p53-dependent transcriptional repression of p21waf1 by hepatitis C virus NS3

Hepatitis C virus (HCV) NS3 protein is known to affect normal cellular functions, such as cell proliferation and cell death, and to be involved, either directly or indirectly, in HCV hepatocarcinogenesis. In this study, we demonstrated that NS3 protein could specifically repress the promoter activity of p21 in a dose-dependent manner. The effect was not cell type-specific and was synergistic when combined with HCV core protein. Repression of the p21 promoter by NS3 was almost completely lost when p53 binding sites present on the p21 promoter were removed. Furthermore, p53 binding sites were sufficient to confer a strong NS3 responsiveness to an heterologous promoter, suggesting that NS3 represses the transcription of p21 by modulating the activity of p53. Although the NS3 protein domain required for the majority of p21 repression was located on the protease domain, the proteinase activity itself does not seem to be necessary for repression. Both transcription and protein stability of p53 were unaffected by NS3, suggesting that NS3 might repress transcription of p21 by inhibiting the regulatory activity of p53 via protein–protein interaction(s). Finally, the growth rate of NS3-expressing cell lines was at least twice as fast as that of the parent NIH 3T3 cells, indicating that the repression of p21 is actually reflected by the stimulation of cell growth.

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

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10.1111/j.1600-0854.2008.00767.x ◽

2008 ◽

Vol 9 (8) ◽

pp. 1268-1282 ◽

Cited By ~ 150

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

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Hepatitis C Virus NS5A Physically Associates with p53 and Regulates p21/waf1 Gene Expression in a p53-Dependent Manner

Journal of Virology ◽

10.1128/jvi.75.3.1401-1407.2001 ◽

2001 ◽

Vol 75 (3) ◽

pp. 1401-1407 ◽

Cited By ~ 177

Author(s):

Mainak Majumder ◽

Asish K. Ghosh ◽

Robert Steele ◽

Ranjit Ray ◽

Ratna B. Ray

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Transcriptional Activation ◽

Transcriptional Repression ◽

Dependent Manner ◽

Downstream Effector ◽

Transcriptional Modulation ◽

P21 Waf1 ◽

Two Hybrid

ABSTRACT We have previously demonstrated that hepatitis C virus (HCV) NS5A protein promotes cell growth and transcriptionally regulates the p21/waf1 promoter, a downstream effector gene of p53. In this study, we investigated the molecular mechanism of NS5A-mediated transcriptional repression of p21/waf1. We observed that transcriptional repression of the p21/waf1 gene by NS5A is p53 dependent by using p53 wild-type (+/+) and null (−/−) cells. Interestingly, p53-mediated transcriptional activation from a synthetic promoter containing multiple p53 binding sites (PG13-LUC) was abrogated following expression of HCV NS5A. Additional studies using pull-down experiments, in vivo coimmunoprecipitation, and mammalian two-hybrid assays demonstrated that NS5A physically associates with p53. Confocal microscopy revealed sequestration of p53 in the perinuclear membrane and colocalization with NS5A in transfected HepG2 and Saos-2 cells. Together these results suggest that an association of NS5A and p53 allows transcriptional modulation of the p21/waf1 gene and may contribute to HCV-mediated pathogenesis.

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

Journal of Virology ◽

10.1128/jvi.73.12.9718-9725.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 9718-9725 ◽

Cited By ~ 117

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.

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

Journal of Virology ◽

10.1128/jvi.74.4.1736-1741.2000 ◽

2000 ◽

Vol 74 (4) ◽

pp. 1736-1741 ◽

Cited By ~ 112

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.

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Ubiquitin-Independent Degradation of Hepatitis C Virus F Protein

Journal of Virology ◽

10.1128/jvi.00832-08 ◽

2008 ◽

Vol 83 (2) ◽

pp. 612-621 ◽

Cited By ~ 43

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.

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Activation of the Interferon-Inducible 2′-5′-Oligoadenylate Synthetase Gene by Hepatitis C Virus Core Protein

Journal of Virology ◽

10.1128/jvi.74.18.8744-8750.2000 ◽

2000 ◽

Vol 74 (18) ◽

pp. 8744-8750 ◽

Cited By ~ 55

Author(s):

Atsushi Naganuma ◽

Akito Nozaki ◽

Torahiko Tanaka ◽

Kazuo Sugiyama ◽

Hitoshi Takagi ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Core Protein ◽

Gene Promoter ◽

Amino Acid Sequences ◽

Expression Vectors ◽

Transcriptional Level ◽

Dependent Manner ◽

Oligoadenylate Synthetase ◽

Hepatocyte Cells

ABSTRACT The effects of hepatitis C virus (HCV) proteins on several signal transduction pathways in human nonneoplastic hepatocyte PH5CH8 cells were investigated using expression vectors encoding HCV proteins derived from HCV-infected human nonneoplastic cultured T-lymphocyte and hepatocyte cells (MT-2C and PH5CH7), which could support HCV replication. The amino acid sequences of HCV proteins obtained from HCV-infected human cells were identical or very close to the consensus sequences of the proteins derived from the original inoculum used for HCV infection. During the course of the study, we found that HCV core protein specifically activated the 40/46-kDa 2′-5′-oligoadenylate synthetase (2′-5′-OAS) gene promoter in a dose-dependent manner in different human hepatocyte cell lines (PH5CH8, HepG2, and PLC/PRF/5). We also found that the activation by core protein was further enhanced in the cells treated with alpha interferon. The expression of E1 or E2 envelope protein or nonstructural NS5A protein did not activate the 2′-5′-OAS gene promoter. We demonstrated that the activation by core protein in the hepatocyte cells was suppressed by antisense RNA complementary to core-encoding RNA. Deletion mutant analysis of core protein and deletion analysis of the 2′-5′-OAS gene promoter have been performed. Finally, we demonstrated that the activation of the 2′-5′-OAS gene occurred at the transcriptional level and furthermore demonstrated that the endogenous 2′-5′-OAS gene was also activated by core protein. This is the first report to show that a viral protein activated the 2′-5′-OAS gene.

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Hepatitis C virus core protein represses the p21 promoter through inhibition of a TGF-β pathway

Journal of General Virology ◽

10.1099/0022-1317-83-9-2145 ◽

2002 ◽

Vol 83 (9) ◽

pp. 2145-2151 ◽

Cited By ~ 32

Author(s):

Mi Nam Lee ◽

Eun Young Jung ◽

Hyun Jin Kwun ◽

Hong Ki Jun ◽

Dae-Yeul Yu ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Cycle Progression ◽

Kinase Inhibitor ◽

Core Protein ◽

3T3 Cells ◽

Hcv Core Protein ◽

Nih 3T3 ◽

P21 Promoter ◽

Nih 3T3 Cells

The increased proliferation rate of hepatocytes is one of the major risk factors for the development of hepatocellular carcinoma. In this study, we investigated the mechanism by which hepatitis C virus (HCV) core protein represses transcription of the universal cyclin-dependent kinase inhibitor p21 gene in murine fibroblast NIH 3T3 cells. From the transient reporter assays of p21 promoter, we found that the TGF-β-responsive element (TβRE) located between −83 and −74 of the p21 promoter is responsible for the effect. The TGF-β-induced p21 promoter activity was specifically decreased by HCV core protein and in the presence of the inhibitory Smad7 the repression effect was almost completely abolished. Furthermore, HCV core protein stimulated the growth rate of NIH 3T3 cells and could overcome growth arrest by TGF-β but not by butyrate, suggesting that HCV core protein stimulates cell cycle progression by repressing p21 transcription through a TGF-β pathway.

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

Journal of Virology ◽

10.1128/jvi.01091-07 ◽

2007 ◽

Vol 81 (24) ◽

pp. 13783-13793 ◽

Cited By ~ 312

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.

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

Journal of Virology ◽

10.1128/jvi.00123-15 ◽

2015 ◽

Vol 89 (8) ◽

pp. 4562-4574 ◽

Cited By ~ 17

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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