core Affect Infectious Virus Production and Major Histocompatibility Complex Class I Molecule Expression

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Megumi Tasaka-Fujita ◽  
Nao Sugiyama ◽  
Wonseok Kang ◽  
Takahiro Masaki ◽  
Asako Murayama ◽  
...  
Keyword(s):  
Major Histocompatibility Complex ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Mhc Class I ◽  
Infectious Virus ◽  
Core Protein ◽  
Virus Production ◽  
Class I ◽  
Histocompatibility Complex

Abstract Amino acid (aa) polymorphisms in the hepatitis C virus (HCV) genotype 1b core protein have been reported to be a potent predictor for poor response to interferon (IFN)-based therapy and a risk factor for hepatocarcinogenesis. We investigated the effects of these polymorphisms with genotype 1b/2a chimeric viruses that contained polymorphisms of Arg/Gln at aa 70 and Leu/Met at aa 91. We found that infectious virus production was reduced in cells transfected with chimeric virus RNA that had Gln at aa 70 (aa70Q) compared with RNA with Arg at aa 70 (aa70R). Using flow cytometry analysis, we confirmed that HCV core protein accumulated in aa70Q clone transfected cells and it caused a reduction in cell-surface expression of major histocompatibility complex (MHC) class I molecules induced by IFN treatment through enhanced protein kinase R phosphorylation. We could not detect any effects due to the polymorphism at aa 91. In conclusion, the polymorphism at aa 70 was associated with efficiency of infectious virus production and this deteriorated virus production in strains with aa70Q resulted in the intracellular accumulation of HCV proteins and attenuation of MHC class I molecule expression. These observations may explain the strain-associated resistance to IFN-based therapy and hepatocarcinogenesis of HCV.

Sign in / Sign up

Export Citation Format

Share Document