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• W2044679901 abstract "Hepatitis C virus (HCV) core protein, a viral nucleocapsid, has been shown to affect various intracellular events including the nuclear factor κB (NF-κB) signaling supposedly associated with inflammatory response, cell proliferation, and apoptosis. In order to elucidate the effect of HCV core protein on the NF-κB signaling in HeLa and HepG2 cells, a reporter assay was utilized. HCV core protein significantly activated NF-κB signaling in a dose-dependent manner not only in HeLa and HepG2 cells transiently transfected with core protein expression plasmid, but also in HeLa cells induced to express core protein under the control of doxycycline. HCV core protein increased the DNA binding affinity of NF-κB in the electrophoretic mobility shift assay. Acetyl salicylic acid, an IKK%-specific inhibitor, and dominant negative form of IKK% significantly blocked NF-κB activation by HCV core protein, suggesting HCV core protein activates the NF-κB pathway mainly through IKK%. Moreover, the dominant negative forms of TRAF2/6 significantly blocked activation of the pathway by HCV core protein, suggesting HCV core protein mimics proinflammatory cytokine activation of the NF-κB pathway through TRAF2/6. In fact, HCV core protein activated interleukin-1% promoter mainly through NF-κB pathway. Therefore, this function of HCV core protein may play an important role in the inflammatory reaction induced by this hepatotropic virus. Hepatitis C virus (HCV) core protein, a viral nucleocapsid, has been shown to affect various intracellular events including the nuclear factor κB (NF-κB) signaling supposedly associated with inflammatory response, cell proliferation, and apoptosis. In order to elucidate the effect of HCV core protein on the NF-κB signaling in HeLa and HepG2 cells, a reporter assay was utilized. HCV core protein significantly activated NF-κB signaling in a dose-dependent manner not only in HeLa and HepG2 cells transiently transfected with core protein expression plasmid, but also in HeLa cells induced to express core protein under the control of doxycycline. HCV core protein increased the DNA binding affinity of NF-κB in the electrophoretic mobility shift assay. Acetyl salicylic acid, an IKK%-specific inhibitor, and dominant negative form of IKK% significantly blocked NF-κB activation by HCV core protein, suggesting HCV core protein activates the NF-κB pathway mainly through IKK%. Moreover, the dominant negative forms of TRAF2/6 significantly blocked activation of the pathway by HCV core protein, suggesting HCV core protein mimics proinflammatory cytokine activation of the NF-κB pathway through TRAF2/6. In fact, HCV core protein activated interleukin-1% promoter mainly through NF-κB pathway. Therefore, this function of HCV core protein may play an important role in the inflammatory reaction induced by this hepatotropic virus. hepatitis C virus amino acid(s) activator protein-1 electrophoretic mobility shift assay IκB kinase interleukin latent membrane protein-1 mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 nuclear factor κB nucleotide(s) nuclear factor κB inducing kinase receptor interacting protein tumor necrosis factor tumor necrosis factor receptor 1 tumor necrosis factor receptor-associated death domain tumor necrosis factor receptor-associated factor hemagglutinin polymerase chain reaction phosphate-buffered saline Hepatitis C virus (HCV),1 a member of theFlaviviridae family, is one of the major causes of chronic hepatitis, which can result in cirrhosis and finally hepatocellular carcinoma (1Saito I. Miyamura T. Ohbayashi A. Harada H. Katayama T. Kikuchi S. Watanabe Y. Koi S. Onji M. Ohta Y. Choo Q.-L. Houghton M. Kuo G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6547-6549Crossref PubMed Scopus (1084) Google Scholar). Its genome consists of a linear, positive-strand RNA molecule of ∼9,500 nucleotides (nt) encoding a single polyprotein precursor of ∼3,000 amino acids (aa) that is processed into three to four structural proteins at the amino terminus (Core, E1, and E2/p7) and six nonstructural proteins at the carboxyl terminus (nonstructural proteins 2, 3, 4a, 4b, 5a, and 5b) (2Hijikata M. Kato N. Ootsuyama Y. Nakagawa M. Shimotohno K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5547-5551Crossref PubMed Scopus (580) Google Scholar, 3Grakoui A. Wychowski C. Lin C.,. Feinstone S.M. Rice C.M. J. Virol. 1993; 67: 1385-1395Crossref PubMed Google Scholar). The genomic region of the putative core protein encodes 191 aa and has an apparent molecular mass of 21 kDa (2Hijikata M. Kato N. Ootsuyama Y. Nakagawa M. Shimotohno K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5547-5551Crossref PubMed Scopus (580) Google Scholar). The core protein, relatively conserved among all identified HCV isolates (4Bukh J. Purcell R.H. Miller R.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8239-8243Crossref PubMed Scopus (267) Google Scholar), may be the fundamental unit for the encapsidation of genomic RNA to help in virus morphogenesis. In addition, previous studies suggested that HCV core protein has various biological properties, one of which is its effect on the nuclear factor κB (NF-κB) pathway (5Shrivastava A. Manna S.K. Ray R. Aggarwal B.B. J. Virol. 1998; 72: 9722-9728Crossref PubMed Google Scholar, 6Zhu N. Khoshnan A. Schneider R. Matsumoto M. Dennert G. Ware C. Lai M.M.C. J. Virol. 1998; 72: 3679-3691Google Scholar, 7Marusawa H. Hijikata M. Chiba T. Shimotohno K. J. Virol. 1999; 73: 4713-4720Crossref PubMed Google Scholar, 8You L.R. Chen C.M. Lee Y.H.W. J. Virol. 1999; 73: 1672-1681Crossref PubMed Google Scholar, 9Kato N. Yoshida H. Ono-Nita S.K. Kato J. Goto T. Otsuka M. Lan K.-H. Matsushima K. Shiratori Y. Omata M. Hepatology. 2000; 32: 405-412Crossref PubMed Scopus (200) Google Scholar). NF-κB belongs to a highly conserved Rel-related protein family, which includes RelA (p65), RelB, c-Rel, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). Of these, the p50/p65 heterodimer, commonly referred to as NF-κB, is the most abundant and ubiquitous. One of the most intensively studied signals to the NF-κB is induced by tumor necrosis factor (TNF), a proinflammatory cytokine associated with inflammation, immune response, and apoptosis. Currently, this signal transduction pathway is understood as follows (10Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4584) Google Scholar, 11Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1739) Google Scholar, 12Stanger B.Z. Leder P. Lee T.-H. Kim E. Seed B. Cell. 1995; 81: 513-523Abstract Full Text PDF PubMed Scopus (861) Google Scholar, 13Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1070) Google Scholar, 14Woronicz J.D. Gao Z. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1065) Google Scholar, 15Nakano H. Shindo M. Sakon S. Nishikawa S. Mihara M. Yagita H. Okumura K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3537-3542Crossref PubMed Scopus (471) Google Scholar); when TNF binds and activates the TNF receptor 1 (TNFR1), TNFR-associated death domain (TRADD), TNFR-associated factor 2 (TRAF2), and receptor interacting protein (RIP) form a complex with TNFR1. Subsequently NF-κB inducing kinase (NIK) and/or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEKK1) are activated. Activated NIK and/or MEKK1 phosphorylate and activate both IκB kinase (IKK) α and IKK%. Activated IKKα/% phosphorylate IκBα, which associates with and sequesters NF-κB in the cytoplasm. Phosphorylated IκBα is ubiquitinated and degraded, and then NF-κB translocates into the nucleus and binds to DNA to initiate the transcription of various genes associated with inflammation, the immune response, cell growth, and survival. There have, however, been conflicting reports up until now about the effect of HCV core protein on this NF-κB pathway. Recently, it was shown that HCV core protein activated the NF-κB pathway (7Marusawa H. Hijikata M. Chiba T. Shimotohno K. J. Virol. 1999; 73: 4713-4720Crossref PubMed Google Scholar, 8You L.R. Chen C.M. Lee Y.H.W. J. Virol. 1999; 73: 1672-1681Crossref PubMed Google Scholar, 9Kato N. Yoshida H. Ono-Nita S.K. Kato J. Goto T. Otsuka M. Lan K.-H. Matsushima K. Shiratori Y. Omata M. Hepatology. 2000; 32: 405-412Crossref PubMed Scopus (200) Google Scholar). On the contrary, it was previously shown that core protein suppressed TNF-induced NF-κB activation (5Shrivastava A. Manna S.K. Ray R. Aggarwal B.B. J. Virol. 1998; 72: 9722-9728Crossref PubMed Google Scholar, 6Zhu N. Khoshnan A. Schneider R. Matsumoto M. Dennert G. Ware C. Lai M.M.C. J. Virol. 1998; 72: 3679-3691Google Scholar). At present, the mechanism for core protein's effect on the NF-κB pathway remains unclear; therefore, we focused our attention on the effect HCV core protein has on the NF-κB pathway and tried to determine how core protein affects NF-κB signaling. Human cervical carcinoma cells (HeLa), human hepatoma cells (HepG2), and monkey kidney cells (COS-7) were obtained from the RIKEN cell bank (Tsukuba Science City, Japan). HeLa Tet-Off cells, which constitutively express the tetracycline-controlled transactivator, were purchased from CLONTECH (Palo Alto, CA). Cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal bovine serum. Type 1b HCV core region (nt 1–575 and aa 1–191 of the prototype HCV type 1b, HCV-J; Ref. 16Kato N. Hijikata M. Ootsuyama Y. Nakagawa N. Ohkoshi S. Sugimura T. Shimotohno K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9524-9528Crossref PubMed Scopus (1087) Google Scholar) was amplified by reverse transcription-polymerase chain reaction (PCR) using the HCV RNA extracted from the sera of a patient with chronic hepatitis C as a template. Reverse transcription-PCR was performed as previously described (17Kato N. Yokosuka O. Omata M. Hosoda K. Ohto M. J. Clin. Invest. 1990; 86: 1764-1767Crossref PubMed Scopus (148) Google Scholar) using the following primers having an XhoI restriction site (underlined) (the nucleotide positions in HCV-J are shown in parentheses): F1 (a sense primer, nt 1–20), 5′-CCGCTCGAGACCATGAGCACGAATCCTAAACC-3′, and R573 (an antisense primer, nt 555–573), 5′-CCGCTCGAGTCAAGCGGAAGCTGGGATGGTC-3′. The amplified product was digested with XhoI and then cloned into theXhoI site of pCXN2 (kindly provided by J. Miyazaki, Osaka University, Osaka, Japan), a mammalian expression vector having a %-actin promoter and cytomegalovirus enhancer (18Niwa H. Yamamura K. Miyazaki J. Gene ( Amst. ). 1991; 108: 193-199Crossref PubMed Scopus (4562) Google Scholar), to generate pCXN2-core. In addition to the plasmid expressing full-length core protein (aa 1–191), we constructed three more plasmids expressing deletion mutant forms of HCV core protein, aa 1–173 (pCXN2-core173), aa 1–151 (pCXN2-HAcore151), and aa 92–191 (pCXN2-HAcore92–191). The core fragments were amplified by PCR using pCXN2-core as a template and then cloned into pCXN2 or pCXN2-HA for the expression of hemagglutinin (HA; YPYDVPDYA)-tagged core protein. For construction of pCXN2-core173, the region of HCV core aa 1–173 was amplified with primers F1 and R519 (an antisense primer, nt 501–519), 5′-CCGCTCGAGTCAAGCGGAAGCTGGGATGGTC-3′, and then cloned into pCXN2. For construction of pCXN2-HAcore151, the region of HCV core aa 1–151 was amplified with F1 and R453 (an antisense primer, nt 438–453), 5′-CCGCTCGAGTCACAGGGCCCTGGCAGCG-3′, and then cloned into pCXN2-HA. Similarly, pCXN2-HAcore92–191, which encodes N-terminal 91-aa deleted core protein, was constructed by using F276 (a sense primer, nt 276–292), 5′-CCGCTCGAGACCATGGGGTGGGCAGGATGGCT-3′, and R573. To generate a tetracycline-regulated HCV core expression plasmid (pTRE2-core), full-length HCV core cDNA was prepared by digesting pCXN2-core with XhoI and then subcloned into theSalI site of pTRE2 (CLONTECH). This construct allowed for the expression of HCV core protein under the transcriptional control of a tetracycline-controlled transactivator-dependent promoter. A series of core expression plasmids were sequenced using a cycle DNA sequencing system (PE Applied Biosystems, Foster City, CA), as described previously (19Togo G. Toda N. Kanai F. Kato N. Shiratori Y. Kishi K. Imazeki F. Makuuchi M. Omata M. Cancer Res. 1996; 56: 5620-5623PubMed Google Scholar), to confirm the integration of core genes. Expression of core proteins was examined by the ECL-plus Western blotting detection system (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), using extracts of COS-7 cells transfected with core expression plasmids and mouse anti-HCV core antigen IgG fraction (Austral Biologicals, San Ramon, CA) or anti-HA polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). To examine the effect of HCV core protein on the NF-κB pathway, NF-κB-inducible reporter plasmid (pNF-κB-Luc) containing the Photinus pyralis(firefly) luciferase reporter gene driven by a basic promoter element (TATA box) plus five repeats of κB cis-enhancer element (TGGGGACTTTCCGC) (Stratagene, La Jolla, CA) was utilized. pFC-MEKK (Stratagene), which expresses constitutively active MEKK1 (amino acids 360–672) driven by a cytomegalovirus promoter, was used as a positive control plasmid to activate the NF-κB pathway. pRL-TK (Promega, Madison, WI), a control plasmid that expresses Renilla reniformis (sea pansy) luciferase driven by the herpes simplex virus thymidine kinase promoter, was used to monitor the efficiency of transfection. To elucidate the mechanism of how core protein affects the NF-κB pathway, the expression vectors for catalytically inactive IKKα (pIKKα(K44A)), containing an alanine substitution of a conserved lysine residue in its kinase domain (13Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1070) Google Scholar), and IKK% (pIKK%(K44A)) (20Schall T.J. Lewis M. Koller K.J. Lee A. Rice G.C. Wong G.H. Gatanaga T. Granger G.A. Lentz R. Raab H. Cell. 1990; 61: 361-370Abstract Full Text PDF PubMed Scopus (844) Google Scholar) (kindly provided by Goeddel DV, Tularik, CA) were utilized. In addition, the expression vectors for the dominant negative form of TRAF2 (pTRAF2-(87–501)), lacking the RING finger motif (21Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (975) Google Scholar), and TRAF6 (pTRAF6-(289–522)), lacking either the entire zinc-binding region or zinc fingers 3–5 (22Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1118) Google Scholar), were kindly provided by Goeddel DV and utilized. Moreover, pCMV-TAK1 (K63W) (kindly provided by K. Matsumoto, University of Tokyo, Tokya, Japan), which expresses catalytically inactive TAK1, was utilized with pCMV-TAB1 (kindly provided by K. Matsumoto), which expresses TAK1 activator (23Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1014) Google Scholar). In addition to the reporter plasmid having synthetic κB cis enhancer element, luciferase reporter plasmids having IL-1% or TNFα promoter containing NF-κB binding sites, were constructed. A fragment of 691 base pairs (positions −585 to +106) of the TNFα gene was amplified by PCR using a primer set of 5′-ggggtaccgcttgtcctgctacccc-3′ and 5′-cccaagcttgtcaggggatgtggcgt-3′. The amplified fragment was digested with KpnI andHindIII, and then cloned into pGL3 basic vector (Promega) (pTNFα-Luc). Similarly, a fragment of 1125 base pairs (−1110 to +15) of IL-1% gene was amplified by PCR using primer set of 5′-ggggtacccctgtagtcccagctg-3′ and 5′-ctagctagctcgaagaggtttggtatct-3′. Those fragments were digested with KpnI and NheI, and then cloned into pGL3 basic vector (pIL-1%-Luc). All cloned plasmids were purified using the Endfree plasmid kit (Qiagen, Hilden, Germany). Nucleotide sequencing of constructed plasmids was performed using an autosequencer (PE Applied Biosystems) and the dye termination method as described previously (19Togo G. Toda N. Kanai F. Kato N. Shiratori Y. Kishi K. Imazeki F. Makuuchi M. Omata M. Cancer Res. 1996; 56: 5620-5623PubMed Google Scholar) to confirm gene expression. HeLa cells induced to express HCV core protein were generated with use of a tetracycline-regulated gene expression system (Tet-Off gene expression system, CLONTECH). HeLa Tet-Off cells were cotransfected with pTRE2-core and pTK-Hyg (CLONTECH), a selection vector that confers hygromycin resistance, followed by selection in culture medium containing 200 μg/ml hygromycin (CLONTECH) and 1 μg/ml doxycycline (Sigma). Hygromycin-resistant clones, termed HeTOC, were examined for expression of the core protein upon withdrawal of doxycycline by Western blotting using mouse anti-HCV core antigen IgG fraction, as described previously. Positive clones were expanded and rescreened by Western blotting of cells grown in the presence and absence of doxycycline. We used the Effectene transfection reagent (Qiagen) for all transfection experiments. Approximately 4 × 105 HeLa cells were plated into the well of a six-well tissue culture plate (Iwaki Glass, Chiba, Japan) 24 h before transfection. To examine the effect of HCV core protein on the NF-κB pathway, HeLa cells were transfected with a total of 0.4 μg of plasmids consisting of 0.19 μg of pNF-κB-Luc, 0.01 μg of pRL-TK, and 0.2 μg of pCXN2 or pCXN2-core. As a positive control, pFC-MEKK was added to the transfection complexes containing pCXN2, or human TNFα (Strathmann Biotech GmbH, Hamburg, Germany) was added to the medium of transfected HeLa cells at a concentration of 20 ng/ml 6 h before harvest. The effect of HCV core protein on the NF-κB pathway in HeLa cells was examined using HepG2 cells with the same protocol as that used for HeLa cells. To examine the dose-dependent effect of core protein on the NF-κB pathway, HeLa cells were transfected with a total of 1.2 μg of plasmids consisting of 0.38 μg of pNF-κB-Luc, 0.02 μg of pRL-TK, 0–0.8 μg of pCXN2-core, and 0–0.8 μg of pCXN2 adjusted to total 1.2 μg. In addition to the HeLa cells, which express core protein transiently, HeTOC cells, which can be induced to express core protein under the control of doxycycline, were used to examine the effect of HCV core protein on the NF-κB pathway. Approximately 4 × 105HeTOC cells were plated into the well of a six-well tissue culture plate containing 1 μg/ml doxycycline 24 h before transfection. Cells were transfected with 0.39 μg of pNF-κB-Luc and 0.01 μg of pRL-TK and cultured in medium with or without doxycycline. To elucidate how HCV core protein affects the NF-κB pathway, dominant negative forms of IKKα/%, TRAF2/6, and TAK1, and an IKK%-specific inhibitor were used. To initially examine the effect of the dominant negative form of components of the NF-κB pathway, HeLa cells were transfected with a total of 1.2 μg of plasmids consisting of 0.38 μg of pNF-κB-Luc, 0.02 μg of pRL-TK, 0.4 μg of pCXN2-core, and 0.4 μg of one of the following pIKKα(K44A), pIKK%(K44A), pTRAF2-(87–501), or pTRAF6-(289–522). Similarly, HeLa cells were transfected with a total of 1.6 μg of plasmids consisting of 0.38 μg of pNF-κB-Luc, 0.02 μg of pRL-TK, 0.4 μg of pCXN2-core, 0.4 μg of pCMV-TAK1(K63W), and 0.4 μg of pCMV-TAB1. Second, we added 5 mm acetyl salicylic acid (Sigma), which inhibits cyclooxygenase (24Vane J. Nature. 1994; 367: 215-216Crossref PubMed Scopus (696) Google Scholar) and IKK% (25Yin M.J. Yamamoto Y. Gaynor R.B. Nature. 1998; 396: 77-80Crossref PubMed Scopus (1422) Google Scholar), to the medium of HeLa cells transfected with 0.19 μg of pNF-κB-Luc, 0.01 μg of pRL-TK, and 0.2 μg of pCXN2 or pCXN2-core. Acetyl salicylic acid was added 1 h after the transfection of plasmids. Instead of acetyl salicylic acid, 25 μm indomethacin (Sigma), a non-steroidal anti-inflammatory drug, which inhibits cyclooxygenase but not IKK% (24Vane J. Nature. 1994; 367: 215-216Crossref PubMed Scopus (696) Google Scholar, 25Yin M.J. Yamamoto Y. Gaynor R.B. Nature. 1998; 396: 77-80Crossref PubMed Scopus (1422) Google Scholar), was added to the medium as a control. The entire cell lysate was collected 36 h after transfection. A luciferase assay was performed with the PikkaGene dual sea pansy system (Toyo Ink, Tokyo, Japan) using a luminometer (Lumat LB9507, EG&G Berthold, Bad Wildbad, Germany). Assays were conducted at least in triplicate. Firefly luciferase activity and sea pansy luciferase activity were measured as a relative light unit. Firefly luciferase activity was then normalized for transfection efficiency based on sea pansy luciferase activity. Indirect immunofluorescence staining was performed as previously described (26Ide Y. Zhang L. Chen M. Inchauspe G. Bahl C. Sasaguri Y. Padmanabhan R. Gene ( Amst. ). 1996; 182: 203-211Crossref PubMed Scopus (96) Google Scholar); COS-7 cells were transfected with pCXN2-core, pCXN2-core173, or pCXN2-HAcore151. Forty-eight hours after transfection, COS-7 cells were washed with phosphate-buffered saline (PBS) and subsequently fixed with 3.7% formaldehyde in PBS for 1 h at room temperature. After washing with PBS, cells were then permeabilized with 0.1% Tween 20 in PBS for 1 h. The cells were then blocked with PBS containing 2% normal rabbit serum for 1 h and incubated with mouse anti-core antigen IgG fraction (1:500) for 1 h at room temperature. After washing three times with PBS, cells were incubated with fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG antibody (1:40) (Dako, Carpinteria, CA) for 1 h at room temperature. Cells were then washed with ice-cold PBS, coated with fluorescent mounting medium (Dako), covered with glass, and observed by microscope using an epifluorescent attachment (AX80, Olympus, Tokyo, Japan). Approximately 2 × 106 HeTOC-22 cells were plated into a 10-cm dish (Iwaki Glass) and cultured in medium with or without 1 μg/ml doxycycline. Forty-eight hours later, the cells were harvested and their nuclear extracts were prepared according to mini-nuclear extraction methods (27Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3913) Google Scholar). The concentration of the nuclear extracts was determined by a Micro BCA protein assay reagent kit (Pierce) and was adjusted to give equal concentrations. Electrophoretic mobility shift assay (EMSA) was performed using a Gel shift assay system (Promega) according to the manufacturer's protocol. Briefly, a synthetic double-stranded oligonucleotide having a κB site (5′-AGTTGAGGGGACTTTCCCAGGC-3′) was end-labeled with [32P]ATP using T4 polynucleotide kinase and incubated with 10 μg of nuclear extracts for 20 min at room temperature. For competition, an unlabeled competitor oligonucleotide or an unlabeled noncompetitor oligonucleotide (5′-GATCGAACTGACCGCCCGCGGCCCGT-3′) was added to the reaction mixture in 100-fold osmolar excess over the labeled probe to examine the binding specificity. Reaction mixtures were loaded onto a 4% polyacrylamide gel and then separated by electrophoresis in 0.5× Tris borate/EDTA electrophoresis buffer (0.045 m Tris borate and 0.001m EDTA). Data were expressed as means ± S.D. Statistical analyses were performed using the t test. Ap value of less than 0.05 was considered statistically significant. Expression of full-length and three deleted core proteins was examined in the soluble protein cell extracts of transiently transfected COS-7 cells by Western blotting (Fig.1, A and B). Full-length (pCXN2-core) and aa 1–173 core (pCXN2-core173) were detected by mouse anti-HCV core antigen IgG fraction (Fig.1 A). HA-tagged core aa 1–151 (pCXN2-HAcore151) and aa 92–191 (pCXN2-HAcore92–191) were detected by anti-HA polyclonal antibody (Fig. 1 B). The size of each core protein was consistent with the expected size. Expression of HCV core protein was detected by Western blotting in HeTOC cells, a clone termed HeTOC-22, which can be induced to express core protein under the control of doxycycline, 48 h after the removal of doxycycline. The induction ratio was greater than 1,000 when the relative amount of expressed HCV core protein with or without doxycycline was analyzed using a LAS-1000 image analyzer (photo film from Fuji, Tokyo, Japan) (data not shown). In HeLa cells, HCV core protein (0.2 μg of pCXN2-core) significantly activated the NF-κB pathway at a value 6.2 ± 3.4 (mean ± S.D.) times higher than that of the control, whereas TNF-α (20 ng/ml) activated the pathway at a value 8.6 ± 2.0 times higher (Fig.2 A). Activation of the NF-κB pathway increased in relation to the amount of plasmid utilized for pCNX2-core (Fig. 3). In HepG2 cells, HCV core protein activated the pathway at a value 4.3 ± 0.9 times higher than the control, whereas TNF-α activated the pathway at a value 4.5 ± 2.5 (Fig. 2 A).Figure 3Dose-dependent activation of the NF-κB pathway by HCV core protein. HeLa cells were transfected with various amounts of pCXN2 or pCXN2-core. Luciferase activities were measured and expressed as described in the legend for Fig. 2. pFC-MEKK, expressing constitutively active MEKK1, served as a positive control for the NF-κB pathway.View Large Image Figure ViewerDownload Hi-res image Download (PPT) HeTOC cells, which can be induced to express core protein under the control of doxycycline, were used to examine activation of the NF-κB pathway. Core-expressing HeTOC-22 cells cultured in a medium without doxycycline showed a pathway activation value 5.2 ± 1.5 times higher than that found with HeTOC-22 cells cultured in a medium with doxycycline. Addition of TNFα to core-expressing HeTOC-22 cells did not affect significantly the activation of NF-κB pathway by core protein (Fig. 2 B). To examine whether core protein enhances NF-κB-DNA binding, EMSA was performed using the nuclear extracts of HeTOC-22 cells, which were induced to express core protein. As shown in Fig. 4, NF-κB-DNA binding activity was enhanced in HeTOC-22 cells expressing core protein (about 2.9 times) as compared with that in HeTOC-22 cells without expressing core protein under the existence of doxycycline. This NF-κB-DNA binding activity observed in these assays was ablated by an excess of unlabeled competitor but not by an excess of unlabeled noncompetitor (Fig. 4). Addition of an antibody directed against p65 or p50 (Santa Cruz Biotechnology) generated a supershifted band, suggesting that this NF-κB-DNA complex was containing p65 and p50 (data not shown). To determine the region of HCV core protein responsible for activation of the NF-κB pathway, we constructed three plasmids, which expressed deletion mutant forms of core protein: pCXN2-core173, pCXN2-HAcore151, and pCXN2-HAcore92–191. HeLa cells were transfected with full-length or each deletion mutant form of HCV core expressing plasmids in combination with pNF-κB-Luc (Fig. 5). None of the deletion mutant forms of core protein activated the NF-κB pathway, whereas full-length HCV core protein did activate the pathway. These results suggest that both the N terminus (aa 1–91) and C terminus (aa 174 to 191) of the core protein may play an important role in activation of the NF-κB pathway. We then examined subcellular localization of full-length and two deletion mutants of HCV core protein by indirect immunofluorescence assay. As shown in Fig. 6, full-length core protein (aa 1–191) was located diffusely in the cytoplasm, in contrast to the perinuclear localization of the C-terminal 18 aa-deleted core protein (aa 1–173) and the nuclear localization of the C-terminal 40 aa-deleted core protein (aa 1–151). To examine whether activation of the NF-κB pathway by HCV core protein is transduced through IKKα or IKK%, HeLa cells were cotransfected with pCXN2/pCXN2-core, pNF-κB-Luc, and pIKKα(K44A)/pIKK%(K44A). Expression of IKK%(K44A), catalytically inactive IKK%, significantly reduced HCV core induced NF-κB activation to about one-tenth, whereas expression of IKKα(K44A), catalytically inactive IKKα, reduced the activation to about two-fifths (Fig.7). To confirm the participation of IKK% in activation of the NF-κB pathway by HCV core protein, we added 5 mm acetyl salicylic acid, an IKK%-specific inhibitor (28Yin M.J. Christerson L.B. Yamamoto Y. Kwak Y.T. Xu S. Mercurio F. Barbosa M. Cobb M.H. Gaynor R.B. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), to the HeLa cells transfected with pCXN2/pCXN2-core and pNF-κB-Luc. Activation of the pathway by core protein was significantly inhibited by acetyl salicylic acid but not by indomethacin, a cyclooxygenase inhibitor (Fig.8). These results suggest that HCV core protein activates the NF-κB pathway through IKK, especially IKK%.Figure 8Acetyl salicylic acid reduced NF-κB activation by HCV core protein.Acetyl salicylic acid, an IKK%-specific inhibitor, was added to the medium of HeLa cells transfected with pCXN2/pCXN2-core and pNF-kB-Luc at a concentration of 5 mm. Luciferase activities were measured and expressed as described in the legend for Fig. 2. Activation of the pathway by HCV core protein was significantly inhibited by acetyl salicylic acid but not indomethacin, a cyclooxygenase inhibitor.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To examine whether activation of the NF-κB pathway by HCV core protein was transduced through TRAF2/6, HeLa cells were cotransfected with pCXN2/pCXN2-core, pNF-κB-Luc, and pTRAF" @default.
• W2044679901 created "2016-06-24" @default.
• W2044679901 creator A5005637906 @default.
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• W2044679901 date "2001-05-01" @default.
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• W2044679901 title "Hepatitis C Virus Core Protein Activates Nuclear Factor κB-dependent Signaling through Tumor Necrosis Factor Receptor-associated Factor" @default.
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• W2044679901 doi "https://doi.org/10.1074/jbc.m006671200" @default.
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