Involvement of the PA28gamma-dependent pathway in insulin resistance induced by hepatitis C virus core protein

The hepatitis C virus (HCV) core protein is a component of nucleocapsids and a pathogenic factor for hepatitis C. Several epidemiological and experimental studies have suggested that HCV infection is associated with insulin resistance, leading to type 2 diabetes. We have previously reported that HCV core gene-transgenic (PA28gamma(+/+)CoreTg) mice develop marked insulin resistance and that the HCV core protein is degraded in the nucleus through a PA28gamma-dependent pathway. In this study, we examined whether PA28gamma is required for HCV core-induced insulin resistance in vivo. HCV core gene-transgenic mice lacking the PA28gamma gene (PA28gamma(-/-)CoreTg) were prepared by mating of PA28gamma(+/+)CoreTg with PA28gamma-knockout mice. Although there was no significant difference in the glucose tolerance test results among the mice, the insulin sensitivity in PA28gamma(-/-)CoreTg mice was recovered to a normal level in the insulin tolerance test. Tyrosine phosphorylation of insulin receptor substrate 1 (IRS1), production of IRS2, and phosphorylation of Akt were suppressed in the livers of PA28gamma(+/+)CoreTg mice in response to insulin stimulation, whereas they were restored in the livers of PA28gamma(-/-)CoreTg mice. Furthermore, activation of the tumor necrosis factor alpha promoter in human liver cell lines or mice by the HCV core protein was suppressed by the knockdown or knockout of the PA28gamma gene. These results suggest that the HCV core protein suppresses insulin signaling through a PA28gamma-dependent pathway.     

Proteomic analysis of hepatitis C virus (HCV) core protein transfection and host regulator PA28γ knockout in HCV pathogenesis: a network-based study

Hepatitis C virus (HCV) causes chronic liver disease worldwide. HCV Core protein (Core) forms the viral capsid and is crucial for HCV pathogenesis and HCV-induced hepatocellular carcinoma, through its interaction with the host factor proteasome activator PA28γ. Here, using BD-PowerBlot high-throughput Western array, we attempt to further investigate HCV pathogenesis by comparing the protein levels in liver samples from Core-transgenic mice with or without the knockout of PA28γ expression (abbreviated PA28γ(-/-)CoreTG and CoreTG, respectively) against the wild-type (WT). The differentially expressed proteins integrated into the human interactome were shown to participate in compact and well-connected cellular networks. Functional analysis of the interaction networks using a newly developed data warehouse system highlighted cellular pathways associated with vesicular transport, immune system, cellular adhesion, and cell growth and death among others that were prominently influenced by Core and PA28γ in HCV infection. Follow-up assays with in vitro HCV cell culture systems validated VTI1A, a vesicular transport associated factor, which was upregulated in CoreTG but not in PA28γ(-/-)CoreTG, as a novel regulator of HCV release but not replication. Our analysis provided novel insights into the Core-PA28γ interplay in HCV pathogenesis and identified potential targets for better anti-HCV therapy and potentially novel biomarkers of HCV infection.     

Direct interaction of hepatitis C virus core protein with the cellular lymphotoxin-beta receptor modulates the signal pathway of the lymphotoxin-beta receptor.

Previous studies suggest that the core protein of hepatitis C virus (HCV) has a pleiotropic function in the replication cycle of the virus. To understand the role of this protein in HCV pathogenesis, we used a yeast two-hybrid protein interaction cloning system to search for cellular proteins physically interacting with the HCV core protein. One such cellular gene was isolated and characterized as the gene encoding the lymphotoxin-beta receptor (LT-betaR). In vitro binding analysis demonstrated that the HCV core protein binds to the C-terminal 98 amino acids within the intracellular domain of the LT-betaR that is involved in signal transduction, although the binding affinity of the full-length HCV core protein was weaker than that of its C-terminally truncated form. Our results also indicated that the N-terminal 40-amino-acid segment of the HCV core protein was sufficient for interaction with LT-betaR and that the core protein could form complexes with the oligomeric form of the intracellular domain of LT-betaR, which is a prerequisite for downstream signaling of this receptor. Similar to other members of the tumor necrosis factor (TNF) receptor superfamily, LT-betaR is involved in the cytotoxic effect of the signaling pathway, and thus we have elucidated the biological consequence of interaction between the HCV core protein and LT-betaR. Our results indicated that in the presence of the synergizing agent gamma interferon, the HCV core protein enhances the cytotoxic effects of recombinant forms of LT-betaR ligand in HeLa cells but not in hepatoma cells. Furthermore, this enhancement of the cytolytic activity was cytokine specific, since in the presence of cycloheximide, the expression of the HCV core protein did not elicit an increase in the cytolytic activity of TNF in both HeLa and hepatoma cells. In summary, the HCV core protein can associate with LT-betaR, and this protein-protein interaction has a modulatory effect on the signaling pathway of LT-betaR in certain cell types. Given the known roles of LT-betaR/LT-alpha1,beta2 receptor-ligand interactions in the normal development of peripheral lymphoid organs and in triggering cytolytic activity and NF-kappaB activation in certain cell types, our finding implies that the HCV core protein may aggravate these biological functions of LT-betaR, resulting in pathogenesis in HCV-infected cells.     

Interaction of hepatitis C virus core protein with viral sense RNA and suppression of its translation

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 beta-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.     

Hepatitis C virus core protein inhibits tumor necrosis factor alpha-mediated apoptosis by a protective effect involving cellular FLICE inhibitory protein

We have previously shown that hepatitis C virus (HCV) core protein modulates multiple cellular processes, including those that inhibit tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis. In this study, we have investigated the signaling mechanism for inhibition of TNF-alpha-mediated apoptosis in human hepatoma (HepG2) cells expressing core protein alone or in context with other HCV proteins. Activation of caspase-3 and the cleavage of DNA repair enzyme poly(ADP-ribose) polymerase were inhibited upon TNF-alpha exposure in HCV core protein-expressing HepG2 cells. In vivo protein-protein interaction studies displayed an association between TNF receptor 1 (TNFR1) and TNFR1-associated death domain protein (TRADD), suggesting that the core protein does not perturb this interaction. A coimmunoprecipitation assay also suggested that HCV core protein does not interfere with the TRADD-Fas-associated death domain protein (FADD)-procaspase-8 interaction. Further studies indicated that HCV core protein expression inhibits caspase-8 activation by sustaining the expression of cellular FLICE (FADD-like interleukin-1beta-converting enzyme)-like inhibitory protein (c-FLIP). Similar observations were also noted upon expression of core protein in context to other HCV proteins expressed from HCV full-length plasmid DNA or a replicon. A decrease in endogenous c-FLIP by specific small interfering RNA induced TNF-alpha-mediated apoptotic cell death and caspase-8 activation. Taken together, our results suggested that the TNF-alpha-induced apoptotic pathway is inhibited by a sustained c-FLIP expression associated with the expression of HCV core protein, which may play a role in HCV-mediated pathogenesis.     

Hepatitis C virus core protein leads to immune suppression and liver damage in a transgenic murine model

Hepatitis C virus (HCV) is remarkably efficient in establishing persistent infection, possibly mediated by an impaired immune response to HCV infection. There is compelling evidence that HCV can infect immune cells, such as macrophages, B cells, and T cells. It has been previously reported that HCV core, the first protein expressed during the early phase of viral infection, contains the immunomodulatory function of suppressing host immune responses. This altered function of immune cells caused by HCV infection may explain the ineffective immune response to HCV. To further characterize the immunomodulatory role of HCV core in vivo, we generated transgenic (TG) mice by directing the expression of core protein to T lymphocytes by using the CD2 promoter. T-lymphocyte responses, including the production of gamma interferon and interleukin-2, were significantly diminished in these mice compared to their non-TG littermates. The inhibition of T-lymphocyte responsiveness may be due to the increased susceptibility of peripheral T lymphocytes to Fas-mediated apoptosis. Surprisingly, significant lymphocyte infiltration was observed in the portal tracts of livers isolated from core TG mice, associated with increasing serum alanine aminotransferase levels. Moreover, no intrahepatic lymphocytes or liver damage was found in non-TG littermates and core TG mice bred to Fas-deficient lpr mice. These results suggest that HCV core drives liver injury by increasing Fas-mediated apoptosis and liver infiltration of peripheral T cells.     

Hepatitis C virus core protein: Its coordinate roles with PA28γ in metabolic abnormality and carcinogenicity in the liver

Hepatitis C virus (HCV) is a major cause of chronic liver diseases, including steatosis, cirrhosis and hepatocellular carcinoma, and epidemiological studies indicate that HCV is also associated with insulin resistance and type 2 diabetes mellitus. The HCV core protein is not only a viral structural component but also a pathogenic factor, since its expression leads to the development of liver steatosis, insulin resistance and hepatocellular carcinoma in mice. The nuclear proteasome activator PA28γ/REGγ, which specifically binds to the core protein, is required for the virulence of the core protein. Elucidation of the mechanisms by which HCV core protein participates in the above conditions may provide clues toward the development of novel therapeutic measures for chronic hepatitis C.     

Identification of a novel phosphatidic acid binding domain in protein phosphatase-1

Phosphatidic acid (PA) has been recognized as a lipid second messenger, yet few cellular targets for PA have been identified. Previous work demonstrated PA as a potent and noncompetitive tight-binding inhibitor of the catalytic subunit (gamma isoform) of protein phosphatase-1 (PP1c gamma) in vitro. The high potency of inhibition, coupled with high specificity for PA over other phospholipids, suggested the presence of a high-affinity PA binding domain on PP1c gamma. In the current study, quantification of the binding interaction and identification of the binding domain were pursued. Surface plasmon resonance was employed to quantitate the interaction between PP1c gamma and immobilized mixed lipid vesicles of PA/phosphatidylcholine (PC) or PC alone. The data disclosed a high-affinity interaction with a KD measured in the low (1-40) nanomolar range, consistent with the range of Ki previously obtained from in vitro enzymatic assays. Next, identification of the segment of PP1 necessary for PA binding was determined using a deletion mutagenesis strategy. Binding assays revealed that PP1c gamma residues between 274 and 299 were required for the interaction with the lipid. When fusions of PP1c gamma fragments with green fluorescent protein (GFP) were generated, it was then determined that PP1c gamma residues 286-296 were sufficient to confer PA binding to GFP, a protein that does not interact with PA. The minimal PA binding domain of PP1c gamma lacked similarity to the previously described PA binding segments of Raf-1 kinase and cyclic-AMP phosphodiesterase 4A1. When these results were taken together with the known crystallographic structure of PP1, they identified a novel PA binding region on PP1c gamma that contains a unique loop-strand structural fold responsible for the interaction with PA.     

Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified.We show here that the aa(109-133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1-173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.     

Hepatitis C virus core protein interacts with 14-3-3 protein and activates the kinase Raf-1

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.     

Memory T-cell-mediated immune responses specific to an alternative core protein in hepatitis C virus infection

In vitro studies have described the synthesis of an alternative reading frame form of the hepatitis C virus (HCV) core protein that was named F protein or ARFP (alternative reading frame protein) and includes a domain coded by the +1 open reading frame of the RNA core coding region. The expression of this protein in HCV-infected patients remains controversial. We have analyzed peripheral blood from 47 chronically or previously HCV-infected patients for the presence of T lymphocytes and antibodies specific to the ARFP. Anti-ARFP antibodies were detected in 41.6% of the patients infected with various HCV genotypes. Using a specific ARFP 99-amino-acid polypeptide as well as four ARFP predicted class I-restricted 9-mer peptides, we show that 20% of the patients display specific lymphocytes capable of producing gamma interferon, interleukin-10, or both cytokines. Patients harboring three different viral genotypes (1a, 1b, and 3) carried T lymphocytes reactive to genotype 1b-derived peptides. In longitudinal analysis of patients receiving therapy, both core and ARFP-specific T-cell- and B-cell-mediated responses were documented. The magnitude and kinetics of the HCV antigen-specific responses differed and were not linked with viremia or therapy outcome. These observations provide strong and new arguments in favor of the synthesis, during natural HCV infection, of an ARFP derived from the core sequence. Moreover, the present data provide the first demonstration of the presence of T-cell-mediated immune responses directed to this novel HCV antigen.     

SOCS1 and SOCS3 are targeted by hepatitis C virus core/gC1qR ligation to inhibit T-cell function

T cells play an important role in the control of hepatitis C virus (HCV) infection. We have previously demonstrated that the HCV core inhibits T-cell responses through interaction with gC1qR. We show here that core proteins from chronic and resolved HCV patients differ in sequence, gC1qR-binding ability, and T-cell inhibition. Specifically, chronic core isolates bind to gC1qR more efficiently and inhibit T-cell proliferation as well as gamma interferon (IFN-gamma) production more profoundly than resolved core isolates. This inhibition is mediated by the disruption of STAT phosphorylation through the induction of SOCS molecules. Silencing either SOCS1 or SOCS3 by small interfering RNA dramatically augments the production of IFN-gamma in T cells, thereby abrogating the inhibitory effect of core. Additionally, the ability of core proteins from patients with chronic infections to induce SOCS proteins and suppress STAT activation greatly exceeds that of core proteins from patients with resolved infections. These results suggest that the HCV core/gC1qR-induced T-cell dysfunction involves the induction of SOCS, a powerful inhibitor of cytokine signaling, which represents a novel mechanism by which a virus usurps the host machinery for persistence.     

Proteasome activator PA28 gamma regulates p53 by enhancing its MDM2-mediated degradation

Downregulation of p53 by MDM2-mediated proteasomal degradation makes cells resistant to apoptosis. The MDM2-p53 interaction is well characterized, but the mechanisms that regulate the interaction are not well understood. Here, we show that PA28gamma, a proteasome activator that inhibits apoptosis and promotes cell cycle progression through unknown mechanisms, exerts an effect as a cofactor in the MDM2-p53 interaction. The polymer form of PA28gamma interacts with both MDM2 and p53 proteins and facilitates their physical interaction. This promotes ubiquitination- and MDM2-dependent proteasomal degradation of p53, limiting its accumulation and resulting in inhibited apoptosis after DNA damage. Elimination of endogenous PA28gamma in human cancer cells abrogates MDM2-mediated p53 degradation, increases the activity of p53, and enhances apoptosis. These findings reveal the mechanism by which PA28gamma affects apoptosis and proliferation. Manipulation of the level of PA28gamma, an approach that would regulate the cellular content of p53, may improve the efficacy of current cancer therapies.     

Immune defects in 28-kDa proteasome activator gamma-deficient mice

Protein complexes of the 28-kDa proteasome activator (PA28) family activate the proteasome and may alter proteasome cleavage specificity. Initial investigations have demonstrated a role for the IFN-gamma-inducible PA28alpha/beta complex in Ag processing. Although the noninducible and predominantly nuclear PA28gamma complex has been implicated in affecting proteasome-dependent signaling pathways, such as control of the mitotic cell cycle, there is no previous evidence demonstrating a role for this structure in Ag processing. We therefore generated PA28gamma-deficient mice and investigated their immune function. PA28gamma(-/-) mice display a slight reduction in CD8+ T cell numbers and do not effectively clear a pulmonary fungal infection. However, T cell responses in two viral infection models appear normal in both magnitude and the hierarchy of antigenic epitopes recognized. We conclude that PA28gamma(-/-) mice, like PA28alpha(-/-)/beta(-/-) mice, are deficient in the processing of only specific Ags.     

丙型肝炎病毒核心蛋白基因表达致人肝细胞恶性转化的研究

丙型肝炎病毒核心（HCV-C）蛋白是维持丙型肝炎病毒结构的重要蛋白质,由于参与调节细胞的生长与凋亡,被认为与HCV感染所致的肝硬化及肝细胞癌的发生有关.为了进一步探索HCV-C蛋白与肝细胞癌发生的关系,首先构建了表达HCV-C蛋白的真核表达载体,脂质体介导转染Chang-liver人肝细胞株,建立表达HCV-C蛋白的人肝细胞模型,RT-PCR方法检测HCV-C基因在人肝细胞内的表达,蛋白质印迹和免疫细胞化学方法鉴定Chang-liver肝细胞内HCV-C蛋白及其在细胞内的分布情况.表达HCV-C蛋白的Chang-liver人肝细胞培养20代以后,与对照组细胞相比,细胞的形态出现长梭形样改变,生长速度显著加快,细胞内DNA含量的均一性变差.接种表达HCV-C蛋白的Chang-liver人肝细胞的6只裸鼠在第20天时全部有肿瘤长出,且肿瘤组织结构符合肝细胞癌病理形态特点,对照组裸鼠未见肿瘤生长.上述结果表明HCV-C基因表达可导致Chang-liver人肝细胞发生恶性转化,提示HCV-C蛋白与HCV感染所致肝细胞癌的发生有直接关系.     

Fcgamma receptor-like activity of hepatitis C virus core protein

We have previously demonstrated that viral particles with the properties of nonenveloped hepatitis C virus (HCV) nucleocapsids occur in the serum of HCV-infected individuals (1). We show here that nucleocapsids purified directly from serum or isolated from HCV virions have FcgammaR-like activity and bind "nonimmune" IgG via its Fcgamma domain. HCV core proteins produced in Escherichia coli and in the baculovirus expression system also bound "nonimmune" IgG and their Fcgamma fragments. Folded conformation was required for IgG binding because the FcgammaR-like site of the core protein was inactive in denaturing conditions. Studies with synthetic core peptides showed that the region spanning amino acids 3-75 was essential for formation of the IgG-binding site. The interaction between the HCV core and human IgG is more efficient in acidic (pH 6.0) than in neutral conditions. The core protein-binding site on the IgG molecule differs from those for C1q, FcgammaRII (CD32), and FcgammaRIII (CD16) but overlaps with that for soluble protein A from Staphylococcus aureus (SpA), which is located in the CH2-CH3 interface of IgG. These characteristics of the core-IgG interaction are very similar to those of the neonatal FcRn. Surface plasmon resonance studies suggested that the binding of an anti-core antibody to HCV core protein might be "bipolar" through its paratope to the corresponding epitope and by its Fcgamma region to the FcgammaR-like motif on this protein. These features of HCV nucleocapsids and HCV core protein may confer an advantage for HCV in terms of survival by interfering with host defense mechanisms mediated by the Fcgamma part of IgG.