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 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 Hepatitis C Virus Core Protein Interacts with 14-3-3 Protein and Activates the Kinase Raf-1

2000 ◽  
Vol 74 (4) ◽  
pp. 1736-1741 ◽  
Author(s):  
Hiroshi Aoki ◽  
Junpei Hayashi ◽  
Mitsuhiko Moriyama ◽  
Yasuyuki Arakawa ◽  
Okio Hino
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Growth Regulation ◽  
Core Protein ◽  
Dependent Manner ◽  
Kinase Activation ◽  
Virus Core ◽  
Hcv Core Protein

ABSTRACT Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver dysfunction in humans and is epidemiologically closely associated with the development of human hepatocellular carcinoma. Among HCV components, core protein has been reported to be implicated in cell growth regulation both in vitro and in vivo, although mechanisms explaining those effects are still unclear. In the present study, we identified that members of the 14-3-3 protein family associate with HCV core protein. 14-3-3 protein bound to HCV core protein in a phosphoserine-dependent manner. Introduction of HCV core protein caused a substantial increase in Raf-1 kinase activity in HepG2 cells and in a yeast genetic assay. Furthermore, the HCV core–14-3-3 interaction was essential for Raf-1 kinase activation by HCV core protein. These results suggest that HCV core protein may represent a novel type of Raf-1 kinase-activating protein through its interaction with 14-3-3 protein and may contribute to hepatocyte growth regulation.

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 Intramembrane Processing by Signal Peptide Peptidase Regulates the Membrane Localization of Hepatitis C Virus Core Protein and Viral Propagation

2008 ◽  
Vol 82 (17) ◽  
pp. 8349-8361 ◽  
Author(s):  
Kiyoko Okamoto ◽  
Yoshio Mori ◽  
Yasumasa Komoda ◽  
Toru Okamoto ◽  
Masayasu Okochi ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Signal Peptide ◽  
Core Protein ◽  
Biological Significance ◽  
Signal Peptidase ◽  
Signal Peptide Peptidase ◽  
Viral Propagation ◽  
The Core ◽  
Hcv Core Protein

ABSTRACT Hepatitis C virus (HCV) core protein has shown to be localized in the detergent-resistant membrane (DRM), which is distinct from the classical raft fraction including caveolin, although the biological significance of the DRM localization of the core protein has not been determined. The HCV core protein is cleaved off from a precursor polyprotein at the lumen side of Ala191 by signal peptidase and is then further processed by signal peptide peptidase (SPP) within the transmembrane region. In this study, we examined the role of SPP in the localization of the HCV core protein in the DRM and in viral propagation. The C terminus of the HCV core protein cleaved by SPP in 293T cells was identified as Phe177 by mass spectrometry. Mutations introduced into two residues (Ile176 and Phe177) upstream of the cleavage site of the core protein abrogated processing by SPP and localization in the DRM fraction. Expression of a dominant-negative SPP or treatment with an SPP inhibitor, L685,458, resulted in reductions in the levels of processed core protein localized in the DRM fraction. The production of HCV RNA in cells persistently infected with strain JFH-1 was impaired by treatment with the SPP inhibitor. Furthermore, mutant JFH-1 viruses bearing SPP-resistant mutations in the core protein failed to propagate in a permissive cell line. These results suggest that intramembrane processing of HCV core protein by SPP is required for the localization of the HCV core protein in the DRM and for viral propagation.

scholarly journals Hepatitis C Virus Genotype 1b Core Protein Does Not Exert Immunomodulatory Effects on Virus-Induced Cellular Immunity

2002 ◽  
Vol 76 (3) ◽  
pp. 990-997 ◽  
Author(s):  
Zhang-Xu Liu ◽  
Hiroshi Nishida ◽  
Jian-Wen He ◽  
Michael M. C. Lai ◽  
Ni Feng ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Liver Injury ◽  
Core Protein ◽  
Virus Genotype ◽  
Hcv Genotype ◽  
Cell Functions ◽  
The Core ◽  
Genotype 1B ◽  
Hcv Core Protein

ABSTRACT The hepatitis C virus (HCV) core protein is among the most conserved proteins in HCV and is known to induce sensitization of cytotoxic T lymphocytes (CTL). Therefore, it is a prime candidate for a component of a potential HCV vaccine. The HCV core protein has, however, been reported to exert multiple effects on cell functions, raising questions as to its suitability for this purpose. This question was investigated here with mice into which replication-deficient adenoviruses expressing core protein of an HCV genotype 1b isolate were injected. We show that induction of cytokines in response to the infection, infiltration of lymphocytes into the infected liver, priming of virus-specific CTL, and liver injury are not modulated by expression of the core protein in the liver. Moreover, no changes in the sensitivity to tumor necrosis factor alpha- or Fas-mediated liver injury are demonstrable. A similar lack of demonstrable effects of the core protein on immune functions has also been obtained using transgenic mice expressing another HCV genotype 1b core protein. It is concluded that the HCV core protein of genotype 1b has no modulatory effects on induction of virus-specific immune responses and may therefore be a suitable component of an HCV vaccine.

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 Ectopic Expression of Hepatitis C Virus Core Protein Differentially Regulates Nuclear Transcription Factors

1998 ◽  
Vol 72 (12) ◽  
pp. 9722-9728 ◽  
Author(s):  
Anju Shrivastava ◽  
Sunil K. Manna ◽  
Ranjit Ray ◽  
Bharat B. Aggarwal
Keyword(s):  
Transcription Factors ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Inflammatory Responses ◽  
Core Protein ◽  
Ectopic Expression ◽  
Cellular Growth ◽  
Mitogen Activated Protein Kinase ◽  
The Core ◽  
Hcv Core Protein

ABSTRACT The putative core protein of hepatitis C virus (HCV) regulates cellular growth and a number of cellular promoters. To further understand its effect, we investigated the role of the core protein in the endogenous regulation of two distinct transcription factors, nuclear factor-κB (NF-κB) and activating protein-1 (AP-1), and the related mitogen-activated protein kinase kinase (MAPKK) and c-Jun N-terminal kinase (JNK). Stable cell transfectants expressing the HCV core protein suppressed tumor necrosis factor (TNF)-induced NF-κB activation. Supershift analysis revealed that NF-κB consists of p50 and p65 subunits. This correlated with inhibition of the degradation of IκBα, the inhibitory subunit of NF-κB. The effect was not specific to TNF, as suppression in core protein-expressing cells was also observed in response to a number of other inflammatory agents known to activate NF-κB. In contrast to the effect on NF-κB, the HCV core protein constitutively activated AP-1, which correlated with the activation of JNK and MAPKK, which are known to regulate AP-1. These observations indicated that the core protein targets transcription factors known to be involved in the regulation of inflammatory responses and the immune system.

scholarly journals Human Apolipoprotein E Is Required for Infectivity and Production of Hepatitis C Virus in Cell Culture

2007 ◽  
Vol 81 (24) ◽  
pp. 13783-13793 ◽  
Author(s):  
Kyung-Soo Chang ◽  
Jieyun Jiang ◽  
Zhaohui Cai ◽  
Guangxiang Luo
Keyword(s):  
Monoclonal Antibodies ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Apolipoprotein E ◽  
Reverse Genetics ◽  
Cultured Cells ◽  
Core Protein ◽  
Hcv Rna ◽  
Dependent Manner ◽  
Small Interfering Rnas

ABSTRACT Recent advances in reverse genetics of hepatitis C virus (HCV) made it possible to determine the properties and biochemical compositions of HCV virions. Sedimentation analysis and characterization of HCV RNA-containing particles produced in the cultured cells revealed that HCV virions cover a large range of heterogeneous densities in sucrose gradient. The fractions of low densities are infectious, while the higher-density fractions containing the majority of HCV virion RNA are not. HCV core protein and apolipoprotein B and apolipoprotein E (apoE) were detected in the infectious HCV virions. The level of apoE correlates very well with HCV infectivity. Both apoE- and HCV E2-specific monoclonal antibodies precipitated HCV, demonstrating that HCV virions contain apoE and E2 proteins. apoE-specific monoclonal antibodies efficiently neutralized HCV infectivity in a dose-dependent manner, resulting in a reduction of infectious HCV by nearly 4 orders of magnitude. The knockdown of apoE expression by specific small interfering RNAs (siRNAs) remarkably reduced the levels of intracellular as well as secreted HCV virions. The apoE siRNA suppressed HCV production by more than 100-fold at 50 nM. These findings demonstrate that apoE is required for HCV virion infectivity and production, suggesting that HCV virions are assembled as apoE-enriched lipoprotein particles. Our findings also identified apoE as a novel target for discovery and development of antiviral drugs and monoclonal antibodies to suppress HCV virion formation and infection.

scholarly journals Detergent-Resistant Membrane Association of NS2 and E2 during Hepatitis C Virus Replication

2015 ◽  
Vol 89 (8) ◽  
pp. 4562-4574 ◽  
Author(s):  
Saravanabalaji Shanmugam ◽  
Dhanaranjani Saravanabalaji ◽  
MinKyung Yi
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Particle Production ◽  
Core Protein ◽  
Virus Assembly ◽  
Hcv Rna ◽  
Dependent Manner ◽  
Short Term Treatment ◽  
Particle Assembly ◽  
Detergent Resistant Membranes

ABSTRACTPreviously, we demonstrated that the efficiency of hepatitis C virus (HCV) E2-p7 processing regulates p7-dependent NS2 localization to putative virus assembly sites near lipid droplets (LD). In this study, we have employed subcellular fractionations and membrane flotation assays to demonstrate that NS2 associates with detergent-resistant membranes (DRM) in a p7-dependent manner. However, p7 likely plays an indirect role in this process, since only the background level of p7 was detectable in the DRM fractions. Our data also suggest that the p7-NS2 precursor is not involved in NS2 recruitment to the DRM, despite its apparent targeting to this location. Deletion of NS2 specifically inhibited E2 localization to the DRM, indicating that NS2 regulates this process. Treatment of cells with methyl-β-cyclodextrin (MβCD) significantly reduced the DRM association of Core, NS2, and E2 and reduced infectious HCV production. Since disruption of the DRM localization of NS2 and E2, either due to p7 and NS2 defects, respectively, or by MβCD treatment, inhibited infectious HCV production, these proteins' associations with the DRM likely play an important role during HCV assembly. Interestingly, we detected the HCV replication-dependent accumulation of ApoE in the DRM fractions. Taking into consideration the facts that ApoE was shown to be a major determinant for infectious HCV particle production at the postenvelopment step and that the HCV Core protein strongly associates with the DRM, recruitment of E2 and ApoE to the DRM may allow the efficient coordination of Core particle envelopment and postenvelopment events at the DRM to generate infectious HCV production.IMPORTANCEThe biochemical nature of HCV assembly sites is currently unknown. In this study, we investigated the correlation between NS2 and E2 localization to the detergent-resistant membranes (DRM) and HCV particle assembly. We determined that although NS2's DRM localization is dependent on p7, p7 was not targeted to these membranes. We then showed that NS2 regulates E2 localization to the DRM, consistent with its role in recruiting E2 to the virus assembly sites. We also showed that short-term treatment with the cholesterol-extracting agent methyl-β-cyclodextrin (MβCD) not only disrupted the DRM localization of Core, NS2, and E2 but also specifically inhibited intracellular virus assembly without affecting HCV RNA replication. Thus, our data support the role of the DRM as a platform for particle assembly process.

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