scholarly journals Ubiquitin-Independent Degradation of Hepatitis C Virus F Protein

2008 ◽  
Vol 83 (2) ◽  
pp. 612-621 ◽  
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.

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

1999 ◽  
Vol 73 (12) ◽  
pp. 9718-9725 ◽  
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.

scholarly journals Hepatitis C Virus Core Protein Induces Lipid Droplet Redistribution in a Microtubule- and Dynein-Dependent Manner

Traffic ◽  
2008 ◽  
Vol 9 (8) ◽  
pp. 1268-1282 ◽  
Author(s):  
Steeve Boulant ◽  
Mark W. Douglas ◽  
Laura Moody ◽  
Agata Budkowska ◽  
Paul Targett-Adams ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Droplet ◽  
Core Protein ◽  
Dependent Manner ◽  
Virus Core

scholarly journals Characterization of Soluble Hepatitis C Virus RNA-Dependent RNA Polymerase Expressed in Escherichia coli

1999 ◽  
Vol 73 (2) ◽  
pp. 1649-1654 ◽  
Author(s):  
Eric Ferrari ◽  
Jacquelyn Wright-Minogue ◽  
Jane W. S. Fang ◽  
Bahige M. Baroudy ◽  
Johnson Y. N. Lau ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Nonstructural Protein ◽  
Full Length ◽  
Dependent Manner ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Hydrophobic Domain ◽  
Hcv Ns5b

ABSTRACT Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BΔCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.

scholarly journals Generation of Infectious Hepatitis C Virus in Immortalized Human Hepatocytes

2006 ◽  
Vol 80 (9) ◽  
pp. 4633-4639 ◽  
Author(s):  
Tatsuo Kanda ◽  
Arnab Basu ◽  
Robert Steele ◽  
Takaji Wakita ◽  
Jan S. Ryerse ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Expression ◽  
Culture Media ◽  
Human Hepatocytes ◽  
Full Length ◽  
Dependent Manner ◽  
Virus Growth ◽  
Hcv Genotype

ABSTRACT Progress in understanding hepatitis C virus (HCV) biology has remained a challenge due to the lack of an efficient cell culture system for virus growth. In this study, we examined HCV core protein-mediated immortalized human hepatocytes (IHH) for growth of HCV. In vitro-transcribed full-length RNA from HCV genotype 1a (clone H77) was introduced into IHH by electroporation. Reverse transcription-PCR of cellular RNA isolated from HCV genome-transfected IHH suggested that viral RNA replication occurred. IHH transfected with the full-length HCV genome also displayed viral protein expression by indirect immunofluorescence. In contrast, cells transfected with polymerase-defective HCV (H77/GND) RNA as a negative control did not exhibit expression of the viral genome. Immunogold labeling demonstrated localization of E1 protein in the rough endoplasmic reticulum of RNA-transfected IHH. Virus-like particles of ∼50 nm were observed in the cytoplasm. After being inoculated with culture media of cells transfected with the full-length HCV genome, naïve IHH displayed NS5a protein expression in a dilution-dependent manner, but expression of NS5a was inhibited by prior incubation of culture medium with HCV-infected patient sera. NS5a-positive immunofluorescence of cell culture media of IHH transfected with full-length H77 RNA yielded ∼4.5 × 104 to 1 × 105 focus-forming units/ml. A similar level of virus growth was observed upon transfection of RNA from HCV genotype 2a (JFH1) into IHH. Taken together, our results suggest that IHH support HCV genome replication and virus assembly.

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.

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 Suppressor of Cytokine Signaling 3 Suppresses Hepatitis C Virus Replication in an mTOR-Dependent Manner

2010 ◽  
Vol 84 (12) ◽  
pp. 6060-6069 ◽  
Author(s):  
Run-Xuan Shao ◽  
Leiliang Zhang ◽  
Lee F. Peng ◽  
Eileen Sun ◽  
Woo Jin Chung ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Full Length ◽  
Cytokine Signaling ◽  
Type I ◽  
Dependent Manner ◽  
Suppressor Of Cytokine Signaling ◽  
Protein Levels ◽  
Infected Cells ◽  
Ifn Treatment

ABSTRACT We and others have observed that hepatic levels of suppressor of cytokine signaling 3 (SOCS3) are significantly higher in persons with chronic hepatitis C, particularly those who are nonresponders to interferon (IFN) treatment, than in healthy individuals. However, the relationship between SOCS3 and hepatitis C virus (HCV) replication remains unclear. Given its putative role, we hypothesized that SOCS3 is permissive for viral replication. We therefore used the OR6 cell line, which harbors a genotype 1b full-length HCV replicon, and the genotype 2a full-length HCV strain JFH1 infection system to analyze the effects of SOCS3 overexpression and short hairpin RNA (shRNA)-mediated knockdown on HCV replication. We further analyzed the role of mTOR in the effects of SOCS3 by treating selected cells with rapamycin. OR6 cells and JFH1-infected Huh7.5.1 cells expressed significantly less SOCS3 than control cells. Furthermore, inhibition of HCV replication with the HCV protease inhibitor BILN 2061 restored SOCS3 protein levels. SOCS3 overexpression in OR6 cells and JFH1-infected Huh7.5.1 cells resulted in significantly lower HCV replication than that in the control cells, despite SOCS3-related inhibition of STAT1 phosphorylation and type I IFN signaling. In contrast, JFH1-infected cells with stable SOCS3 knockdown expressed higher levels of HCV proteins and RNA than did control cells. SOCS3-targeting shRNA also knocked down mTOR and phospho-mTOR. The mTOR inhibitor rapamycin reversed the inhibitory effects of SOCS3. In independent investigations, SOCS3 unexpectedly suppressed HCV replication in an mTOR-dependent manner. These findings suggest that increased SOCS3 levels consistently observed in chronic IFN nonresponders may reflect a compensatory host antiviral response to persistent infection and that manipulation of SOCS3/mTOR may offer benefit against HCV infection.

Sign in / Sign up

Export Citation Format

Share Document