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DENG LinGraduate School of Medicine / Center for Infectious Diseases (CID)Associate Professor
Research activity information
■ Award- Oct. 2018 一般社団法人神緑会, 第6回神緑会ヤングインベスティゲーターアワード（YIA) 優秀賞, B型肝炎ウイルスXタンパク質の新規結合因子Prdx1とエキソソーム構成因子Exosc5によるHBV RNA分解とHBV増殖制御Others
- Oct. 2014 一般社団法人神緑会, 第2回神緑会ヤングインベスティゲーターアワード（YIA) 優秀賞, Ｃ型肝炎ウイルスによるミトコンドリア介在性アポトーシス誘導機構の解明Others
- Oct. 2011 社団法人日本肝臓学会, 社団法人日本肝臓学会第10回 MSD Award, Ｃ型肝炎ウイルスによる糖代謝異常の分子機序の解明
- Jun. 2011 社団法人日本肝臓学会, 第４７回日本肝臓学会総会優秀演題賞, Ｃ型肝炎ウイルスは酸化ストレスを介して糖新生を亢進し糖尿病発症に関与する
- A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes mild-to-severe respiratory symptoms, including acute respiratory distress. Despite remarkable efforts to investigate the virological and pathological impacts of SARS-CoV-2, many of the characteristics of SARS-CoV-2 infection still remain unknown. The interferon-inducible ubiquitin-like protein ISG15 is covalently conjugated to several viral proteins to suppress their functions. It was reported that SARS-CoV-2 utilizes its papain-like protease (PLpro) to impede ISG15 conjugation, ISGylation. However, the role of ISGylation in SARS-CoV-2 infection remains unclear. We aimed to elucidate the role of ISGylation in SARS-CoV-2 replication. We observed that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation in cultured cells. Site-directed mutagenesis reveals that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation, alongside conserved lysine residue in MERS-CoV (K372) and SARS-CoV (K375). We also observed that the nucleocapsid-ISGylation results in the disruption of nucleocapsid oligomerization, thereby inhibiting viral replication. Knockdown of ISG15 mRNA enhanced SARS-CoV-2 replication in the SARS-CoV-2 reporter replicon cells, while exogenous expression of ISGylation components partially hampered SARS-CoV-2 replication. Taken together, these results suggest that SARS-CoV-2 PLpro inhibits ISGylation of the nucleocapsid protein to promote viral replication by evading ISGylation-mediated disruption of the nucleocapsid oligomerization.IMPORTANCEISG15 is an interferon-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation in many viruses. However, the role of ISGylation in SARS-CoV-2 infection remains largely unclear. Here, we demonstrated that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation. We also found that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation. We obtained evidence suggesting that nucleocapsid-ISGylation results in the disruption of nucleocapsid-oligomerization, thereby suppressing SARS-CoV-2 replication. We discovered that SARS-CoV-2 papain-like protease inhibits ISG15 conjugation of nucleocapsid protein via its de-conjugating enzyme activity. The present study may contribute to gaining new insight into the roles of ISGylation-mediated anti-viral function in SARS-CoV-2 infection and may lead to the development of more potent and selective inhibitors targeted to SARS-CoV-2 nucleocapsid protein.Sep. 2024, Journal of virology, 98(9) (9), e0085524, English, International magazine[Refereed]Scientific journal
- Statins, such as lovastatin, have been known to inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Statins were reported to moderately suppress hepatitis C virus (HCV) replication in cultured cells harboring HCV RNA replicons. We report here using an HCV cell culture (HCVcc) system that high concentrations of lovastatin (5-20 μg/mL) markedly enhanced the release of HCV infectious particles (virion) in the culture supernatants by up to 40 times, without enhancing HCV RNA replication, HCV protein synthesis, or HCV virion assembly in the cells. We also found that lovastatin increased the phosphorylation (activation) level of extracellular-signal-regulated kinase 5 (ERK5) in both the infected and uninfected cells in a dose-dependent manner. The lovastatin-mediated increase of HCV virion release was partially reversed by selective ERK5 inhibitors, BIX02189 and XMD8-92, or by ERK5 knockdown using small interfering RNA (siRNA). Moreover, we demonstrated that other cholesterol-lowering statins, but not dehydrolovastatin that is incapable of inhibiting HMG-CoA reductase and activating ERK5, enhanced HCV virion release to the same extent as observed with lovastatin. These results collectively suggest that statins markedly enhance HCV virion release from infected cells through HMG-CoA reductase inhibition and ERK5 activation.Jul. 2024, Microbiology and immunology, English, International magazine[Refereed]Scientific journal
- Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus that causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The release of infectious HCV particles from infected hepatocytes is a crucial step in viral dissemination and disease progression. While the exact mechanisms of HCV particle release remain poorly understood, emerging evidence suggests that HCV utilizes intracellular membrane trafficking and secretory pathways. These pathways include the Golgi secretory pathway and the endosomal trafficking pathways, such as the recycling endosome pathway and the endosomal sorting complex required for transport (ESCRT)-dependent multivesicular bodies (MVBs) pathway. This review provides an overview of recent advances in understanding the release of infectious HCV particles, with a particular focus on the involvement of the host cell factors that participate in HCV particle release. By summarizing the current knowledge in this area, this review aims to contribute to a better understanding of endosomal pathways involved in the extracellular release of HCV particles and the development of novel antiviral strategies.Lead, Dec. 2023, Viruses, 15(12) (12), English, International magazine[Refereed]Scientific journal
- Hepatitis B virus (HBV) infection promotes reactive oxygen species production while paradoxically inducing the expression of antioxidant enzymes. HBV-induced disorders of redox homeostasis are closely associated with the development of hepatic diseases. However, the molecular mechanisms underlying the HBV-induced antioxidant response are poorly understood. The NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway is an intrinsic defense mechanism against oxidative stress. We here aim to elucidate the role of the Nrf2/ARE signaling pathway in the HBV life cycle. ARE-driven reporter assays revealed that expression of HBV X protein (HBx), but not HBV core, large HBV surface, or HBV polymerase, strongly enhanced ARE-luciferase activity, suggesting that HBx plays an important role in the HBV-induced antioxidant response. Knockdown of Nrf2 resulted in a marked decrease in HBx-induced ARE-luciferase activity. Immunoblot analysis and immunofluorescence staining suggested that HBx activates Nrf2 by increasing Nrf2 protein levels and enhancing Nrf2 nuclear localization. The oxidative stress sensor Kelch-like ECH-associated protein 1 (Keap1) is required for the ubiquitin-dependent degradation of Nrf2. Coimmunoprecipitation analysis revealed that HBx interacted with Keap1, suggesting that HBx competes with Nrf2 for interaction with Keap1. A cell-based ubiquitylation assay showed that HBx promoted polyubiquitylation of Nrf2 via K6-linked polyubiquitin chains, and that this action may be associated with Nrf2 stabilization. A chromatin immunoprecipitation assay suggested that Nrf2 interacts with the HBV core promoter. Overexpression of Nrf2 strongly suppressed HBV core promoter activity, resulting in a marked reduction in viral replication. Based on the above, we propose that Keap1 recognizes HBx to activate the Nrf2/ARE signaling pathway upon HBV infection, thereby inhibiting HBV replication.IMPORTANCEThe Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway is one of the most important defense mechanisms against oxidative stress. We previously reported that a cellular hydrogen peroxide scavenger protein, peroxiredoxin 1, a target gene of transcription factor Nrf2, acts as a novel HBV X protein (HBx)-interacting protein and negatively regulates hepatitis B virus (HBV) propagation through degradation of HBV RNA. This study further demonstrates that the Nrf2/ARE signaling pathway is activated during HBV infection, eventually leading to the suppression of HBV replication. We provide evidence suggesting that Keap1 interacts with HBx, leading to Nrf2 activation and inhibition of HBV replication via suppression of HBV core promoter activity. This study raises the possibility that activation of the Nrf2/ARE signaling pathway is a potential therapeutic strategy against HBV. Our findings may contribute to an improved understanding of the negative regulation of HBV replication by the antioxidant response.Oct. 2023, Journal of virology, 97(10) (10), e0128723, English, International magazine[Refereed]Scientific journal
- Norovirus (NoV) is a leading cause of epidemic and sporadic gastroenteritis in people of all ages. Humans are the primary source of NoV and household contact is one of the risk factors for NoV transmission. However, the mechanisms underlying person-to-person NoV transmission are poorly understood. Here we conducted a survey to profile the frequency and characteristics of intrafamily NoV transmission. Stool samples were collected every week from three households between 2016 and 2020; the total number of samples was 1105. The detection of NoV and the genotyping were performed by reverse transcription-polymerase chain reaction targeting the capsid region and direct sequencing methods. NoV was detected in 3.4% of all samples. Eight NoV genotypes were identified. The most common genotype was GII.17, followed in order by GII.6, GI.6, GII.4, GI.3, and GI.2/GI.8/GI.9. Most NoV-positive samples were obtained from asymptomatic individuals. The highest number of NoV transmissions was found in household 3 (6 infections), followed by household 2 (2 infections), while household 1 had no NoV transmission, suggesting that asymptomatic NoV carriers play a major role in infection as NoV reservoirs in the households. Further clarification of the mode of infection will contribute to improved understanding and an appropriate prevention.Oct. 2023, Journal of medical virology, 95(10) (10), e29164, English, International magazine[Refereed]Scientific journal
- Transcription Factor JunB Suppresses Hepatitis C Virus Replication.We previously reported that hepatitis C virus (HCV) infection activates the reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK) signaling pathway. Activation of JNK contributes to the development of liver diseases, including metabolic disorders, steatosis, liver cirrhosis and hepatocellular carcinoma. JNK is known to have numerous target genes, including JunB, a member of activator protein-1 transcription factor family. However, the roles of JunB in the HCV life cycle and HCV-associated pathogenesis remain unclear. To clarify a physiological role of JunB in HCV infection, we investigated the phosphorylation of JunB in HCV J6/JFH1-infected Huh-7.5 cells. Immunoblot analysis revealed that HCV-induced ROS/JNK activation promoted phosphorylation of JunB. The small interfering RNA (siRNA) knockdown of JunB significantly increased the amount of intracellular HCV RNA as well as the intracellular and extracellular HCV infectivity titers. Conversely, overexpression of JunB significantly reduced the amount of intracellular HCV RNA and the intracellular and extracellular HCV infectivity titers. These results suggest that JunB plays a role in inhibiting HCV propagation. Additionally, HCV-mediated JunB activation promoted hepcidin promoter activity and hepcidin mRNA levels, a key factor in modulating iron homeostasis, suggesting that JunB is involved in HCV-induced transcriptional upregulation of hepcidin. Taken together, we propose that the HCV-induced ROS/JNK/JunB signaling pathway plays roles in inhibiting HCV replication and contributing to HCV-mediated iron metabolism disorder.Aug. 2023, The Kobe journal of medical sciences, 69(3) (3), E86-E95, English, Domestic magazineScientific journal
- Annexins (ANXs) comprise a family of calcium- and phospholipid-binding proteins and are implicated in the hepatitis C virus (HCV) life cycle. Here, we demonstrate a novel role of ANX5 in the HCV life cycle. Comparative analysis by quantitative PCR in human hepatoma cells revealed that ANX2, ANX4, and ANX5 were highly expressed among the ANX family proteins. Knockdown of ANX5 mRNA resulted in marked enhancement of HCV RNA replication but had no effect on either HCV translation or assembly. Using the HCV pseudoparticle (HCVpp) system, we observed enhancement of HCVpp infectivity in ANX5 knockdown Huh-7OK1 cells, suggesting that ANX5 is involved in suppression of HCV entry. Additionally, we observed that subcellular localizations of tight-junction proteins, such as claudin 1 (CLDN1) and occludin (OCLN), were disrupted in the ANX5 knockdown cells. It was reported that HCV infection was facilitated by disruption of OCLN distribution and that proper distribution of OCLN was regulated by its phosphorylation. Knockdown of ANX5 resulted in a decrease of OCLN phosphorylation, thereby disrupting OCLN distribution and HCV infection. Further analysis revealed that protein kinase C (PKC) isoforms, including PKCα and PKCη, play important roles in the regulation of ANX5-mediated phosphorylation and distribution of OCLN and in the restriction of HCV infection. HCV infection reduced OCLN phosphorylation through the downregulation of PKCα and PKCη expression. Taken together, these results suggest that ANX5, PKCα, and PKCη contribute to restriction of HCV infection by regulating OCLN integrity. We propose a model that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting HCV propagation. IMPORTANCE Host cells have evolved host defense machinery to restrict viral infection. However, viruses have evolved counteracting strategies to achieve their infection. In the present study, we obtained results suggesting that ANX5 and PKC isoforms, including PKCα and PKCη, contribute to suppression of HCV infection by regulating the integrity of OCLN. The disruption of OCLN integrity increased HCV infection. We also found that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting viral infection. We propose that HCV disrupts ANX5-mediated OCLN integrity to establish a persistent infection. The disruption of tight-junction assembly may play important roles in the progression of HCV-related liver diseases.Jun. 2023, Journal of virology, 97(6) (6), e0065523, English, International magazine[Refereed]Scientific journal
- Rotavirus A (RVA) is a major viral cause of acute gastroenteritis worldwide. G12 RVA strains have emerged globally since 2007. There has been no report of the whole genome sequences of G12 RVAs in Indonesia. We performed the complete genome analysis by the next-generation sequencing of five G12 strains from hospitalized children with acute gastroenteritis in Surabaya from 2017-2018. All five G12 strains were Wa-like strains (G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1) and were clustered into lineage-III of VP7 gene phylogenetic tree. STM430 sample was observed as a mixed-infection between G12 and G1 strains: G12/G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. A phylogenetic tree analysis revealed that all five Indonesian G12 strains (SOEP379, STM371, STM413, STM430, and STM433) were genetically close to each other in all 11 genome segments with 98.0-100% nucleotide identities, except VP3 and NSP4 of STM430, suggesting that these strains have originated from a similar ancestral G12 RVA. The VP3 and NSP4 genome segments of STM430-G12P[8] were separated phylogenetically from those of the other four G12 strains, probably due to intra-genotype reassortment between the G12 and G1 Wa-like strains. The change from G12P[6] lineage-II in 2007 to G12P[8] lineage-III 2017-2018 suggests the evolution and diversity of G12 RVAs in Indonesia over the past approximately 10 years. This article is protected by copyright. All rights reserved.Jan. 2023, Journal of medical virology, 95(2) (2), e28485, English, International magazine[Refereed]Scientific journal
- Informa UK Limited, Jul. 2022, Autophagy Reports, 1(1) (1), 264 - 285Scientific journal
- We previously reported that hepatitis C virus (HCV) infection activates the reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK) signaling pathway. However, the roles of ROS/JNK activation in the HCV life cycle still remain unclear. We sought to identify a novel role of ROS/JNK signaling pathway in the HCV life cycle. Immunoblot analysis revealed that HCV-induced ROS/JNK activation promoted phosphorylation of Itch, a HECT-type E3 ubiquitin ligase, leading to activation of Itch. The siRNA-knockdown of Itch significantly reduced the extracellular HCV infectivity titers, HCV RNA, and HCV core protein without affecting intracellular HCV infectivity titers, HCV RNA, and HCV proteins, suggesting that Itch is involved in release of HCV particles. HCV-mediated JNK/Itch activation specifically promoted polyubiquitylation of an AAA-type ATPase VPS4A, but not VPS4B, required to form multivesicular bodies. Site-directed mutagenesis revealed that two lysine residues (K23 and K121) on VPS4A were important for VPS4A polyubiquitylation. The siRNA-knockdown of VPS4A, but not VPS4B, significantly reduced extracellular HCV infectivity titers. Co-immunoprecipitation analysis revealed that HCV infection specifically enhanced the interaction between CHMP1B, a subunit of endosomal sorting complexes required for transport (ESCRT)-III complex, and VPS4A, but not VPS4B, whereas VPS4A K23R/K121R greatly reduced the interaction with CHMP1B. HCV infection significantly increased ATPase activity of VPS4A, but not VPS4A K23R/K121R or VPS4B, suggesting that HCV-mediated polyubiquitylation of VPS4A contributes to activation of VPS4A. Taken together, we propose that HCV-induced ROS/JNK/Itch signaling pathway promotes VPS4A polyubiquitylation, leading to enhanced VPS4A-CHMP1B interaction and promotion of VPS4A ATPase activity, thereby promoting the release of HCV particles. IMPORTANCE ROS/JNK signaling pathway contributes to liver diseases, including steatosis, metabolic disorders, and hepatocellular carcinoma. We previously reported that HCV activates the ROS/JNK signaling pathway, leading to the enhancement of hepatic gluconeogenesis and apoptosis induction. This study further demonstrates that HCV-induced ROS/JNK signaling pathway activates the E3 ubiquitin ligase Itch to promote release of HCV particles via polyubiquitylation of VPS4A. We provide evidence suggesting that HCV infection promotes the ROS/JNK/Itch signaling pathway and ESCRT/VPS4A machinery to release infectious HCV particles. Our results may lead to a better understanding of the mechanistic details of HCV particle release.Lead, Jan. 2022, Journal of virology, 96(6) (6), JVI0181121, English, International magazine[Refereed]Scientific journal
- Ubiquitin and ubiquitin-like protein modification play important roles in modulating the functions of viral proteins in many viruses. Here we demonstrate that hepatitis B virus (HBV) X protein (HBx) is modified by ISG15, which is a type I IFN-inducible, ubiquitin-like protein; this modification is called ISGylation. Immunoblot analyses revealed that HBx proteins derived from four different HBV genotypes accepted ISGylation in cultured cells. Site-directed mutagenesis revealed that three lysine residues (K91, K95 and K140) on the HBx protein, which are well conserved among all the HBV genotypes, are involved in acceptance of ISGylation. Using expression plasmids encoding three known E3 ligases involved in the ISGylation to different substrates, we found that HERC5 functions as an E3 ligase for HBx-ISGylation. Treatment with type I and type III IFNs resulted in the limited suppression of HBV replication in Hep38.7-Tet cells. When cells were treated with IFN-α, silencing of ISG15 resulted in a marked reduction of HBV replication in Hep38.7-Tet cells, suggesting a role of ISG15 in the resistance to IFN-α. In contrast, the silencing of USP18 (an ISG15 de-conjugating enzyme) increased the HBV replication in Hep38.7-Tet cells. Taken together, these results suggest that the HERC5-mediated ISGylation of HBx protein confers pro-viral functions on HBV replication and participates in the resistance to IFN-α-mediated antiviral activity.Oct. 2021, The Journal of general virology, 102(10) (10), English, International magazine[Refereed]Scientific journal
- NS5A-ISGylation via Lysine 26 Has a Critical Role for Efficient Propagation of Hepatitis C Virus Genotype 2a.We previously reported that hepatitis C virus (HCV) NS5A (1b, Con1) protein accepts covalent ISG15 conjugation at specific lysine (Lys) residues (K44, K68, K166, K215 and K308), exhibiting proviral effects on HCV RNA replication. Here we investigated a role of NS5A-ISGylation via Lys residues in HCV propagation using HCV infectious clone. The alignment of amino acid sequences revealed that 5 Lys residues (K20, K26, K44, K139, and K166) of the 13 Lys residues within NS5A (genotype 2a, JFH1 strain) were conserved compared to those of HCV (genotype 1b, Con1 strain). The cell-based ISGylation assay revealed that the K26 residue in the amphipathic helix (AH) domain and the K139 residue in domain I of NS5A (2a, JFH1) had the potential to accept ISGylation. Use of the HCV replicon carrying luciferase gene revealed that the K26 residue but not K139 residue of NS5A (2a, JFH1) was important for HCV RNA replication. Furthermore, cell culture HCV revealed that the mutation with the K26 residue in combination with K139 or K166 on NS5A (2a, JFH1) resulted in complete abolishment of viral propagation, suggesting that the K26 residue collaborates with either the K139 residue or K166 residue for efficient HCV propagation. Taken together, these results suggest that HCV NS5A protein has the potential to accept ISGylation via specific Lys residues, involving efficient viral propagation in a genotype-specific manner.Sep. 2021, The Kobe journal of medical sciences, 67(2) (2), E38-E47, English, Domestic magazineScientific journal
- Noroviruses are recognized as a leading cause of outbreaks and sporadic cases of acute gastroenteritis (AGE) among individuals of all ages worldwide, especially in children <5 years old. We investigated the epidemiology of noroviruses among hospitalized children at two hospitals in East Java, Indonesia. Stool samples were collected from 966 children with AGE during September 2015-July 2019. All samples were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) for the amplification of both the RNA-dependent RNA polymerase (RdRp) and the capsid genes of noroviruses. The genotypes were determined by phylogenetic analyses. In 2015-2019, noroviruses were detected in 12.3% (119/966) of the samples. Children <2 years old showed a significantly higher prevalence than those ≥2 years old (P = 0.01). NoV infections were observed throughout the year, with the highest prevalence in December. Based on our genetic analyses of RdRp, GII.[P31] (43.7%, 31/71) was the most prevalent RdRp genotype, followed by GII.[P16] (36.6%, 26/71). GII.[P31] was a dominant genotype in 2016 and 2018, whereas GII.[P16] was a dominant genotype in 2015 and 2017. Among the capsid genotypes, the most predominant norovirus genotype from 2015 to 2018 was GII.4 Sydney_2012 (33.6%, 40/119). The most prevalent genotype in each year was GII.13 in 2015, GII.4 Sydney_2012 in 2016 and 2018, and GII.3 in 2017. Based on the genetic analyses of RdRp and capsid sequences, the strains were clustered into 13 RdRp/capsid genotypes; 12 of them were discordant, e.g., GII.4 Sydney[P31], GII.3[P16], and GII.13[P16]. The predominant genotype in each year was GII.13[P16] in 2015, GII.4 Sydney[P31] in 2016, GII.3[P16] in 2017, and GII.4 Sydney[P31] in 2018. Our results demonstrate high detection rates and genetic diversity of norovirus GII genotypes in pediatric AGE samples from Indonesia. These findings strengthen the importance of the continuous molecular surveillance of emerging norovirus strains.Elsevier BV, Mar. 2021, Infection, Genetics and Evolution, 88, 104703 - 104703, English, International magazine[Refereed]Scientific journal
- Lysosome incorporate and degrade proteins in a process known as autophagy. There are three types of autophagy; macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Although autophagy is considered a nonselective degradation process, CMA is known as a selective degradation pathway. All proteins internalized in the lysosome via CMA contain a pentapeptide KFERQ-motif, also known as a CMA-targeting motif, which is necessary for selectivity. CMA directly delivers a substrate protein into the lysosome lumen using the cytosolic chaperone HSC70 and the lysosomal receptor LAMP-2A for degradation. Hepatitis C virus (HCV) NS5A protein interacts with hepatocyte-nuclear factor 1α (HNF-1α) together with HSC70 and promotes the lysosomal degradation of HNF-1α via CMA, resulting in HCV-induced pathogenesis. HCV NS5A promotes recruitment of HSC70 to the substrate protein HNF-1α. HCV NS5A plays a crucial role in HCV-induced CMA. Further investigations of HCV NS5A-interacting proteins containing CMA-targeting motifs may help to elucidate HCV-induced pathogenesis.2021, Frontiers in cellular and infection microbiology, 11, 796664 - 796664, English, International magazine[Refereed]Scientific journal
- Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that is covalently conjugated to many substrate proteins in order to modulate their functions; this conjugation is called 'ISGylation'. Several groups reported that the ISGylation of hepatitis C virus (HCV) NS5A protein affects HCV replication. However, ISG15 conjugation sites on NS5A are not well determined, and it is unclear whether the role of NS5A-ISGylation in HCV replication is pro-viral or anti-viral. Here we investigated the role of NS5A-ISGylation in HCV replication by using HCV RNA replicons that have a mutation at each lysine (Lys) residue of NS5A protein. Immunoblot analyses revealed that five Lys residues (K44, K68, K166, K215, and K308) of 14 Lys residues within NS5A (1b, Con1) have the potential to accept ISGylation. We tested the NS5A-ISGylation among different HCV genotypes and observed that the NS5A of all of the HCV genotypes accept ISGylation at the multiple Lys residues. Using an HCV luciferase reporter replicon assay revealed that the residue K308 of NS5A is important for HCV (1b, Con1) RNA replication. We observed that K308, one of the Lys residues for NS5A-ISGylation, is located within the binding region of cyclophilin A (CypA), which is the critical host factor for HCV replication. We obtained evidence suggesting that NS5A-ISGylation derived from all of HCV genotypes enhances the interaction between NS5A and CypA. Taken together, these results suggest that NS5A-ISGylation functions as a pro-viral factor and promotes HCV replication via the recruitment of CypA.IMPORTANCEHost cells have evolved host defense machinery (such as innate immunity) to eliminate viral infections. Viruses have evolved several counteracting strategies for achieving an immune escape from host defense machinery, including type-I interferons (IFNs) and inflammatory cytokines. ISG15 is an IFN-inducible, ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation. Here we demonstrate that HCV NS5A protein accepts ISG15-conjugation at specific Lys residues and that the HERC5 E3 ligase specifically promotes NS5A-ISGylation. We obtained evidence suggesting that NS5A-ISGylation facilitates the recruitment of CypA, which is the critical host factor for HCV replication, thereby promoting HCV replication. These findings indicate that E3 ligase HERC5 is a potential therapeutic target for HCV infection. We propose that HCV hijacks an intracellular ISG15 function to escape the host defense machinery in order to establish a persistent infection.Jul. 2020, Journal of virology, 94(20) (20), English, International magazine[Refereed]Scientific journal
- Hepatitis B virus (HBV) infection is a major risk factor for the development of chronic liver diseases, including cirrhosis and hepatocellular carcinoma (HCC). A growing body of evidence suggests that HBV X protein (HBx) plays a crucial role in viral replication and HCC development. Here, we identified peroxiredoxin 1 (Prdx1), a cellular hydrogen peroxide scavenger, as a novel HBx-interacting protein. Coimmunoprecipitation analysis coupled with site-directed mutagenesis revealed that the region from amino acids 17 to 20 of the HBx, particularly HBx Cys17, is responsible for the interaction with Prdx1. Knockdown of Prdx1 by siRNA significantly increased the levels of intracellular HBV RNA, HBV antigens, and extracellular HBV DNA, whereas knockdown of Prdx1 did not increase the activities of HBV core, enhancer I (Enh1)/X, preS1, and preS2/S promoters. Kinetic analysis of HBV RNA showed that knockdown of Prdx1 inhibited HBV RNA decay, suggesting that Prdx1 reduces HBV RNA levels posttranscriptionally. The RNA coimmunoprecipitation assay revealed that Prdx1 interacted with HBV RNA. The exosome component 5 (Exosc5), a member of the RNA exosome complexes, was coimmunoprecipitated with Prdx1, suggesting its role in regulation of HBV RNA stability. Taken together, these results suggest that Prdx1 and Exosc5 play crucial roles in host defense mechanisms against HBV infection. IMPORTANCE Hepatitis B virus (HBV) infection is a major global health problem. HBx plays important roles in HBV replication and viral carcinogenesis through its interaction with host factors. In this study, we identified Prdx1 as a novel HBx-binding protein. We provide evidence suggesting that Prdx1 promotes HBV RNA decay through interaction with HBV RNA and Exosc5, leading to downregulation of HBV RNA. These results suggest that Prdx1 negatively regulates HBV propagation. Our findings may shed new light on the roles of Prdx1 and Exosc5 in host defense mechanisms in HBV infection.Mar. 2019, J Virol, 93(6) (6), e0220318, English, International magazine[Refereed]Scientific journal
- Group A rotavirus (RVA) is the most important cause of severe gastroenteritis among children worldwide, and effective RVA vaccines have been introduced in many countries. Here we performed a molecular epidemiological analysis of RVA infection among pediatric patients in East Java, Indonesia, during 2015-2018. A total of 432 stool samples were collected from hospitalized pediatric patients with acute gastroenteritis. None of the patients in this cohort had been immunized with an RVA vaccine. The overall prevalence of RVA infection was 31.7% (137/432), and RVA infection was significantly more prevalent in the 6- to 11-month age group than in the other age groups (P < 0.05). Multiplex reverse transcription-PCR (RT-PCR) revealed that the most common G-P combination was equine-like G3P[8] (70.8%), followed by equine-like G3P[6] (12.4%), human G1P[8] (8.8%), G3P[6] (1.5%), and G1P[6] (0.7%). Interestingly, the equine-like strains were exclusively detected until May 2017, but in July 2017 they were completely replaced by a typical human genotype (G1 and G3), suggesting that the dynamic changes in RVA genotypes from equine-like G3 to typical human G1/G3 in Indonesia can occur even in the country with low RVA vaccine coverage rate. The mechanism of the dynamic changes in RVA genotypes needs to be explored. Infants and children with RVA-associated gastroenteritis presented more frequently with some dehydration, vomiting, and watery diarrhea, indicating a greater severity of RVA infection compared to those with non-RVA gastroenteritis. In conclusion, a dynamic change was found in the RVA genotype from equine-like G3 to a typical human genotype. Since severe cases of RVA infection were prevalent, especially in children aged 6 to 11 months or more generally in those less than 2 years old, RVA vaccination should be included in Indonesia's national immunization program.2019, Frontiers in microbiology, 10, 940 - 940, English, International magazine[Refereed]Scientific journal
- Rotavirus A (RVA) is a major cause of acute gastroenteritis in humans and animals worldwide. As a result of the segmented nature of the rotavirus genome, genetic reassortment commonly occurs. This study aims to clarify the genetic characteristics of RVAs circulating in Indonesia. From June 2015 through August 2016, stool samples were collected from 134 children aged <5 years (71 male and 63 female) with acute gastroenteritis who were inpatients at a private hospital in Surabaya, Indonesia. All stool samples were screened for RVA antigen using immunochromatography. Forty-two samples (31.3%, 42/134) were RVA antigen-positive. All RVA positive samples tested showed the unusual combinations of G3P[8] (n = 36) and G3P[6] (n = 3) with a short RNA pattern by G/P typing and polyacrylamide gel electrophoresis (PAGE). Whole genome analysis by next-generation sequencing (NGS) was performed for 11 strains to determine the RVA genotypes. Eleven rotavirus strains were found to carry a DS-like genetic backbone; nine strains showed a G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 genome constellation, which was recently reported in Australia, Hungary, Spain and Brazil; as well, two strains showed a G3-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2 genome constellation. The phylogenetic tree based on the VP7 gene showed that all 11 strains were classified as equine-like G3, which is genetically distinct and different in origin from typical human G3 strains. The phylogenetic tree based on the NSP4 gene showed that six strains were classified as bovine-like strain and the remaining five were classified as human strain. In conclusion, we identified the strains which are intergenogroup reassortants containing an equine-like G3 VP7, a P[8])/P[6] VP4, with a DS-1-like genetic backbone. These findings suggest that equine-like G3P[8] and P[6] RVA strains have been circulating in the Indonesian population for at least 1 year and probably longer, indicating a diversity of RVAs in this area.Jul. 2018, Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 61(1) (1), 224 - 228, English, International magazine[Refereed]Scientific journal
- Hepatitis C virus (HCV) infection is closely associated with type 2 diabetes. We reported that HCV infection induces the lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) via interaction with HCV nonstructural protein 5A (NS5A) protein, thereby suppressing GLUT2 gene expression. The molecular mechanisms of selective degradation of HNF-1α caused by NS5A are largely unknown. Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway. Here, we investigated whether CMA is involved in the selective degradation of HNF-1α in HCV-infected cells and observed that the pentapeptide spanning from amino acid (aa) 130 to aa 134 of HNF-1α matches the rule for the CMA-targeting motif, also known as KFERQ motif. A cytosolic chaperone protein, heat shock cognate protein of 70 kDa (HSC70), and a lysosomal membrane protein, lysosome-associated membrane protein type 2A (LAMP-2A), are key components of CMA. Immunoprecipitation analysis revealed that HNF-1α was coimmunoprecipitated with HSC70, whereas the Q130A mutation (mutation of Q to A at position 130) of HNF-1α disrupted the interaction with HSC70, indicating that the CMA-targeting motif of HNF-1α is important for the association with HSC70. Immunoprecipitation analysis revealed that increasing amounts of NS5A enhanced the association of HNF-1α with HSC70. To determine whether LAMP-2A plays a role in the degradation of HNF-1α protein, we knocked down LAMP-2A mRNA by RNA interference; this knockdown by small interfering RNA (siRNA) recovered the level of HNF-1α protein in HCV J6/JFH1-infected cells. This result suggests that LAMP-2A is required for the degradation of HNF-1α. Immunofluorescence study revealed colocalization of NS5A and HNF-1α in the lysosome. Based on our findings, we propose that HCV NS5A interacts with HSC70 and recruits HSC70 to HNF-1α, thereby promoting the lysosomal degradation of HNF-1α via CMA.IMPORTANCE Many viruses use a protein degradation system, such as the ubiquitin-proteasome pathway or the autophagy pathway, for facilitating viral propagation and viral pathogenesis. We investigated the mechanistic details of the selective lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) induced by hepatitis C virus (HCV) NS5A protein. Using site-directed mutagenesis, we demonstrated that HNF-1α contains a pentapeptide chaperone-mediated autophagy (CMA)-targeting motif within the POU-specific domain of HNF-1α. The CMA-targeting motif is important for the association with HSC70. LAMP-2A is required for degradation of HNF-1α caused by NS5A. We propose that HCV NS5A interacts with HSC70, a key component of the CMA machinery, and recruits HSC70 to HNF-1α to target HNF-1α for CMA-mediated lysosomal degradation, thereby facilitating HCV pathogenesis. We discovered a role of HCV NS5A in CMA-dependent degradation of HNF-1α. Our results may lead to a better understanding of the role of CMA in the pathogenesis of HCV.Jun. 2018, J Virol, 92(13) (13), e0063918, English, International magazine[Refereed]Scientific journal
- 生命科学系学会合同年次大会運営事務局, Dec. 2017, 生命科学系学会合同年次大会, 2017年度, [3LBA - 019], Japanese脱ユビキチン化酵素USP15阻害剤候補分子の機能解析
- Norovirus (NoV) is a major cause of nonbacterial acute gastroenteritis worldwide in all age groups, and asymptomatic individuals may contribute to NoV transmission as a reservoir. Nonetheless, little information is available regarding asymptomatic NoV infection in Indonesia. We performed an epidemiological analysis of NoV infection among asymptomatic healthy volunteers in the city of Surabaya, Indonesia (population similar to 2.75 million). A total of 512 stool samples from 18 individuals (age range 20-42 years) collected from July 2015 to June 2016 were examined. The detection of NoV and the genotype classification were carried out by a reverse transcription-polymerase chain reaction (RT-PCR) direct sequencing method. NoV was detected in 14 of the 512 stool samples (2.7%), with 7 individuals (38.9%) having at least 1 positive stool sample. All 14 of the NoV strains detected belonged to genogroup GII. The phylogenetic analysis indicated that 10 strains (71.4%) were grouped with GII.2, 2 (14.3%) were GII.17, 1 was GII.4 Sydney 2012, and 1 was GII.1. The circulation of GII.Pg/GII.1 and GII.Pe/GII.4 Sydney 2012 recombinant variants was detected among an asymptomatic population in Surabaya, Indonesia. Of the 7 positive individuals, 2 were repeatedly infected with the same strain and heterogenous strains. Taken together, our results suggest that the excretion of NoV from healthy individuals is one of the sources of NoV outbreak.ELSEVIER SCIENCE BV, Nov. 2017, INFECTION GENETICS AND EVOLUTION, 55, 1 - 7, English[Refereed]Scientific journal
- Hepatitis C virus (HCV), dengue virus (DENV) and Japanese encephalitis virus (JEV) belong to the family Flaviviridae. Their viral particles have the envelope composed of viral proteins and a lipid bilayer acquired from budding through the endoplasmic reticulum (ER). The phospholipid content of the ER membrane differs from that of the plasma membrane (PM). The phospholipase A(2) (PLA(2)) superfamily consists of a large number of members that specifically catalyse the hydrolysis of phospholipids at a particular position. Here we show that the CM-II isoform of secreted PLA(2) obtained from Naja mossambica mossambica snake venom (CM-II-sPLA(2)) possesses potent virucidal (neutralising) activity against HCV, DENV and JEV, with 50% inhibitory concentrations (IC50) of 0.036, 0.31 and 1.34 ng/ ml, respectively. In contrast, the IC50 values of CM-II-sPLA(2) against viruses that bud through the PM (Sindbis virus, influenza virus and Sendai virus) or trans-Golgi network (TGN) (herpes simplex virus) were > 10,000 ng/ml. Moreover, the 50% cytotoxic (CC50) and haemolytic (HC50) concentrations of CMII- sPLA(2) were > 10,000 ng/ml, implying that CM-II-sPLA(2) did not significantly damage the PM. These results suggest that CM-II-sPLA(2) and its derivatives are good candidates for the development of broadspectrum antiviral drugs that target viral envelope lipid bilayers derived from the ER membrane.NATURE PUBLISHING GROUP, Nov. 2017, SCIENTIFIC REPORTS, 7(1) (1), 15931, English[Refereed]Scientific journal
- Interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein, is induced by type I INF. Although several groups have reported ISGylation of the HCV NS5A protein, it is still unclear whether ISGylation of NS5A has anti- or pro-viral effects in hepatitis C virus (HCV) infection. In the present study, the role of ISGylation-independent, unconjugated ISG15 in HCV infection was examined. Immunoprecipitation analyses revealed that ISG15 interacts specifically with NS5A domain I. ISG15 mutants lacking the C-terminal glycine residue that is essential for ISGylation still interacted with NS5A protein. Taken together, these results suggest that unconjugated ISG15 affects the functions of HCV NS5A through protein-protein interaction.WILEY, Jul. 2017, MICROBIOLOGY AND IMMUNOLOGY, 61(7) (7), 287 - 292, English[Refereed]Scientific journal
- Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multifunctional protein that is involved in the HCV life cycle and pathogenesis. In this study, a host protein(s) interacting with NS5A by tandem affinity purification were searched for with the aim of elucidating the role of NS5A. An NS5A-interacting protein, SET and MYND domain-containing 3 (SMYD3), a lysine methyltransferase reportedly involved in the development of cancer, was identified. The interaction between NS5A and SMYD3 was confirmed in ectopically expressing, HCV RNA replicon-harboring and HCV-infected cells. The other HCV proteins did not bind to SMYD3. SMYD3 bound to NS5A of HCV genotypes 1b and 2a. Deletion mutational analysis revealed that domains II and III of NS5A (amino acids [aa] 250 to 447) and the MYND and N-SET domains of SMYD3 (aa 1 to 87) are involved in the full extent of NS5A-SMYD3 interaction. NS5A co-localized with SMYD3 exclusively in the cytoplasm, thereby inhibiting nuclear localization of SMYD3. Moreover, NS5A formed a complex with SMYD3 and heat shock protein 90 (HSP90), which is a positive regulator of SMYD3. The intensity of binding between SMYD3 and HSP90 was enhanced by NS5A. Luciferase reporter assay demonstrated that NS5A significantly induces activator protein 1 (AP-1) activity, this being potentiated by co-expression of SMYD3 with NS5A. Taken together, the present results suggest that NS5A interacts with SMYD3 and induces AP-1 activation, possibly by facilitating binding between HSP90 and SMYD3. This may be a novel mechanism of AP-1 activation in HCV-infected cells.Corresponding, WILEY-BLACKWELL, Jun. 2016, MICROBIOLOGY AND IMMUNOLOGY, 60(6) (6), 407 - 417, English[Refereed]Scientific journal
- Hepatitis B virus (HBV) is a widespread human pathogen that often causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The detailed mechanisms underlying HBV pathogenesis remain poorly understood. The HBV X protein (HBx) is a multifunctional regulator that modulates viral replication and host cell functions, such as cell cycle progression, apoptosis and protein degradation through interaction with a variety of host factors. Recently, the nonstructural protein 5A (NS5A) of hepatitis C virus has been reported to interact with methyltransferase SET and MYND domain-containing 3 (SMYD3), which is implicated in chromatin modification and development of cancer. Because HBx shares fundamental regulatory functions concerning viral replication and pathogenesis with NS5A, it was decided to examine whether HBx interacts with SMYD3. In the present study, it was demonstrated by co-immunoprecipitation analysis that HBx interacts with both ectopically and endogenously expressed SMYD3 in Huh-7.5 cells. Deletion mutation analysis revealed that the C-terminal region of HBx (amino acids [aa] 131-154) and an internal region of SMYD3 (aa 269-288) are responsible for their interaction. Immunofluorescence and proximity ligation assays showed that HBx and SMYD3 co-localize predominantly in the cytoplasm. Luciferase reporter assay demonstrated that the interaction between HBx and SMYD3 activates activator protein 1 (AP-1) signaling, but not that of nuclear factor-kappa B (NF-kappa B). On the other hand, neither overexpression nor knockdown of SMYD3 altered production of HBV transcripts and HBV surface antigen (HBsAg). In conclusion, a novel HBx-interacting protein, SMYD3, was identified, leading to proposal of a novel mechanism of AP-1 activation in HBV-infected cells.Corresponding, WILEY-BLACKWELL, Jan. 2016, MICROBIOLOGY AND IMMUNOLOGY, 60(1) (1), 17 - 25, English[Refereed]Scientific journal
- (公社)日本生化学会, Dec. 2015, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 88回・38回, [1P0487] - [1P0487], Japanese脱ユビキチン化酵素USP15阻害剤の探索
- We previously reported that hepatitis C virus (HCV) infection induces Bax-triggered, mitochondrion-mediated apoptosis by using the HCV J6/JFH1 strain and Huh-7.5 cells. However, it was still unclear how HCV-induced Bax activation. In this study, we showed that the HCV-induced activation and mitochondrial accumulation of Bax were significantly attenuated by treatment with a general antioxidant, N-acetyl cysteine (NAC), or a specific c-Jun N-terminal kinase (JNK) inhibitor, SP600125, with the result suggesting that the reactive oxygen species (ROS)/JNK signalling pathway is upstream of Box activation in HCV-induced apoptosis. We also demonstrated that HCV infection transcriptionally activated the gene for the proapoptotic protein Bim and the protein expression of three major splice variants of Bim (Bim(EL), Bim(L) and Bim(S)). The HCV-induced increase in the Bim mRNA and protein levels was significantly counteracted by treatment with NAC or SP600125, suggesting that the ROS/JNK signalling pathway is involved in Bim upregulation. Moreover, HCV infection led to a marked accumulation of Bim on the mitochondria to facilitate its interaction with Bax. On the other hand, downregulation of Bim by siRNA (small interfering RNA) significantly prevented HCV-mediated activation of Bax and caspase 3. Taken together, these observations suggest that HCV-induced ROS/JNK signalling transcriptionally activates Bim expression, which leads to Box activation and apoptosis induction.SOC GENERAL MICROBIOLOGY, Sep. 2015, JOURNAL OF GENERAL VIROLOGY, 96(9) (9), 2670 - 2683, English[Refereed]Scientific journal
- Hepatitis C virus (HCV) NS5A protein plays crucial roles in viral RNA replication, virus assembly, and viral pathogenesis. Although NS5A has no known enzymatic activity, it modulates various cellular pathways through interaction with cellular proteins. HCV NS5A (and other HCV proteins) are reportedly degraded through the ubiquitin-proteasome pathway; however, the physiological roles of ubiquitylation and deubiquitylation in HCV infection are largely unknown. To elucidate the role of deubiquitylation in HCV infection, an attempt was made to identify a deubiquitinase (DUB) that can interact with NS5A protein. An ovarian tumor protein (OTU), deubiquitinase 7B (OTUD7B), was identified as a novel NS5A-binding protein. Co-immunoprecipitation analyses showed that NS5A interacts with OTUD7B in both Huh-7 and HCV RNA replicon cells. Immunofluorescence staining revealed that HCV NS5A protein colocalizes with OTUD7B in the cytoplasm. Moreover, HCV infection was found to enhance the nuclear localization of OTUD7B. The OTUD7B-binding domain on NS5A was mapped using a series of NS5A deletion mutants. The present findings suggest that the domain I of NS5A is important and the region from amino acid 121 to 126 of NS5A essential for the interaction. Either V121A or V124A mutation in NS5A disrupts the NS5A-OTUD7B interaction. The results of this in vivo ubiquitylation assay suggest that HCV NS5A enhances OTUD7B DUB activity. Taken together, these results suggest that HCV NS5A protein interacts with OTUD7B, thereby modulating its DUB activity.WILEY-BLACKWELL, Aug. 2015, MICROBIOLOGY AND IMMUNOLOGY, 59(8) (8), 466 - 476, English[Refereed]Scientific journal
- Hepatitis C virus (HCV) infection often causes extrahepatic manifestations, such as type 2 diabetes. We previously reported that HCV infection induces the lysosomal degradation of the transcription factor HNF-1 alpha via an interaction with viral NS5A, thereby suppressing GLUT2 gene expression. However, the molecular mechanism of NS5A-induced degradation of HNF-1 alpha is largely unknown. We aimed to identify the determinants necessary for the degradation of HNF-1 alpha induced by NS5A. Coimmunoprecipitation analysis revealed that the POU specific (POUs) domain spanning from aa 91 to 181 of HNF-1 alpha is responsible for the interaction of NS5A. We also found that the region from aa 121 to 126 of NS5A, which is known as the binding motif of the HCV replication factor FKBP8, is important for the degradation of HNF-1 alpha. A NS5A V121A mutation disrupted the NS5A-HNF-1 alpha interaction as well as the degradation of HNF-1 alpha. Our findings suggest that NS5A Val(121) is crucial for viral pathogenesis.SOC GENERAL MICROBIOLOGY, Aug. 2015, JOURNAL OF GENERAL VIROLOGY, 96(8) (8), 2200 - 2205, English[Refereed]Scientific journal
- Background: Hepatitis C virus (HCV) is a major global health problem, causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Development of well-tolerated regimens with high cure rates and fewer side effects is still much needed. Recently, natural antimicrobial peptides (AMPs) are attracting more attention as biological compounds and can be a good template to develop therapeutic agents, including antiviral agents against a variety of viruses. Various AMPs have been characterized from the venom of different venomous animals including scorpions. Methods: The possible antiviral activities of crude venoms obtained from five Egyptian scorpion species (Leiurus quinquestriatus, Androctonus amoreuxi, A. australis, A. bicolor and Scorpio maurus palmatus) were evaluated by a cell culture method using Huh7.5 cells and the J6/JFH1-P47 strain of HCV. Time-of-addition experiments and inactivation of enzymatic activities of the venoms were carried out to determine the characteristics of the anti-HCV activities. Results: S. maurus palmatus and A. australis venoms showed anti-HCV activities, with 50% inhibitory concentrations (IC50) being 6.3 +/- 1.6 and 88.3 +/- 5.8 mu g/ml, respectively. S. maurus palmatus venom (30 mu g/ml) impaired HCV infectivity in culture medium, but not inside the cells, through virocidal effect. The anti-HCV activity of this venom was not inhibited by a metalloprotease inhibitor or heating at 60 degrees C. The antiviral activity was directed preferentially against HCV. Conclusions: S. maurus palmatus venom is considered as a good natural source for characterization and development of novel anti-HCV agents targeting the entry step. To our knowledge, this is the first report describing antiviral activities of Egyptian scorpion venoms against HCV, and may open a new approach towards discovering antiviral compounds derived from scorpion venoms.BIOMED CENTRAL LTD, Mar. 2015, VIROLOGY JOURNAL, 12, 47, English[Refereed]Scientific journal
- [Hepatitis C virus-induced glucose metabolic disorder].Hepatitis C virus (HCV) infection often causes intrahepatic diseases, such as chronic hepatitis, liver chirrohsis, and hepatocellular carcinoma (HCC). Moreover, HCV infection exhibits various extrahepatic manifestations, such as thyroiditis, glucose and lipid metabolic disorder, and iron metabolic disorder. HCV infection is often associated with type 2 diabetes, involving hepatic fibrosis and poor prognosis. Type 2 diabetes increases the risk of HCC. We have been investigating molecular mechanisms of HCV-induced glucose metabolic disorder and we reported that HCV infection promotes hepatic gluconeogenesis through forkhead box O1 (FoxO1)-dependent pathway and that HCV infection suppresses the cell surface expression of glucose transporter 2 (GLUT2), resulting in suppression of glucose uptake. We have found that HCV NS5A protein plays important roles in these two independent pathways. Here we discuss the roles of HCV NS5A in HCV-induced glucose metabolic disorder.2015, Uirusu, 65(2) (2), 263 - 268, Japanese, Domestic magazineScientific journal
- Hepatitis C virus (HCV) induces cytopathic effects in the form of hepatocytes apoptosis thought to be resulted from the interaction between viral proteins and host factors. Using pathway specific PCR array, we identified 9 apoptosis-related genes that are dysregulated during HCV infection, of which the BH3- only pro-apoptotic Bcl-2 family protein, BIK, was consistently up-regulated at the mRNA and protein levels. Depletion of BIK protected host cells from HCV-induced caspase-3/7 activation but not the inhibitory effect of HCV on cell viability. Furthermore, viral RNA replication and release were significantly suppressed in BIK-depleted cells and over-expression of the RNA-dependent RNA polymerase, NS5B, was able to induce BIK expression. Immunofluorescence and co-immunoprecipitation assays showed co-localization and interaction of BIK and NS5B, suggesting that BIK may be interacting with the HCV replication complex through NS5B. These results imply that BIK is essential for HCV replication and that NS5B is able to induce BIK expression. (C) 2014 Elsevier Inc. All rights reserved.ACADEMIC PRESS INC ELSEVIER SCIENCE, Jan. 2015, VIROLOGY, 474, 41 - 51, English[Refereed]Scientific journal
- The NS5A protein of hepatitis C virus transcriptionally upregulates the AGR3 gene expressionThe non-structural protein 5A (NS5A) of hepatitis C virus (HCV) is a multifunctional protein involved in the HCV lifecycle and pathogenesis. The precise molecular mechanisms of NS5A-mediated pathogenesis still remain to be clarified. In this study, we performed cDNA microarray analysis on NS5A-expressing HEK293 cells and the non-expressing control to screen the possible cellular genes dysregulated by NS5A. Subsequent quantitative real time PCR (qRT-PCR) analysis on NS5A-expressing cells and the control confirmed that NS5A upregulated the anterior gradient homolog 3 (AGR3) mRNA expression. The domain III of NS5A was responsible for the activation of AGR3 gene expression. AGR3 mRNA expression levels were upregulated also in Huh7.5 cells harboring a full-genome HCV-1b RNA replicon (FGR) and in those infected with HCV-2a. Moreover, AGR3 promoter activity was activated in NS5A-expressing cells, FGR-harboring cells and HCV-infected cells. Taken together, our present results suggest that HCV NS5A transcriptionally activates the cancer-associated AGR3 gene. This may be a novel mechanism of HCV-mediated pathogenesis, especially hepatocarcinogenesis.Kobe University School of Medicine, 2015, Kobe Journal of Medical Sciences, 61(1) (1), E27 - E35, English[Refereed]Scientific journal
- Effective therapeutic vaccines against virus infection must induce sufficient levels of cell-mediated immune responses against the target viral epitopes and also must avoid concomitant risk factors, such as potential carcinogenic properties. The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) carries a variety of CD4(+) and CD8(+) T cell epitopes, and induces strong HCV-specific T cell responses, which are correlated with viral clearance and resolution of acute HCV infection. On the other hand, NS3 possesses serine protease and nucleoside triphosphatase (NTPase)/RNA helicase activities, which not only play important roles in viral life cycle but also concomitantly interfere with host defense mechanisms by deregulating normal cellular functions. In this study, we constructed a series of DNA vaccines that express NS3 of HCV. To avoid the potential harm of NS3, we introduced mutations to the catalytic triad of the serine protease (H57A, D81A and S139A) and the NTPase/RNA helicase domain (K210N, F444A, R461Q and W501A) to eliminate the enzymatic activities. Immunization of BALB/c mice with each of the DNA vaccine candidates (pNS3[S139A/K210N], pNS3[S139A/F444A], pNS3[S139A/R461Q] and pNS3[S139A/W501A]) that expresses an NS3 mutant lacking both serine protease and NTPase/helicase activities induced T cell immune responses to the degree comparable to that induced by the wild type NS3 and the NS3/4A complex, as demonstrated by interferon-gamma production and cytotoxic T lymphocytes activities against NS3. The present study has demonstrated that plasmids expressing NS3 mutants, NS3(S139A/K210N), NS3(S139A/F444A), NS3(S139A/R461Q) and NS3(S139A/W501A), which lack both serine protease and NTPase/RNA helicase activities, would be good candidates for safe and efficient therapeutic DNA vaccines against HCV infection.PUBLIC LIBRARY SCIENCE, Jun. 2014, PLOS ONE, 9(6) (6), e98877, English[Refereed]Scientific journal
- The development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still needed. Antiviral compounds in medicinal plants are potentially good targets to study. Morinda citrifolia is a common plant distributed widely in Indo-Pacific region; its fruits and leaves are food sources and are also used as a treatment in traditional medicine. In this study, using a HCV cell culture system, it was demonstrated that a methanol extract, its n-hexane, and ethyl acetate fractions from M. citrifolia leaves possess anti-HCV activities with 50%-inhibitory concentrations (IC50) of 20.6, 6.1, and 6.6 mu g/mL, respectively. Bioactivity-guided purification and structural analysis led to isolation and identification of pheophorbide a, the major catabolite of chlorophyll a, as an anti-HCV compound present in the extracts (IC50 = 0.3 mu g/mL). It was also found that pyropheophorbide a possesses anti-HCV activity (IC50 = 0.2 mu g/mL). The 50%-cytotoxic concentrations (CC50) of pheophorbide a and pyropheophorbide a were 10.0 and 7.2 mu g/mL, respectively, their selectivity indexes being 33 and 36, respectively. On the other hand, chlorophyll a, sodium copper chlorophyllin, and pheophytin a barely, or only marginally, exhibited anti-HCV activities. Time-of-addition analysis revealed that pheophorbide a and pyropheophorbide a act at both entry and the post-entry steps. The present results suggest that pheophorbide a and its related compounds would be good candidates for seed compounds for developing antivirals against HCV.WILEY-BLACKWELL, Mar. 2014, MICROBIOLOGY AND IMMUNOLOGY, 58(3) (3), 188 - 194, English[Refereed]Scientific journal
- Development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still much needed from clinical and economic points of view. Antiviral substances obtained from medicinal plants are potentially good targets to study. Glycyrrhiza uralensis and G. glabra have been commonly used in both traditional and modern medicine. In this study, extracts of G. uralensis roots and their components were examined for anti-HCV activity using an HCV cell culture system. It was found that a methanol extract of G. uralensis roots and its chloroform fraction possess anti-HCV activity with 50%-inhibitory concentrations (IC50) of 20.0 and 8.0 mu g/mL, respectively. Through bioactivity-guided purification and structural analysis, glycycoumarin, glycyrin, glycyrol and liquiritigenin were isolated and identified as anti-HCV compounds, their IC50 being 8.8, 7.2, 4.6 and 16.4 mu g/mL, respectively. However, glycyrrhizin, the major constituent of G. uralensis, and its monoammonium salt, showed only marginal anti-HCV activity. It was also found that licochalcone A and glabridin, known to be exclusive constituents of G. inflata and G. glabra, respectively, did have anti-HCV activity, their IC50 being 2.5 and 6.2 mu g/mL, respectively. Another chalcone, isoliquiritigenin, also showed anti-HCV activity, with an IC50 of 3.7 mu g/mL. Time-of-addition analysis revealed that all Glycyrrhiza-derived anti-HCV compounds tested in this study act at the post-entry step. In conclusion, the present results suggest that glycycoumarin, glycyrin, glycyrol and liquiritigenin isolated from G. uralensis, as well as isoliquiritigenin, licochalcone A and glabridin, would be good candidates for seed compounds to develop antivirals against HCV.WILEY, Mar. 2014, MICROBIOLOGY AND IMMUNOLOGY, 58(3) (3), 180 - 187, English[Refereed]Scientific journal
- Hepatocellular carcinoma (HCC) is one of the common sequelae of hepatitis C virus (HCV) infection. It remains controversial, however, whether HCV itself plays a direct role in the development of HCC. Although HCV core, NS3, and NS5A proteins were reported to display tumorigenic activities in cell culture and experimental animal systems, their clinical impact on HCC development in humans is still unclear. In this study we investigated sequence polymorphisms in the core protein, NS3, and NS5A of HCV genotype 1b (HCV-1b) in 49 patients who later developed HCC during a follow-up of an average of 6.5 years and in 100 patients who did not develop HCC after a 15-year follow-up. Sequence analysis revealed that Gln at position 70 of the core protein (core-Gln70), Tyr at position 1082 plus Gln at 1112 of NS3 (NS3-Tyr1082/Gln1112), and six or more mutations in the interferon/ribavirin resistance-determining region of NS5A (NS5A-IRRDR≥6) were significantly associated with development of HCC. Multivariate analysis identified core-Gln70, NS3-Tyr1082/Gln1112, and α-fetoprotein (AFP) levels (> 20 ng/L) as independent factors associated with HCC. Kaplan-Meier analysis revealed a higher cumulative incidence of HCC for patients infected with HCV isolates with core-Gln70, NS3-Tyr1082/Gln1112 or both than for those with non-(Gln70 plus NS3-Tyr1082/Gln1112). In most cases, neither the residues at position 70 of the core protein nor positions 1082 and 1112 of the NS3 protein changed during the observation period. Conclusion: HCV isolates with core-Gln70 and/or NS3-Tyr1082/Gln1112 are more closely associated with HCC development compared to those with non-(Gln70 plus NS3-Tyr1082/Gln1112). © 2012 American Association for the Study of Liver Diseases.Aug. 2013, Hepatology, 58(2) (2), 555 - 563, English[Refereed]Scientific journal
- Background: Hepatitis C virus (HCV) is a major cause of liver disease and a potential cause of substantial morbidity and mortality worldwide. The overall prevalence of HCV infection is 2%, representing 120 million people worldwide. Current standard treatment using pegylated interferon and ribavirin is effective in only 50% of the patients infected with HCV genotype 1, and is associated with significant side effects. Therefore, it is still of importance to develop new drugs for treatment of HCV. Antiviral substances obtained from natural products, including medicinal plants, are potentially good targets to study. In this study, we evaluated Indonesian medicinal plants for their anti-HCV activities. Methods: Ethanol extracts of 21 samples derived from 17 species of medicinal plants explored in the East Java region were tested. Anti-HCV activities were determined by a cell culture method using Huh7.5 cells and HCV strains of 9 different genotypes (1a to 7a, 1b and 2b). Results: Four of the 21 samples tested showed antiviral activities against HCV: Toona sureni leaves (TSL) with 50% inhibitory concentrations (IC50) of 13.9 and 2.0 mu g/ml against the HCV J6/JFH1-P47 and -P1 strains, respectively, Melicope latifolia leaves (MLL) with IC50 of 3.5 and 2.1 mu g/ml, respectively, Melanolepis multiglandulosa stem (MMS) with IC50 of 17.1 and 6.2 mu g/ml, respectively, and Ficus fistulosa leaves (FFL) with IC50 of 15.0 and 5.7 mu g/ml, respectively. Time-of-addition experiments revealed that TSL and MLL inhibited both at the entry and post-entry steps while MMS and FFL principally at the entry step. TSL and MLL inhibited all of 11 HCV strains of all the genotypes tested to the same extent. On the other hand, FFL showed significantly weaker inhibitory activities against the HCV genotype 1a strain, and MMS against the HCV strains of genotypes 2b and 7a to a lesser extent, compared to the other HCV genotypes. Conclusions: Ethanol extracts of TSL, MLL, MMS and FFL showed antiviral activities against all the HCV genotypes tested with the exception that some genotype(s) showed significant resistance to FFL and to MMS to a lesser extent. These plant extracts may be good candidates for the development of anti-HCV drugs.BIOMED CENTRAL LTD, Aug. 2013, VIROLOGY JOURNAL, 10(1) (1), 259, English[Refereed]Scientific journal
- Hepatitis C virus genotype 4 (HCV-4) is the cause of approximately 20% of the 180 million cases of chronic hepatitis C in the world. HCV-4 infection is common in the Middle East and Africa, with an extraordinarily high prevalence in Egypt. Viral genetic polymorphisms, especially within core and NS5A regions, have been implicated in influencing the response to pegylated-interferon and ribavirin (PEG-IFN/RBV) combination therapy in HCV-1 infection. However, this has not been confirmed in HCV-4 infection. Here, we investigated the impact of heterogeneity of NS5A and core proteins of HCV-4, mostly subtype HCV-4a, on the clinical outcomes of 43 Egyptian patients treated with PEG-IFN/RBV. Sliding window analysis over the carboxy terminus of NS5A protein identified the IFN/RBV resistance-determining region (IRRDR) as the most prominent region associated with sustained virological response (SVR). Indeed, 21 (84%) of 25 patients with SVR, but only 5 (28%) of 18 patients with non-SVR, were infected with HCV having IRRDR with 4 or more mutations (IRRDR >= 4) (P = 0.0004). Multivariate analysis identified IRRDR >= 4 as an independent SVR predictor. The positive predictive value of IRRDR >= 4 for SVR was 81% (21/26; P = 0.002), while its negative predictive value for non-SVR was 76% (13/17; P = 0.02). On the other hand, there was no significant correlation between core protein polymorphisms, either at residue 70 or at residue 91, and treatment outcome. In conclusion, the present results demonstrate for the first time that IRRDR >= 4, a viral genetic heterogeneity, would be a useful predictive marker for SVR in HCV-4 infection when treated with PEG-IFN/RBV.AMER SOC MICROBIOLOGY, Dec. 2012, JOURNAL OF CLINICAL MICROBIOLOGY, 50(12) (12), 3886 - 3892, English[Refereed]Scientific journal
- Hepatitis C virus (HCV) infection causes not only intrahepatic diseases but also extrahepatic manifestations, including type 2 diabetes. We previously reported that HCV replication suppresses cellular glucose uptake by downregulation of cell surface expression of glucose transporter 2 (GLUT2) (D. Kasai et al., J. Hepatol. 50:883-894, 2009). GLUT2 mRNA levels were decreased in both HCV RNA replicon cells and HCV J6/JFH1-infected cells. To elucidate molecular mechanisms of HCV-induced suppression of GLUT2 gene expression, we analyzed transcriptional regulation of the GLUT2 promoter using a series of GLUT2 promoter-luciferase reporter plasmids. HCV-induced suppression of GLUT2 promoter activity was abrogated when the hepatocyte nuclear factor 1 alpha (HNF-1 alpha)-binding motif was deleted from the GLUT2 promoter. HNF-1 alpha mRNA levels were significantly reduced in HCV J6/JFH1-infected cells. Furthermore, HCV infection remarkably decreased HNF-1 alpha protein levels. We assessed the effects of proteasome inhibitor or lysosomal protease inhibitors on the HCV-induced reduction of HNF-1 alpha protein levels. Treatment of HCV-infected cells with a lysosomal protease inhibitor, but not with a proteasome inhibitor, restored HNF-1 alpha protein levels, suggesting that HCV infection promotes lysosomal degradation of HNF-1 alpha protein. Overexpression of NS5A protein enhanced lysosomal degradation of HNF-1 alpha protein and suppressed GLUT2 promoter activity. Immunoprecipitation analyses revealed that the region from amino acids 1 to 126 of the NS5A domain I physically interacts with HNF-1 alpha protein. Taken together, our results suggest that HCV infection suppresses GLUT2 gene expression via downregulation of HNF-1 alpha expression at transcriptional and posttranslational levels. HCV-induced downregulation of HNF-1 alpha expression may play a crucial role in glucose metabolic disorders caused by HCV.AMER SOC MICROBIOLOGY, Dec. 2012, JOURNAL OF VIROLOGY, 86(23) (23), 12903 - 12911, English[Refereed]Scientific journal
- Hepatitis C virus (HCV) infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A) protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV) implied that NS5A was tyrosine phosphorylated by pervanadate (PV) treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST)-fusion proteins of various Src homology 2 (SH2) domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3) domain. Substitution of Arg(176) to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr(334) was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.PUBLIC LIBRARY SCIENCE, Oct. 2012, PLOS ONE, 7(10) (10), English[Refereed]Scientific journal
- This study explores pretreatment predictive factors for ultimate virological responses to pegylated interferon-alpha (1.5 mu g/kg/week) and ribavirin (600-1000 mg/day) (PEG-IFN/RBV) combination therapy for patients infected with hepatitis C virus (HCV)-1b and a high viral load. A total of 75 patients underwent PEG-IFN/RBV combination therapy for 48 weeks. HCV amino acid (aa) substitutions in non-structural protein 5a, including those in the IFN/RBV resistance-determining region (IRRDR) and the IFN sensitivity-determining region and the core regions, as well as the genetic variation (rs8099917) near the interleukin 28B (IL28B) gene (genotype TT) were analyzed. Of the 75 patients, 49 % (37/75) achieved a sustained virological response (SVR), 27 % (20/75) showed relapse, and 24 % (18/75) showed null virological response (NVR). Multivariate logistic regression analysis identified IRRDR with 6 or more mutations (IRRDR a parts per thousand yen6) [odds ratio (OR) 11.906, p < 0.0001] and age < 60 years (OR 0.228, p = 0.015) as significant determiners of SVR and IL28B minor (OR 14.618, p = 0.0019) and platelets < 15 x 10(4)/mm(3) (OR 0.113, p = 0.0096) as significant determiners of NVR. A combination of IRRDR a parts per thousand yen6 and age < 60 years improved SVR predictability (93.3 %), and that of IRRDR a parts per thousand currency sign5 and age a parts per thousand yen60 years improved non-SVR predictability (84.0 %). Similarly, a combination of IL28B minor and platelets < 15 x 10(4)/mm(3) improved NVR predictability (85.7 %), and that of IL28B major and platelets a parts per thousand yen15 x 10(4)/mm(3) improved non-NVR (response) (97.1 %) predictability. IRRDR a parts per thousand yen6 and age < 60 years were significantly associated with SVR. IL28B minor and platelets < 15 x 10(4)/mm(3) were significantly associated with NVR. Certain combinations of these factors improved SVR and NVR predictability and could, therefore, be used to design therapeutic strategies.SPRINGER JAPAN KK, Oct. 2012, JOURNAL OF GASTROENTEROLOGY, 47(10) (10), 1143 - 1151, English[Refereed]Scientific journal
- WILEY-BLACKWELL, Sep. 2012, FEBS JOURNAL, 279(10) (10), 161 - 162, EnglishHCV NS5A protein containing potential ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src family kinase Fyn in B cells[Refereed]Scientific journal
- Pegylated-interferon plus ribavirin (PEG-IFN/RBV) therapy is a current standard treatment for chronic hepatitis C. We previously reported that the viral sequence heterogeneity of part of NS5A, referred to as the IFN/RBV resistance-determining region (IRRDR), and a mutation at position 70 of the core protein of hepatitis C virus genotype 1b (HCV-1b) are significantly correlated with the outcome of PEG-IFN/RBV treatment. Here, we aimed to investigate the impact of viral genetic variations within the NS5A and core regions of other genotypes, HCV-2a and HCV-2b, on PEG-IFN/RBV treatment outcome. Pretreatment sequences of NS5A and core regions were analyzed in 112 patients infected with HCV-2a or HCV-2b, who were treated with PEG-IFN/RBV for 24 weeks and followed up for another 24 weeks. The results demonstrated that HCV-2a isolates with 4 or more mutations in IRRDR (IRRDR[2a]>= 4) was significantly associated with rapid virological response at week 4 (RVR) and sustained virological response (SVR). Also, another region of NS5A that corresponds to part of the IFN sensitivity-determining region (ISDR) plus its carboxy-flanking region, which we referred to as ISDR/+C[2a], was significantly associated with SVR in patients infected with HCV-2a. Multivariate analysis revealed that IRRDR[2a]>= 4 was the only independent predictive factor for SVR. As for HCV-2b infection, an N-terminal half of IRRDR having two or more mutations (IRRDR[2b]/N >= 2) was significantly associated with RVR, but not with SVR. No significant correlation was observed between core protein polymorphism and PEG-IFN/RBV treatment outcome in HCV-2a or HCV-2b infection. Conclusion: The present results suggest that sequence heterogeneity of NS5A of HCV-2a (IRRDR[2a]>= 4 and ISDR/+C[2a]), and that of HCV-2b (IRRDR[2b]/N >= 2) to a lesser extent, is involved in determining the viral sensitivity to PEG-IFN/RBV therapy.PUBLIC LIBRARY SCIENCE, Feb. 2012, PLOS ONE, 7(2) (2), English[Refereed]Scientific journal
- The molecular basis of antibody neutralization against hepatitis C virus (HCV) is poorly understood. The E2 glycoprotein of HCV is critically involved in viral infectivity through specific binding to the principal virus receptor component CD81, and is targeted by anti-HCV neutralizing antibodies. A previous study showed that a mutation at position 534 (N534H) within the sixth N-glycosylation motif of E2 of the J6/JFH1 strain of HCV genotype 2a (HCV-2a) was responsible for more efficient access of E2 to CD81 so that the mutant virus could infect the target cells more efficiently. The purpose of this study was to analyze the sensitivity of the parental J6/JFH1, its cell culture-adapted variant P-47 possessing 10 amino acid mutations and recombinant viruses with the adaptive mutations to neutralization by anti-HCV antibodies in sera of HCV-infected patients. The J6/JFH1 virus was neutralized by antibodies in sera of patients infected with HCV-2a and -1b, with mean 50% neutralization titers being 1:670 and 1:200, respectively (P < 0.00001). On the other hand, the P-47 variant showed 50- to 200-times higher sensitivity to antibody neutralization than the parental J6/JFH1 without genotype specificity. The N534H mutation, and another one at position 416 (T416A) near the first N-glycosylation motif to a lesser extent, were shown to be responsible for the enhanced sensitivity to antibody neutralization. The present results suggest that the residues 534, and 416 to a lesser extent, of the E2 glycoprotein are critically involved in the HCV infectivity and antibody neutralization. J. Med. Virol. 84:229-234, 2012. (C) 2011 Wiley Periodicals, Inc.WILEY-BLACKWELL, Feb. 2012, JOURNAL OF MEDICAL VIROLOGY, 84(2) (2), 229 - 234, English[Refereed]Scientific journal
- The lack of a culture system that efficiently produces progeny virus has hampered hepatitis C virus (HCV) research. Recently, the discovery of a novel HCV isolate JFH1 and its chimeric derivative J6/JFH1 has led to the development of an efficient virus productive culture system. To construct an easy monitoring system for the viral life cycle of HCV, we generated bicistronic luciferase reporter virus genomes based on the JFH1 and J6/JFH1 isolates, respectively. Transfection of the J6/JFH1-based reporter genome to Huh7.5 cells produced significantly greater levels of progeny virus than transfection of the JFH1 genome. Furthermore, the expression of dominant-negative Vps4, a key molecule of the endosomal sorting complex required for transport machinery, inhibited the virus production of JFH1, but not that of J6/JFH1. These results may account for the different abilities to produce progeny virus between JFH1 and J6/JFH1. Using the J6/JFH1/Luc system, we showed that the two polyanions heparin and polyvinyl sulfate decreased the infectivity of J6/JFH1/Luc virus in a dose-dependent manner. We also analyzed the function of microRNA on HCV replication and found that miR-34b could affect the replication of HCV. The reporter virus generated in this study will be useful for investigating the nature of the HCV life cycle and for identification of HCV inhibitors. (C) 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.ELSEVIER SCIENCE BV, Jan. 2012, MICROBES AND INFECTION, 14(1) (1), 69 - 78, English[Refereed]Scientific journal
- Objective: Hepatitis C virus (HCV genome) polymorphisms are thought to influence the outcome of pegylated-interferon/ribavirin (PEG-IFN/RBV) therapy. This study aimed to examine non-structural protein 5A (NS5A) polymorphisms, e.g. IFN/RBV resistance-determining region (IRRDR) and IFN sensitivity- determining region (ISDR), and core protein polymorphism as predictive therapeutic markers. Methods: Pretreatment sequences of NS5A and core regions were analyzed in 68 HCV-1b-infected patients treated with PEG-IFN/RBV. Results: Of 24 patients infected with HCV having an IRRDR with 6 or more mutations (IRRDR >= 6), 18 (75%) patients achieved sustained virological response (SVR), where as only 11 (25%) of 44 patients infected with HCV having IRRDR <= 5 did. IRRDR >= 6 was significantly associated with SVR (p < 0.0001). On the other hand, ISDR >= 2 was significantly associated with relapse (either before [breakthrough] or after end-of-treatment response [ETR-relapse]) (p < 0.05) and a point mutation of the core protein from Arg to Gln at position 70 (Gln(70)) was significantly associated with null-response (p < 0.05). Multivariate analysis identified IRRDR >= 6 as the only viral genetic factor that independently predicted SVR. Conclusion: NS5A (IRRDR and ISDR) and core protein polymorphisms are associated with the outcome of PEG-IFN/ RBV therapy for chronic hepatitis C. In particular, IRRDR >= 6 is a useful marker for prediction of SVR. Copyright (C) 2011 S. Karger AG, BaselKARGER, 2012, INTERVIROLOGY, 55(1) (1), 1 - 11, English[Refereed]Scientific journal
- Chronic hepatitis C virus (HCV) infection is often associated with type 2 diabetes. However, the precise mechanism underlying this association is still unclear. Here, using Huh-7.5 cells either harboring HCV-1b RNA replicons or infected with HCV-2a, we showed that HCV transcriptionally upregulated the genes for phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), the rate-limiting enzymes for hepatic gluconeogenesis. In this way, HCV enhanced the cellular production of glucose 6-phosphate (G6P) and glucose. PEPCK and G6Pase gene expressions are controlled by the transcription factor forkhead box O1 (FoxO1). We observed that although neither the mRNA levels nor the protein levels of FoxO1 expression were affected by HCV, the level of phosphorylation of FoxO1 at Ser319 was markedly diminished in HCV-infected cells compared to the control cells, resulting in an increased nuclear accumulation of FoxO1, which is essential for sustaining its transcriptional activity. It was unlikely that the decreased level of FoxO1 phosphorylation was mediated through Akt inactivation, as we observed an increased phosphorylation of Akt at Ser473 in HCV-infected cells compared to control cells. By using specific inhibitors of c-Jun N-terminal kinase (JNK) and reactive oxygen species (ROS), we demonstrated that HCV infection induced JNK activation via increased mitochondrial ROS production, resulting in decreased FoxO1 phosphorylation, FoxO1 nuclear accumulation, and, eventually, increased glucose production. We also found that HCV NS5A mediated increased ROS production and JNK activation, which is directly linked with the FoxO1-dependent increased gluconeogenesis. Taken together, these observations suggest that HCV promotes hepatic gluconeogenesis through an NS5A-mediated, FoxO1-dependent pathway.Lead, AMER SOC MICROBIOLOGY, Sep. 2011, JOURNAL OF VIROLOGY, 85(17) (17), 8556 - 8568, English[Refereed]Scientific journal
- Both host and viral factors have been implicated in influencing the response to pegylated-interferon/ribavirin (PEG-IFN/RBV) therapy for hepatitis C virus (HCV) infection. Among the viral factors, sequence heterogeneity within NS5A and core regions has been proposed. This study aimed to clarify the relationship between virological responses to PEG-IFN/RBV therapy and sequence heterogeneity within NS5A, including the IFN/RBV resistance-determining region (IRRDR), the interferon sensitivity-determining region (ISDR) and the core region. Pretreatment sequences of NS5A and the core regions were analyzed in 57 HCV-1b-infected patients who were to be treated with PEG-IFN/RBV. Of 40 patients infected with HCV having an IRRDR with four or more mutations (IRRDR ≥ 4), 28 (70%) patients achieved a sustained virological response (SVR). On the other hand, only 4 (24%) of 17 patients infected with HCV having an IRRDR with three or fewer mutations (IRRDR ≤ 3) achieved a SVR (P= 0.001). Similarly, 22 (71%) of 31 patients infected with HCV and having an ISDR with one or more mutations (ISDR ≥ 1) achieved a SVR while 10 (38%) of 26 patients infected with HCV and having an ISDR without any mutations (ISDR = 0) achieved a SVR (P= 0.014). As for the core region, there was significant correlation between a single mutation at position 70 (Gln70) and non-SVR (P= 0.02). Notably, Gln70 was more prominently associated with the null response (P= 0.0007). In conclusion, sequence heterogeneity within the IRRDR and ISDR, and a single point mutation at position 70 of the core region of HCV-1b are likely to be correlated with virological responses to PEG-IFN/RBV therapy. © 2011 The Societies and Blackwell Publishing Asia Pty Ltd.Jun. 2011, Microbiology and Immunology, 55(6) (6), 418 - 426, English[Refereed]Scientific journal
- Persistent infection with hepatitis C virus causes serious liver diseases, such as chronic hepatitis, hepatic cirrhosis and hepatocellular carcinoma. The male gender is one of the critical factors in progression of hepatic fibrosis due to chronic HCV infection; thus female hormones may play a role in delaying the progression of hepatic fibrosis. It has also been reported that women are more likely than men to clear HCV in the acute phase of infection. These observations lead the present authors to the question: do female hormones inhibit HCV infection? In this study using HCV J6/JFH1 and Huh-7.5 cells, the possible inhibitory effect(s) of female hormones such as 17 beta-estradiol (the most potent physiological estrogen) and progesterone on HCV RNA replication, HCV protein synthesis and production of HCV infectious particles (virions) were analyzed. It was found that E(2), but not P(4), significantly inhibited production of the HCV virion without inhibiting HCV RNA replication or HCV protein synthesis. E(2)-mediated inhibition of HCV virion production was abolished by a nuclear estrogen receptor (ER) antagonist ICI182780. Moreover, treatment with the ER alpha-selective agonist 4, 4', 4 '- (4-propyl-[1H]-pyrazole-1, 3, 5-triyl)trisphenol (PPT), but not with the ER beta-selective agonist 2, 3-bis (4-hydroxyphenyl)-propionitrile (DPN) or the G protein-coupled receptor 30 (GPR30)-selective agonist 1-(4-[6-bromobenzo 1, 3 dioxol-5-yl]-3a, 4, 5, 9b-tetrahydro-3H-cyclopenta [c] quinolin-8-yl)-ethanone (G-1), significantly inhibited HCV virion production. Taken together, the present results suggest that the most potent physiological estrogen, E(2), inhibits the production of HCV infectious particles in an ER alpha-dependent manner.WILEY-BLACKWELL, Nov. 2010, MICROBIOLOGY AND IMMUNOLOGY, 54(11) (11), 684 - 690, English[Refereed]Scientific journal
- The aim of the study was to identify a predictive marker for the virological response in hepatitis C virus 1b (HCV-1b)-infected patients treated with pegylated interferon plus ribavirin therapy. A total of 139 patients with chronic hepatitis C who received therapy for 48 weeks were enrolled. The secondary structure of the 120 residues of the amino-terminal HCV-1b non-structural region 3 (NS3) deduced from the amino acid sequence was classified into two major groups: A and B. The association between HCV NS3 protein polymorphism and virological response was analyzed in patients infected with group A (n=28) and B (n=40) isolates who had good adherence to both pegylated interferon and ribavirin administration (>95% of the scheduled dosage) for 48 weeks. A sustained virological response (SVR) representing successful HCV eradication occurred in 33 (49%) in the 68 patients. Of the 28 patients infected with the group A isolate, 18 (64%) were SVR, whereas of the 40 patients infected with the group B isolate only 15 (38%) were SVR. The proportion of virological responses differed significantly between the two groups (P<0.05). These results suggest that polymorphism in the secondary structure of the HCV-1b NS3 amino-terminal region influences the virological response to pegylated interferon plus ribavirin therapy, and that virus grouping based on this polymorphism can contribute to prediction of the outcome of this therapy. J. Med. Virol. 82:1364-1370, 2010. (C) 2010 Wiley-Liss, Inc.WILEY-LISS, Aug. 2010, JOURNAL OF MEDICAL VIROLOGY, 82(8) (8), 1364 - 1370, English[Refereed]Scientific journal
- Analysis of neutralizing antibodies against hepatitis C virus in patients who were treated with pegylated-interferon plus ribavirinThe role of neutralizing antibodies (NAb) in determining responses to antiviral therapy has not been defined well. By using hepatitis C virus (HCV) cell culture system with the J6/JFH1 strain of HCV genotype 2a, we analyzed NAb responses in patients with chronic hepatitis C who received pegylated-interferon plus ribavirin (PEG-IFN/RBV) antiviral therapy. A total of 65 patients chronically infected with HCV genotype 1b were enrolled in this study. Of all the 65 patients, 34 (52%) patients achieved early virological response (EVR), with the remaining 31 patients (48%) being Non-EVR. Twenty-seven patients (42%) achieved sustained virological response (SVR), with the remaining 38 patients (58%) being Non-SVR. Thus, NAb titers were significantly higher in sera of patients who achieved EVR and SVR than those of Non-EVR and Non-SVR, respectively. Rather unexpectedly, NAb titers did not significantly decrease when measured even one year after disappearance of HCV RNA. On the other hand, when change ratios of NAb titers before and after disappearance of HCV RNA were compared between patients with different treatment outcomes, we noticed that the change ratio of NAb titers of patients who achieved an EVR was significantly lower than that of Non-SVR. In conclusion, our present results suggest that NAb titers were significantly associated with clinical responses to PEG-IFN/RBV therapy.2010, Kobe Journal of Medical Sciences, 56(2) (2), E60 - E66, English[Refereed]Scientific journal
- The hepatitis C virus (HCV) core protein is known to modulate apoptosis and contribute to viral replication and pathogenesis. In this study, we have identified a Bcl-2 homology 3 (BH3) domain in the core protein that is essential for its proapoptotic property. Coimmunoprecipitation experiments showed that the core protein interacts specifically with the human myeloid cell factor 1 (Mcl-1), a prosurvival member of the Bcl-2 family, but not with other prosurvival members (Bcl-X-L and Bcl-w). Moreover, the overexpression of Mcl-1 protects against core-induced apoptosis. By using peptide mimetics, core was found to release cytochrome c from isolated mitochondria when complemented with Bad. Thus, core is a bona fide BH3-only protein having properties similar to those of Noxa, a BH3-only member of the Bcl-2 family that binds preferentially to Mcl-1. There are three critical hydrophobic residues in the BH3 domain of the core protein, and they are essential for the proapoptotic property of the core protein. Furthermore, the genotype 1b core protein is more effective than the genotype 2a core protein in inducing apoptosis due to a single-amino-acid difference at one of these hydrophobic residues (residue 119). Replacing this residue in the J6/JFH-1 infectious clone (genotype 2a) with the corresponding amino acid in the genotype 1b core protein produced a mutant virus, J6/JFH-1(V119L), which induced significantly higher levels of apoptosis in the infected cells than the parental J6/JFH-1 virus. Furthermore, the core protein of J6/JFH-1(V119L), but not that of J6/JFH-1, interacted with Mcl-1 in virus-infected cells. Taken together, the core protein is a novel BH3-only viral homologue that contributes to the induction of apoptosis during HCV infection.AMER SOC MICROBIOLOGY, Oct. 2009, JOURNAL OF VIROLOGY, 83(19) (19), 9993 - 10006, English[Refereed]Scientific journal
- Robust production of infectious hepatitis C virus (HCV) in cell culture was realized by using the JFH1 strain and the homologous chimeric J6/JFH1 strain in Huh-7.5 cells, a highly HCV-permissive subclone of Huh-7 cells. In this study, we aimed to establish a more efficient HCV-production system and to gain some insight into the adaptation mechanisms of efficient HCV production. By serial passaging of J6/JFH1-infected Huh-7.5 cells, we obtained culture-adapted J6/JFH1 variants, designated P-27, P-38 and P-47. Sequence analyses revealed that the adaptive mutant viruses P-27, P-38 and P-47 possessed eight mutations [four in E2, two in NS2, one in NS5A and one in NS5B), 10 mutations [two additional mutations in the 5'-untranslated region (5'-UTR) and core] and 11 mutations (three additional mutations in 5'-UTR, core and NS5B), respectively. We introduced amino acid substitutions into the wild-type J6/JFH1 clone, generated recombinant viruses with adaptive mutations and analysed their infectivity and ability to produce infectious viruses. The viruses with the adaptive mutations exhibited higher expression of HCV proteins than did the wild type in Huh-7.5 cells. Moreover, we provide evidence suggesting that the mutation N534H in the E2 glycoprotein of the mutant viruses conferred an advantage at the entry level. We thus demonstrate that an efficient HCV-production system could be obtained by introducing adaptive mutations into the J6/JFH1 genome. The J6/JFH1-derived mutant viruses presented here would be a good tool for producing HCV particles with enhanced infectivity and for studying the molecular mechanism of HCV entry.SOC GENERAL MICROBIOLOGY, Jul. 2009, JOURNAL OF GENERAL VIROLOGY, 90, 1681 - 1691, English[Refereed]Scientific journal
- Background/Aims: Persistent infection with hepatitis C virus (HCV) causes extrahepatic diseases, including diabetes. We investigated the possible effect(s) of HCV replication on cellular glucose uptake and expression of the facilitative glucose transporter (GLUT) 2 and 1. Methods: We used Huh-7.5 cells harboring either an HCV subgenomic RNA replicon (SGR) or an HCV full-genomic RNA replicon (FGR), HCV-infected cells, and the respective cells treated with interferon (1FN). We also used liver tissue samples obtained from patients with or without HCV infection. Results:Glucose uptake and surface expression of GLUT2 and GLUT1 were suppressed in SGR, FGR and HCV-infected cells compared to the control cells. Expression levels of GLUT2 mRNA, but not GLUT1 mRNA, were lower in SGR, FGR and HCV-infected cells than in the control. Luciferase reporter assay demonstrated decreased GLUT2 promoter activities in SGR, FGR and HCV-infected cells. IFN treatment restored glucose uptake, GLUT2 surface expression, GLUT2 mRNA expression and GLUT2 promoter activities. Also, GLUT2 expression was reduced in hepatocytes of liver tissues obtained from HCV-infected patients. Conclusions: HCV replication down-regulates cell surface expression of GLUT2 partly at the transcriptional level, and possibly at the intracellular trafficking level as suggested for GLUT1, thereby lowering glucose uptake by hepatocytes. (c) 2009 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, May 2009, JOURNAL OF HEPATOLOGY, 50(5) (5), 883 - 894, English[Refereed]Scientific journal
- We previously reported that cells harboring the hepatitis C virus (HCV) RNA replicon as well as those expressing HCV NS3/4A exhibited increased sensitivity to suboptimal doses of apoptotic stimuli to undergo mitochondrion-mediated apoptosis (Y. Nomura-Takigawa, et al., J. Gen. Virol. 87: 1935-1945, 2006). Little is known, however, about whether or not HCV infection induces apoptosis of the virus-infected cells. In this study, by using the chimeric J6/JFH1 strain of HCV genotype 2a,we demonstrated that HCV infection induced cell death in Huh7.5 cells. The cell death was associated with activation of caspase 3, nuclear translocation of activated caspase 3, and cleavage of DNA repair enzyme poly(ADP-ribose) polymerase, which is known to be an important substrate for activated caspase 3. These results suggest that HCV-induced cell death is, in fact, apoptosis. Moreover, HCV infection activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change and its increased accumulation on mitochondrial membranes. Concomitantly, HCV infection induced disruption of mitochondrial transmembrane potential, followed by mitochondrial swelling and release of cytochrome c from mitochondria. HCV infection also caused oxidative stress via increased production of mitochondrial superoxide. On the other hand, HCV infection did not mediate increased expression of glucose-regulated protein 78 (GRP78) or GRP94, which are known as endoplasmic reticulum (ER) stress-induced proteins; this result suggests that ER stress is not primarily involved in HCV-induced apoptosis in our experimental system. Taken together, our present results suggest that HCV infection induces apoptosis of the host cell through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway(s).Lead, AMER SOC MICROBIOLOGY, Nov. 2008, JOURNAL OF VIROLOGY, 82(21) (21), 10375 - 10385, English[Refereed]Scientific journal
- Non-structural protein 4A (NS4A) of Hepatitis C virus (HCV) functions as a cofactor for NS3 by forming a complex with it to augment its enzymic activities. NS4A also forms a complex with other HCV proteins, such as NS4B/NS5A, to facilitate the formation of the viral RNA replication complex on the encloplasmic reticulum (ER) membrane. In addition to its essential role in HCV replication, NS4A is thought to be involved in viral pathogenesis by affecting cellular functions. In this study, it was demonstrated that NS4A was localized not only on the ER, but also on mitochondria when expressed either alone or together with NS3 in the form of the NS3/4A polyprotein and in the context of HCV RNA replication in Huh7 cells harbouring an HCV RNA replicon. Moreover, NS4A expression altered the intracellular distribution of mitochondria significantly and caused mitochondrial damage, as evidenced by the collapsed mitochondrial transmembrane potential and release of cytochrome c into the cytoplasm, which led ultimately to induction of apoptosis through activation of caspase-3, but not caspase-8. Consistently, Huh7 cells expressing NS3/4A and those harbouring an HCV RNA replicon were shown to be more prone to undergoing actinomycin D-induced, mitochondria-mediated apoptosis, compared with the control Huh7 cells. Taken together, these results suggest the possibility that HCV exerts cytopathic effect (CPE) on the infected cells under certain conditions and that NS4A is responsible, at least in part, for the conditional CPE in HCV-infected cells.SOC GENERAL MICROBIOLOGY, Jul. 2006, JOURNAL OF GENERAL VIROLOGY, 87(Pt 7) (Pt 7), 1935 - 1945, English[Refereed]Scientific journal
- The N-terminal 198 residues of NS3 (NS3-N) of Hepatitis C virus (HCV) subtype 1b obtained from 29 patients, as well as full-length NS3 (NS3-Full), were analysed for their subcellular localization, interaction with the tumour suppressor p53 and serine protease activity in the presence and absence of the viral cofactor NS4A. Based on the subcellular-localization patterns in the absence of NS4A, NS3-N sequences were classified into three groups, with each group exhibiting either dot-like, diffuse or a mixed type of localization. Chimeric NS3-Full sequences, each consisting of an individual NS3-N and a shared C-terminal sequence, showed the same localization patterns as those of the respective NS3-N. Site-directed mutagenesis experiments revealed that a single or a few amino acid substitutions at a particular position(s) of NS3-N altered the localization pattern. Interestingly, NS3 of the dot-like type, either NS3-N or NS3-Full, interacted with p53 more strongly than that of the diffuse type, in both the presence and the absence of NS4A. Moreover, NS3-N of the dot-like type suppressed trans-activating activity of p53 more strongly than that of the diffuse type. Serine protease activity did not differ significantly between the two types of NS3. In HCV RNA replicon-harbouring cells, physical interaction between NS3 and p53 was observed consistently and p53-mediated transcriptional activation was suppressed significantly compared with HCV RNA-negative control cells. Our results collectively suggest the possibility that NS3 plays an important role in the hepatocarcinogenesis of HCV by interacting differentially with p53 in an NS3 sequence-dependent manner.Lead, SOC GENERAL MICROBIOLOGY, Jun. 2006, JOURNAL OF GENERAL VIROLOGY, 87, 1703 - 1713, English[Refereed]Scientific journal
- The N-terminal domain of NS3 of hepatitis C virus (HCV) possesses serine protease activity, which is essential for virus replication. This portion is also implicated in malignant transformation of hepatocytes. We previously demonstrated that an N-terminal portion of NS3 formed a complex with the tumor suppressor p53 and suppressed actinomycin D-induced apoptosis. We report here that single-point mutations of NS3 at position 106 from Leu to Ala (L106A), and position 43 from Phe to Ala (F43A) to a lesser extent, significantly impaired complex formation with p53. Moreover, the L106A Mutation impaired an otherwise more distinct anti-apoptotic activity of NS3. F43A and L106A mutations also inhibited serine protease activity of NS3. These results collectively suggest the possibility that Leu(106) and Phe(43) are involved in p53 interaction and serine protease activity, and therefore, can be a good target for certain low-molecular-weight compound(s) to inhibit both oncogenic and replicative abilities of HCV. (c) 2005 Elsevier Inc. All rights reserved.ACADEMIC PRESS INC ELSEVIER SCIENCE, Feb. 2006, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 340(3) (3), 792 - 799, English[Refereed]Scientific journal
- The NS3 protein of hepatitis C virus (HCV) has a serine protease activity in its N-terminal region, which plays a crucial role in virus replication. This region has also been reported to interact not only with its viral cofactor NS4A, but also with a number of host-cell proteins, which suggests a multifunctional feature of NS3. By means of yeast two-hybrid screening using an N-terminal region of NS3 as bait, a human cDNA encoding a region of ELKS-delta, a member of a novel family of proteins involved in intracellular transport and secretory pathways, was molecularly cloned. Using co-immunoprecipitation, GST pull-down and confocal and immunoelectron microscopic analyses, it was shown that full-length NS3 interacted physically with full-length ELKS-delta and its splice variant, ELKS-alpha, both in the absence and presence of NS4A, in cultured human cells, including Huh-7 cells harbouring an HCV subgenomic RNA replicon. The degree of binding to ELKS-delta varied with different sequences of the N-terminal 180 residues of NS3. Interestingly, NS3, either full-length or N-terminal fragments, enhanced secretion of secreted alkaline phosphatase (SEAP) from the cells, and the increase in SEAP secretion correlated well with the degree of binding between NS3 and ELKS-delta. Taken together, these results suggest the possibility that NS3 plays a role in modulating host-cell functions such as intracellular transport and secretion through its binding to ELKS-delta and ELKS-alpha, which may facilitate the virus life cycle and/or mediate the pathogenesis of HCV.SOC GENERAL MICROBIOLOGY, Aug. 2005, JOURNAL OF GENERAL VIROLOGY, 86(8) (8), 2197 - 2208, English[Refereed]Scientific journal
- Nonstructural proteins 4A and 4B of hepatitis C virus transactivate the interleukin 8 promoterInterleukin 8 (IL-8) is induced in many cell types by various stimuli including virus infection. It was reported that nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) was involved in induction of IL-8 expression at both mRNA and protein levels in cultured human cells. In this study, we aimed to determine whether or not another HCV protein(s) transactivates the IL-8 gene expression, by means of an IL-8 promoter-driven luciferase reporter assay and measurement of endogenous IL-8 mRNA and secreted IL-8 protein levels. We observed that NS4B, and NS4A to a lesser extent, significantly transactivated the IL-8 promoter, which resulted in enhanced production of IL-8 protein. Also, the IL-8 expression was augmented in Huh-7 cells harboring an HCV subgenomic RNA replicon, compared with the control cells. Deletion mutational analysis of the IL-8 promoter revealed the possible involvement of the transcription factor AP-1 in both NS4A- and NS4B-mediated IL-8 gene activation. In addition, the IL-8 gene activation by NS4B, but not that by NS4A, was likely to involve NF-kappa B and/or NFIL-6. The degree of the transactivation by NS4B and NS4A varied with different human cell lines, with HeLa cells showing the strongest activation followed by Huh-7 cells, and with HepG2 cells exhibiting a marginal level of activation. Taken together, our present results suggest the possibility that NS4B and NS4A play an important role in inducing the IL-8 gene expression under certain cellular conditions, which might be one of the strategies to establish persistent HCV infection.WILEY-BLACKWELL, 2005, MICROBIOLOGY AND IMMUNOLOGY, 49(3) (3), 265 - 273, English[Refereed]Scientific journal
- Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of the viral genomeRNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA), an RNA duplex 21 to 23 nucleotides (nt) long, or short hairpin RNA (shRNA) resembling siRNA, mediates degradation of the target RNA molecule in a sequence-specific manner. RNAi is now expected to be a useful therapeutic strategy for hepatitis C virus (HCV) infection. In the present study we compared the efficacy of a number of shRNAs directed against different target regions of the HCV genome, such as 5'-untranslated region (5'UTR) (nt 286 to 304), Core (nt 371 to 389), NS3-1 (nt 2052 to 2060), NS3-2 (nt 2104 to 2122), and NS5B (nt 7326 to 7344), all of which except for NS5B are conserved among most, if not all, HCV subtype 1b (HCV-1b) isolates in Japan. We utilized two methods to express shRNAs, one utilizing an expression plasmid (pAVU6+27) and the other utilizing a recombinant lentivirus harboring the pAVU6+27-derived expression cassette. Although 5'UTR has been considered to be the most suitable region for therapeutic siRNA and/or shRNA because of its extremely high degree of sequence conservation, we observed only a faint suppression of an HCV subgenomic replicon by shRNA against 5'UTR. In both plasmid- and lentivirus-mediated expression systems, shRNAs against NS3-1 and NS5B suppressed most efficiently the replication of the HCV replicon without suppressing host cellular gene expression. Synthetic siRNA against NS3-1 also inhibited replication of the HCV replicon in a dose-dependent manner. Taken together, the present results imply the possibility that the recombinant lentivirus expressing shRNA against NS3-1 would be a useful tool to inhibit HCV-1b infection.CENTER ACADEMIC PUBL JAPAN, 2004, MICROBIOLOGY AND IMMUNOLOGY, 48(8) (8), 591 - 598, English[Refereed]Scientific journal
- Cleavage of the hepatitis C virus NS5A protein by caspase-3 in the interferon sensitivity-determining region in a sequence-dependent mannerHepatitis C virus (HCV) nonstructural protein 5A (NS5A) has versatile functions and has been implicated in viral pathogenesis, including interferon (IFN) resistance and hepatocarcinogenesis. It has been reported that NS5A is cleaved into a few fragments by a caspase(s) or caspase-like enzyme(s) under certain conditions. Two cleavage sites have been mapped to the Asp residues at positions 154 and 398 (D154 and D398). However, other possible cleavage sites were not determined yet so far. In this study, we demonstrated caspase-3-mediated NS5A cleavage upon apoptotic stimuli and identified a new site as the third cleavage target. This site was mapped to D251, which lies within IFN sensitivity-determining region (ISDR). Although D251 was conserved among all HCV subtype 1b (HCV-1b) strains tested, the consensus caspase-3 recognition sequence (D248-X-X- D251) was not conserved due to the sequence variation at position 248 in ISDR. Furthermore, A252 was found to be necessary for efficient cleavage of NS5A. The virological significance of the HCV strain-dependent NS5A cleavage at this site awaits further investigation.2004, Kobe Journal of Medical Sciences, 50(5) (5), 153 - 166, English[Refereed]Scientific journal
- The NS3 protein of hepatitis C virus subtype 1b (HCV-1b) isolates obtained from 89 patients with hepatocellular carcinoma (HCC) and 78 patients without HCC were analyzed. On the basis of the secondary structure of the amino-terminal 120 residues of NS3, HCV-1b isolates were classified into group A, group B, and an indeterminate group, each of which was further divided into a number of subgroups, such as A1-1, A1-2, A2-1, A2-2, B1-1, B1-2, B2-1, B2-2, C-1, C-2, and C-3. HCV-1b isolates of subgroup B1-1 were found in 53 (59.6%) of 89 patients with HCC and 19 (24.4%) of 78 patients without HCC, with the difference between the two patient groups being statistically significant (P < 0.00001). Although the number of isolates was small, subgroup B2-1 was also highly associated with HCC, with all five isolates in that subgroup being found in patients with HCC (P < 0.05). On the other hand, HCV-1b isolates of subgroup A1-1 were associated only weakly with HCC; they were found in 6 (6.7%) of 89 patients with HCC and in 25 (32.1%) of 78 patients without HCC, with the difference between the two patient groups being statistically significant (P < 0.0001). The other subgroups, such as A1-2, A2-1, B1-2, C-1, C-2, and C-3, were moderately associated with HCC; their distribution patterns among patients with HCC did not differ significantly from those among patients without HCC. Taken together, our results suggest that HCV-1b isolates of subgroups B1-1 and B2-1 are highly associated with HCC and that this secondary structure analysis may be useful for predicting the relative risk of developing HCC.AMER SOC MICROBIOLOGY, Jul. 2003, JOURNAL OF CLINICAL MICROBIOLOGY, 41(7) (7), 2835 - 2841, EnglishScientific journal
- Hepatitis C virus core protein selectively inhibits synthesis and accumulation of p21/Waf1 and certain nuclear proteinsBy using a vaccinia virus-T7 expression system, possible effects of hepatitis C virus (HCV) core protein on synthesis and accumulation of host cellular proteins transiently expressed in cultured cells were analyzed. Immunoblot and immunofluorescence analyses revealed that synthesis and accumulation of certain nuclear proteins, such as p21/Waf1, p53, proliferating cell nuclear antigen and c-Fos, were strongly inhibited by HCV core protein. On the other hand, synthesis and accumulation of cytoplasmic proteins, such as 2'-5'-oligoadenylate synthetase (2'-5'-OAS), RNase L and MEK1, were barely affected by HCV core protein. Northern blot analysis showed that the degrees of mRNA expression for those proteins did not differ between HCV core protein-expressing cells and the control, suggesting that the inhibition occurred at the post-transcription level. Pulse-labeling analysis suggested that HCV core protein strongly inhibited synthesis of p21/Waf1 at the translation level. Once being accumulated in the nucleus, p21/Waf1 stability was not significantly affected by HCV core protein. Mutants of HCV core protein C-terminally deleted by 18 or 41 amino acids (aa), which were localized almost exclusively in the nucleus, lost their ability to inhibit synthesis/accumulation of p21/Waf1 whereas another mutant C-terminally deleted by 8 aa still maintained the same properties (subcellular localization and the inhibitory effect) as the full-length HCV core protein of 191 aa. Taken together, our present results suggest that expression of HCV core protein in the cytoplasm selectively inhibits synthesis of p21/Waf1 and some other nuclear proteins at the translation level.CENTER ACADEMIC PUBL JAPAN, 2003, MICROBIOLOGY AND IMMUNOLOGY, 47(6) (6), 429 - 438, English[Refereed]Scientific journal
- Physical interaction between hepatitis C virus NS4B protein and CREB-RP/ATF6 betaBy using a yeast two-hybrid assay, cyclic AMP-response-element-binding protein-related protein (CREB-RP), also called activating transcription factor 60 (ATF6beta), was identified-as a cellular protein that interacts with the NS4B protein of hepatitis C virus. An N-terminal half of NS4B and a central portion of CREB-RP/ATF6beta containing the basic leucine zipper (bZIP) domain were involved in the interaction. The interaction between NS4B and CREB-RP/ATF6beta was demonstrated also in mammalian cells by coimmunoprecipitation and confocal microscopic analyses using specific antibodies. The bZIP domain of ATF6alpha, which shares high sequence similarity with CREB-RP/ATF6beta, was also shown to interact with NS4B in yeast although the interaction was weaker than that between NS4B and CREB-RP/ATF6beta. CREB-RP/ATF6beta and ATF6alpha are known as endoplasmic reticulum, (ER) stress-induced transcription factors. Collectively, our results imply the possibility that NS4B modulates certain cellular responses upon ER stress through the physical interaction with CREB-RP/ATF6beta and ATF6a. (C) 2002 Elsevier Science (USA). All rights reserved.ACADEMIC PRESS INC ELSEVIER SCIENCE, Dec. 2002, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 299(3) (3), 366 - 372, English[Refereed]Scientific journal
- 2023, 日本ウイルス学会学術集会プログラム・予稿集(Web), 70thUnusual G9P[4] rotavirus emerged after the dynamic changes in rotavirus genotypes from equine-like G3 to typical human G1/G3 in Indonesia
- 2020, 日本分子生物学会年会プログラム・要旨集(Web), 43rd細胞内アネキシン5はC型肝炎ウイルスの増殖抑制に関与する
- 2020, 日本分子生物学会年会プログラム・要旨集(Web), 43rdHCV NS3/4AプロテアーゼはSPG20を選択的に切断し脂肪滴の巨大化を促進する
- 2019, 日本ウイルス学会学術集会プログラム・予稿集(Web), 67thHCV NS3/4AプロテアーゼはSPG20を選択的に切断し脂肪滴の巨大化を促進する
- 2019, 日本ウイルス学会学術集会プログラム・予稿集(Web), 67thC型肝炎ウイルスNS5A蛋白質のISGylation修飾反応におけるウイルス遺伝子型間の解析
- 2017, 日本生化学会大会(Web), 90th脱ユビキチン化酵素USP15阻害剤候補分子の機能解析
- 2016, 日本分子生物学会年会プログラム・要旨集(Web), 39th脱ユビキチン化酵素USP15阻害剤の探索と機能解析
- Hepatitis C virus (HCV) infection often causes intrahepatic diseases, such as chronic hepatitis, liver chirrohsis, and hepatocellular carcinoma (HCC). Moreover, HCV infection exhibits various extrahepatic manifestations, such as thyroiditis, glucose and lipid metabolic disorder, and iron metabolic disorder. HCV infection is often associated with type 2 diabetes, involving hepatic fibrosis and poor prognosis. Type 2 diabetes increases the risk of HCC. We have been investigating molecular mechanisms of HCV-induced glucose metabolic disorder and we reported that HCV infection promotes hepatic gluconeogenesis through forkhead box O1 (FoxO1)-dependent pathway and that HCV infection suppresses the cell surface expression of glucose transporter 2 (GLUT2), resulting in suppression of glucose uptake. We have found that HCV NS5A protein plays important roles in these two independent pathways. Here we discuss the roles of HCV NS5A in HCV-induced glucose metabolic disorder.The Japanese Society for Virology, Dec. 2015, ウイルス, 65(2) (2), 263 - 268, Japanese[Refereed][Invited]Introduction scientific journal
- 2014, 日本ウイルス学会学術集会プログラム・抄録集, 62ndC型肝炎ウイルス感染によるHepatocyte nuclear factor(HNF)-1α蛋白質の選択的分解機構
- 2014, 日本分子生物学会年会プログラム・要旨集(Web), 37thC型肝炎ウイルスによるHNF-1α蛋白質の選択的分解機構の解析
- 2013, 日本ウイルス学会学術集会プログラム・抄録集, 61stHCV NS5AとHepatocyte nuclear factor(HNF)-1αの相互作用と病態生理
- 2013, 日本ウイルス学会学術集会プログラム・抄録集, 61stHCV感染による糖代謝障害の分子機序
- 31 Oct. 2012, 日本ウイルス学会学術集会プログラム・抄録集, 60th, 244, JapaneseC型肝炎ウイルスNS5AはB細胞のチロシンキナーゼFynを活性化する
- 2012, 日本ウイルス学会学術集会プログラム・抄録集, 60thC型肝炎ウイルスによるGLUT2遺伝子発現抑制の分子機構
- 2012, 日本分子生物学会年会プログラム・要旨集(Web), 35thC型肝炎ウイルス感染はHNF-1αの発現を負に制御しGLUT2遺伝子発現を抑制する
- 2012, 日本分子生物学会年会プログラム・要旨集(Web), 35th, 1LBA-0769 (WEB ONLY), JapaneseC型肝炎ウイルスNS5A蛋白質のSrc homology2/3ドメイン結合能とB細胞での発現によるSrcファミリーキナーゼFynの活性化
- Hepatitis C virus (HCV) infection causes not only intrahepatic diseases but also extrahepatic manifestations, including metabolic disorders. Chronic HCV infection is often associated with type 2 diabetes. However, the precise mechanism underlying this association is still unclear. Glucose is transported into hepatocytes via glucose transporter 2 (GLUT2). Hepatocytes play a crucial role in maintaining plasma glucose homeostasis via the gluconeogenic and glycolytic pathways. We have been investigating the molecular mechanism of HCV-related type 2 diabetes using HCV RNA replicon cells and HCV J6/JFH1 system. We found that HCV replication down-regulates cell surface expression of GLUT2 at the transcriptional level. We also found that HCV infection promotes hepatic gluconeogenesis in HCV J6/JFH1-infected Huh-7.5 cells. HCV infection transcriptionally up-regulated the genes for phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), the rate-limiting enzymes for hepatic gluconeogenesis. Gene expression of PEPCK and G6Pase was regulated by the transcription factor forkhead box O1 (FoxO1) in HCV-infected cells. Phosphorylation of FoxO1 at Ser319 was markedly diminished in HCV-infected cells, resulting in increased nuclear accumulation of FoxO1. HCV NS5A protein was directly linked with the FoxO1-dependent increased gluconeogenesis. This paperwill discuss the current model of HCV-induced glucose metabolic disorders.FRONTIERS RESEARCH FOUNDATION, 2012, FRONTIERS IN MICROBIOLOGY, 3, English[Refereed]Book review
- 2009, 日本ウイルス学会学術集会プログラム・抄録集, 57thC型肝炎ウイルス感染によるインスリン抵抗性誘導の分子機序について
- 2009, 日本分子生物学会年会講演要旨集, 32nd(Vol.1) (Vol.1)C型肝炎ウイルスコア蛋白質の安定性調節因子E6AP及びPA28γの相互作用解析
- ELSEVIER SCIENCE BV, Dec. 2003, JOURNAL OF CLINICAL VIROLOGY, 28, S52 - S52, EnglishSequence variation of Hepatitis C virus subtype 1B and development of hepatocellular carcinomaSummary international conference
- 第68回日本ウイルス学会学術集会, Nov. 2021, JapaneseHCV-induced ROS/JNK signaling pathway activates the E3 ubiquitin ligase Itch to promote the release of HCV particles via polyubiquitylation of VPS4A.Oral presentation
- 2021 International HBV Meeting, Sep. 2021, EnglishActivation of Nrf2/ARE signaling pathway upon hepatitis B virus infection exerts an inhibitory effect on viral replicationPoster presentation
- the 27th International Symposium on Hepatitis C Virus and Related Viruses, Jul. 2021, EnglishHCV-induced ROS/JNK signaling pathway activates the E3 ubiquitin ligase Itch to promote the release of HCV particles via polyubiquitylation of VPS4APoster presentation
- 17th ISVHLD GHS (Global Hepatitis Summit) 2021, Jun. 2021, EnglishHepatitis B virus X protein activates Nrf2/ARE signaling pathway to suppress viral replicationOral presentation
- 第43回日本分子生物学会年会, Dec. 2020ISGylation of HBx protein confers the pro-viral effects on HBV replication and resistance to interferon (IFN)-responsePoster presentation
- 第43回日本分子生物学会年会, Dec. 2020Screening for anti-hepatitis B virus activity among heterocyclic compounds using HBV-NanoLuc reporter genePoster presentation
- 第43回日本分子生物学会年会, Dec. 2020Annexin 5 participates in the negative regulation of HCV propagationPoster presentation
- The 43rd Annual Meeting of the Molecular Biology Society of Japan, Dec. 2020Hepatitis B virus X protein induces antioxidant response via activation of Nrf2/ARE signaling pathwayPoster presentation
- The 43rd Annual Meeting of the Molecular Biology Society of Japan, Dec. 2020, Japanese, The Molecular Biology Society of Japan, Japan, Domestic conferenceHCV-induced ROS/JNK signaling pathway activates the E3 ubiquitin ligase Itch to promote the release of HCV particles via polyubiquitylation of VPS4A.Poster presentation
- 2018 International HBV Meeting, Oct. 2018, English, Hepatitis B Foundation, シシリー, International conferenceThe role of ISG15-conjugation on the Hepatitis B virus HBx proteinPoster presentation
- 第66回日本ウイルス学会学術集会, Oct. 2018, English, 日本ウイルス学会, 京都, Domestic conferenceHepatitis C virus induces the lysosomal degradation of the HNF-1β transcription factor via chaperone-mediated autophagyOral presentation
- The 25th International Symposium on Hepatitis C Virus and Related Viruses, Oct. 2018, English, Organising Committee of the 25th International Symposium on Hepatitis C Virus and Related Viruses, ダブリン, International conferenceHCV-induced activation of JNK/Itch signaling pathway is involved in the release of infectious viral particles.Oral presentation
- 第66回日本ウイルス学会学術集会, Oct. 2018, English, 日本ウイルス学会, 京都, Domestic conferenceHCV-induced activation of JNK/Itch signaling pathway is involved in the release of infectious viral particles.Public symposium
- The 25th International Symposium on Hepatitis C Virus and Related Viruses, Oct. 2018, English, Organising Committee of the 25th International Symposium on Hepatitis C Virus and Related Viruses, ダブリン, International conferenceDegradation of HCV-induced HNF-1β protein by chaperone-mediated autophagyPoster presentation
- The 25th International Symposium on Hepatitis C Virus and Related Viruses, Oct. 2018, English, Organising Committee of the 25th International Symposium on Hepatitis C Virus and Related Viruses, ダブリン, International conferenceCritical role of NS5A-ISGylation in the efficient HCV RNA replicationPoster presentation
- 第66回日本ウイルス学会学術集会, Oct. 2018, English, 日本ウイルス学会, 京都, Domestic conferenceCritical role of NS5A-ISGylation in the efficient HCV RNA replicationOral presentation
- 第38回近畿腸管微生物研究会, Jun. 2018, Japanese, 近畿腸管微生物研究会, 大阪, Domestic conferenceC型肝炎ウイルスによる脂肪滴形成機構の解析Oral presentation
- Consortium of Biological Sciences 2017, Dec. 2017, English, The Molecular Biology Society of Japan, 神戸, Domestic conferenceCharacterization of deubiquitinating enzyme USP15 inhibitor candidatesPoster presentation
- The 65th Annual Meeting of the Japanese Society for Virology, Oct. 2017, English, Japanese Society for Virology, 大阪, Domestic conferenceNovel equine-like G3 rotavirus strains among children in Surabaya, Indonesia, 2015-2016Poster presentation
- The 65th Annual Meeting of the Japanese Society for Virology, Oct. 2017, English, Japanese Society for Virology, 大阪, Domestic conferenceNorovirus infection in an asymptomatic population in IndonesiaPoster presentation
- The 65th Annual Meeting of the Japanese Society for Virology, Oct. 2017, English, The Japanese Society for Virology, 大阪, Domestic conferenceInterferon-stimulated gene 15 (ISG15) regulates HCV RNA replication via different ISGylation sites on HCV NS5APublic symposium
- The 65th Annual Meeting of the Japanese Society for Virology, Oct. 2017, Japanese, Japanese Society for Virology, 大阪, Domestic conferenceHepatitis C virus NS3/4A protease cleaves SGP20 and promotes lipid droplet growthPoster presentation
- The 65th Annual Meeting of the Japanese Society for Virology, Oct. 2017, English, The Japanese Society for Virology, 大阪, Domestic conferenceHCV infection promotes Itch ubiquitin ligase activity via activation of JNK and modulates release of infectious viral particles.Public symposium
- 2017 International HBV Meeting., Sep. 2017, English, Organising Committee of 2017 International Meeting on Molecular Biology of Hepatitis B Viruses, Washington DC, USA, International conferencePeroxiredoxin 1, a novel HBx-interacting protein, negatively regulates HBV replication through binding to HBV RNA for HBV RNA degradationOral presentation
- The 24th International Symposium on Hepatitis C virus and Related Viruses, Sep. 2017, English, Organising Committee of the 24th International Symposium on Hepatitis C Virus and Related Viruses, Massachusetts, USA, International conferenceInterferon-stimulated gene 15 (ISG15) regulates HCV RNA replication via different ISGylation sites on HCV NS5APoster presentation
- The 24th International Symposium on Hepatitis C Virus and Related Viruses, Sep. 2017, English, Organising Committee of the 24th International Symposium on Hepatitis C Virus and Related Viruses, Washington DC, USA, International conferenceHCV infection promotes Itch ubiquitin ligase activity via activation of JNK and modulates release of infectious viral particles.Poster presentation
- 24th International Symposium on Hepatitis C Virus and Related Viruses, Sep. 2017, English, Organising Committee of the 24th International Symposium on Hepatitis C Virus and Related Viruses, Cape Cod, USA, International conferenceA novel pathway for lipid droplet formation induced by hepatitis C virusPoster presentation
- 2nd Molecular, Cellular, and Life Sciences 2017, Jul. 2017, English, Osaka University and Universitas Airlangga, Surabaya, Indonesia, International conferenceNorovirus and Rotavirus Infections among Hospitalized Children with Acute Gastroenteritis in Surabaya, East Java, IndonesiaOral presentation
- 第37回近畿腸管微生物研究会, Jun. 2017, Japanese, 近畿腸管微生物研究会, 大阪, Domestic conferenceSelective protein degradation via HCV-induced chaperone mediated autophagyOral presentation
- The 39th Annual Meeting of the Molecular Biology Society of Japan, Nov. 2016, English, The Molecular Biology Society of Japan, 横浜, Domestic conferenceScreening for deubiquitinating enzyme USP15 inhibitors and their characterizationPoster presentation
- The 23rd International Symposium on Hepatitis C Virus and Related Viruses, Oct. 2016, English, Organising Committee of the 23rd International Symposium on Hepatitis C Virus and Related Viruses, Kyoto, Japan, International conferenceUpregulation of MAPK phosphatase 3 is involved in HCV-induced dephosphorylation of FoxO1.Poster presentation
- 23rd International Symposium on Hepatitis C Virus and Related Viruses, Oct. 2016, English, Kyoto, International conferenceUpregulation of MAPK phosphatase 3 is involved in HCV-induced dephosphorylation of FoxO1.Poster presentation
- The 64th Annual Meeting of the Japanese Society for Virology, Oct. 2016, English, The Japanese Society for Virology, 札幌, Domestic conferenceTwo different roles of ISG15 in HCV infectionPoster presentation
- The 64th Annual Meeting of the Japanese Society for Virology, Oct. 2016, English, The Japanese Society for Virology, 札幌, Domestic conferencePeroxiredoxin 1, a novel HBx-interacting protein, negatively regulates HBV replication through acceleration of HBV RNA degradation.Public symposium
- The 23rd International symposium on Hepatitis C Virus and Related Viruses, Oct. 2016, English, Organising Committee of the 23rd International Symposium on Hepatitis C Virus and Related Viruses, Kyoto, Japan, International conferenceMolecular mechanism of HCV-induced lysosomal degradation of HNF-1α proteinPoster presentation
- The 64th Annual Meeting of the Japanese Society for Virology, Oct. 2016, English, The Japanese Society for Virology, 札幌, Domestic conferenceC型肝炎ウイルスによるHNF-1α蛋白質の選択的分解機構Oral presentation
- 23rd International Symposium on Hepatitis C Virus and Related Viruses, Oct. 2016, English, Kyoto, International conferenceAntiviral activities of the scorpine-like peptide Smp 76 isolated from Scorpio maurus palmatus against dengue virus and hapatitis C virus.Poster presentation
- 23rd International Symposium on Hepatitis C Virus and Related Viruses, Oct. 2016, English, Kyoto, International conferenceAnti-hepatitis C virus compounds from Ruta angustifolia mediate a synergistic effect in combination with current direct-acting antiviral agents.Poster presentation
- 2016 International HBV Meeting., Sep. 2016, English, Organising Committee of 2016 International Meeting on Molecular Biology of Hepatitis B Viruses, Seoul, Korea, International conferencePeroxiredoxin 1 is a guardian of the host cell against HBV infection.Poster presentation
- 2016 International HBV Meeting. The Molecular Biology of Hepatitis B Viruses, Sep. 2016, English, Seoul, International conferencePeroxiredoxin 1 is a guardian of the host cell against HBV infection.Poster presentation
- Intenational seminar Global Strategy to Combat Emerging Infectious Diseases in Borderless Era, Aug. 2016, English, Faculty of Medicine and Institute of Tropical Disease, Universitas Airlangga, スラバヤ, インドネシア, International conferenceOccurrence of norovirus infection in an asymptomatic population in IndonesiaOral presentation
- Intenational seminar Global Strategy to Combat Emerging Infectious Diseases in Borderless Era, Aug. 2016, English, Faculty of Medicine and Institute of Tropical Disease, Universitas Airlangga, スラバヤ, インドネシア, International conferenceA preliminary survey of norovirus and rotavirus infections among children in Surabaya, Indonesia[Invited]Invited oral presentation
- The 11th Japan-China International Conference of Virology, Jul. 2016, English, 日中国際ウイルス学会世話人, 観音時, International conferenceUp-regulation of MAPK phosphatase 3 is involved in HCV-induced dephosphorylation of FoxO1.Oral presentation
- The 11th Japan-China International Conference of Virology, Jul. 2016, English, Organising Committee of The 11th Japan-China International Conference of Virology, Kanonji, Japan, International conferenceUp-regulation of MAPK phosphatase 3 is involved in HCV-induced dephosphorylaion of FoxO1.Oral presentation
- The 4th JAPAN-TAIWAN Research Symposium on Hepatitis B Virus, Apr. 2016, English, Organising Committee of The 4th JAPAN-TAIWAN Research Symposium on Hepatitis B Virus, Taipei, Taiwan, International conferencePeroxiredoxin 1 negatively regulates hepatitis B virus replication through interaction with HBx.Public symposium
- 4th JAPAN-TAIWAN Research Symposium on Hepatitis B Virus, Apr. 2016, English, Taiwan Association for the study of the Liver, Taipei, Taiwan, International conferencePeroxiredoxin 1 negatively regulates hepatitis B virus replication through interaction with HBx.[Invited]Invited oral presentation
- BMB2015 Biochemistry and Molecular Biology, Dec. 2015, Japanese, 日本分子生物学会、日本生化学会, 神戸, Domestic conferenceScreening for small molecule inhibitors of deubiquitinating enzyme USP15Poster presentation
- The 63rd Annual Meeting of the Japanese Society for Virology, Nov. 2015, English, The Japanese Society for Virology, 福岡, Domestic conferencePeroxiredoxin 1, a novel binding partner of HBx, is a negative regulator of HBV replicationOral presentation
- The 63rd Annual Meeting of the Japanese Society for Virology, Nov. 2015, English, The Japanese Society for Virology, 福岡, Domestic conferenceA novel pathway for lipid droplet formation induced by hepatitis C virusOral presentation
- 22th International Symposium on Hepatitis C Virus and Related Viruses, Oct. 2015, English, Organising Committee of the22th International Symposium on Hepatitis C Virus and Related Viruses, Strasbourg, France, International conferencePhysical and functional interaction between hepatitis C virus NS5A protein and ovarian tumor protein deubiquitinase 7BPoster presentation
- 2015 International Meeting on Molecular Biology of Hepatitis B Viruses, Oct. 2015, English, Organising Committee of 2015 International Meeting on Molecular Biology of Hepatitis B Viruses, Bad Nauheim, Germany, International conferencePeroxiredoxin 1 negatively regulates hepatitis B virus replication through interaction with HBxPoster presentation
- 22th International Symposium on Hepatitis C Virus and Related Viruses, Oct. 2015, English, Organising Committee of the22th International Symposium on Hepatitis C Virus and Related Viruses, Strasbourg, France, International conferenceMAPK phosphatase 3 is involved in HCV-induced dephosphorylaion of FoxO1Poster presentation
- 22nd International symposium on Hepatitis C Virus and Related Viruses, Oct. 2015, English, HCV2015 Organising committee, Strasbourg, France, International conferenceA single-amino-acid mutation in hepatitis C virus NS5A disrupts physical and functional interaction with the transcription factor HNF-1αPoster presentation
- 第62回日本ウイルス学会学術集会., Nov. 2014, Japanese, 日本ウイルス学会, 横浜, Domestic conferenceＣ型肝炎ウイルスによるミトコンドリア介在性アポトーシス誘導機構の解明.Oral presentation
- 第62回日本ウイルス学会学術集会., Nov. 2014, English, 日本ウイルス学会, 横浜, Domestic conferenceHCV NS5A protein physically and functionally interacts with an OTU deubiquitinase.Poster presentation
- 第62回日本ウイルス学会学術集会., Nov. 2014, English, 日本ウイルス学会, 横浜, Domestic conferenceHCV NS5A interacts with lysine methyltransferase SMYD3 and transcriptionally activates the protein disulfide isomerase gene AGR3.Oral presentation
- 第62回日本ウイルス学会学術集会., Nov. 2014, Japanese, 日本ウイルス学会, 横浜, Domestic conferenceC型肝炎ウイルス感染によるTGF-βスーパーファミリーにおけるSmad2/3とSmad1/5/9経路の脱制御とその分子機序の解明.Oral presentation
- 第62回日本ウイルス学会学術集会., Nov. 2014, Japanese, 日本ウイルス学会, 横浜, Domestic conferenceC型肝炎ウイルス感染によるHepatocyte nuclear factor (HNF) -1α蛋白質の選択的分解機構.Poster presentation
- 第37回日本分子生物学会年会, Nov. 2014, Japanese, 日本分子生物学会, 横浜, Domestic conferenceC型肝炎ウイルスによるHNF-1α蛋白質の選択的分解機構の解析Poster presentation
- 第62回日本ウイルス学会学術集会., Nov. 2014, Japanese, 日本ウイルス学会, 横浜, Domestic conferenceB型肝炎ウイルスXタンパク質の新規結合因子抗酸化酵素ペルオキシレドキシン1(Prdx1)の同定と機能解析.Oral presentation
- 第62回日本ウイルス学会学術集会., Nov. 2014, Japanese, 日本ウイルス学会, 横浜, Domestic conferenceB型肝炎ウイルスXタンパク質とヒストンメチル基転移酵素SMYD3の相互作用の解析.Poster presentation
- 21th International Symposium on Hepatitis C Virus and Related Viruses., Sep. 2014, English, Organising Committee of the 21th International Symposium on Hepatitis C Virus and Related Viruses, Banff, Banff, International conferencePhysical and functional interaction between an OTU deubiquitinase and HCV NS5A protein.Poster presentation
- 2014 International Meeting on Molecular Biology of Hepatitis B Viruses., Sep. 2014, English, Organising Committee of the 2014 International Meeting on Molecular Biology of Hepatitis B Viruses., Los Angeles, International conferencePeroxiredoxin 1 is a novel binding partner of HBx and a positive regulator of hepatitis B virus transcription.Oral presentation
- 21th International Symposium on Hepatitis C Virus and Related Viruses., Sep. 2014, English, Organising Committee of the 21th International Symposium on Hepatitis C Virus and Related Viruses, Banff, Banff, International conferenceNS5B induces up-regulation of the BH3-only protein, BIK, essential for the Hepatitis C virus RNA replication and viral release.Poster presentation
- 2014 International Meeting on Molecular Biology of Hepatitis B Viruses., Sep. 2014, English, Organising Committee of the 2014 International Meeting on Molecular Biology of Hepatitis B Viruses., Los Angeles, International conferenceInteraction between HBx and lysine methyltransferase SMYD3, a novel HBx-interacting protein.Poster presentation
- 21th International Symposium on Hepatitis C Virus and Related Viruses., Sep. 2014, English, Organising Committee of the 21th International Symposium on Hepatitis C Virus and Related Viruses, Banff, Canada, International conferenceHCV induces Bim/Bax-mediated apoptosis through the ROS/JNK signaling pathway.Poster presentation
- 21th International Symposium on Hepatitis C Virus and Related Viruses., Sep. 2014, English, Organising Committee of the 21th International Symposium on Hepatitis C Virus and Related Viruses, Banff, Banff, International conferenceHCV dysregulates Smad2/3- and Smad1/5-signaling pathways of the TGF-β superfamily.Poster presentation
- 21th International Symposium on Hepatitis C Virus and Related Viruses., Sep. 2014, English, Organising Committee of the 21th International Symposium on Hepatitis C Virus and Related Viruses, Banff, Banff, International conferenceDeterminants of specific interaction between hepatitis C virus NS5A and HNF-1α protein.Poster presentation
- 2014 TASL-Japan Hepatitis B workshop., Apr. 2014, English, Taiwan Association for the study of the Liver, Taipei, Taiwan, International conferenceA tandem affinity purification analysis of HBx-interacting proteins and identification of two novel interactors Prdx1 and SMYD3.[Invited]Invited oral presentation
- 第61回日本ウイルス学会学術集会, Nov. 2013, Japanese, 日本ウイルス学会, 神戸, Domestic conferenceＣ型肝炎ウイルス感染によるBax活性化の分子機序の解析Oral presentation
- 第61回日本ウイルス学会学術集会, Nov. 2013, Japanese, 日本ウイルス学会, 神戸, Domestic conferenceHCV感染による糖代謝障害の分子機序[Invited]Nominated symposium
- 第61回日本ウイルス学会学術集会, Nov. 2013, Japanese, 日本ウイルス学会, 神戸, Domestic conferenceHCV NS5AとHepatocyte nuclear factor (HNF) -1αの相互作用と病態生理Poster presentation
- 第61回日本ウイルス学会学術集会, Nov. 2013, Japanese, 日本ウイルス学会, 神戸, Domestic conferenceC型肝炎ウイルス非構造蛋白質NS5AにおけるFyn-SH2ドメインとの結合に重要なチロシン残基同定の試みPoster presentation
- 第61回日本ウイルス学会学術集会, Nov. 2013, Japanese, 日本ウイルス学会, 神戸, Domestic conferenceC型肝炎ウイルス感染によるSmad1/Smad5経路の脱制御とその分子機序についてPoster presentation
- 第61回日本ウイルス学会学術集会, Nov. 2013, Japanese, 日本ウイルス学会, 神戸, Domestic conferenceChlorophill分解産物Pheophorbide a、Chlorin e6及び半合成誘導体Mono-L-aspartyl chlorin e6 (NPe6) はC型肝炎ウイルス増殖を阻害するPoster presentation
- 20th International Symposium on Hepatitis C Virus and Related Viruses., Oct. 2013, English, Organising Committee of the20th International Symposium on Hepatitis C Virus and Related Viruses, Melbourne, Australia, International conferenceRegulation of hepatocyte nuclear factor 1α by hepatitis C virus NS5A proteinPoster presentation
- 20th International Symposium on Hepatitis C Virus and Related Viruses., Oct. 2013, English, Organising Committee of the20th International Symposium on Hepatitis C Virus and Related Viruses, Melbourne, Australia, International conferenceHCV upregulates Bim through ROS/JNK signaling pathway leading to Bax-mediated apoptosisOral presentation
- 20th International Symposium on Hepatitis C Virus and Related Viruses., Oct. 2013, English, Organising Committee of the20th International Symposium on Hepatitis C Virus and Related Viruses, Melbourne, Australia, International conferenceHCV NS5A interacts with SMYD3 and upregulates SMYD3-mediated expression of AGR3 mRNAPoster presentation
- 20th International Symposium on Hepatitis C Virus and Related Viruses., Oct. 2013, English, Organising Committee of the20th International Symposium on Hepatitis C Virus and Related Viruses, Melbourne, Australia, International conferenceDevelopment of a prophylactic and therapeutic vaccine against Hepatitis C virusPoster presentation
- 20th International Symposium on Hepatitis C Virus and Related Viruses., Oct. 2013, English, Organising Committee of the20th International Symposium on Hepatitis C Virus and Related Viruses, Melbourne, Australia, International conferenceAntiviral activity of chlorophyll derivatives, pheophorbide a, chlorin e6 and mono-L-aspartyl chlorin e6 (NPe6), against hepatitis C virusPoster presentation
- The 35th Annual Meeting of the Molecular Biology Society of Japan, Dec. 2012, Japanese, The Molecular Biology Society of Japan, 福岡, Hepatitis C virus (HCV) infection causes not only intrahepatic diseases but also extrahepatic manifestations, including type 2 diabetes. We previously reported that HCV replication suppresses cellular glucose uptake by down-regulation of cell surface expr, Domestic conferenceHepatitis C virus infection suppresses GLUT2 gene expression via down-regulation of hepatocyte nuclear factor 1αOral presentation
- 第35回日本分子生物学会年会, Dec. 2012, Japanese, 日本分子生物学会, 福岡, C 型肝炎ウイルス（HCV）は肝細胞以外にB 細胞にも感染し、慢性肝炎の合併症として混合型クリオグロブリン血症や非ホジキンリンパ腫を引き起こすが、発症機序は不明である。今回、我々は、HCV 非構造蛋白質NS5A のB 細胞に対する影響を検討するため、エピトープタグを付加したNS5A を安定発現するB 細胞株を樹立した。この細胞を蛋白質チロシンフォスファターゼ阻害剤であるPervanadate で処理したところ、NS5A がチロシンリン酸化されることを見出した。チロシンリン酸化蛋白質と結合する蛋白質ドメインとしてSrc homology 2 (SH2) が知られており、B 細胞受容体を介するシグナル伝達経路中にもこのドメインを持つ蛋白質がいくつかある。そこで、これらのNS5A 結合能をプルダウンアッセイ法で検討し、Src ファミリーキナーゼFyn 由来, Domestic conferenceC 型肝炎ウイルスNS5A 蛋白質のSrc homology 2/3 ドメイン結合能とB 細胞での発現によるSrc ファミリーキナーゼFyn の活性化Poster presentation
- 第60回日本ウイルス学会学術集会, Nov. 2012, Japanese, 日本ウイルス学会, 大阪, 【目的と意義】C型肝炎ウイルス（HCV）の慢性感染は2型糖尿病の発症に関与することが知られているが、その分子機序は不明な点が多い。我々は昨年本会において、1) HCV感染が酸化ストレス誘導を介してJNKを活性化し、糖新生制御因子である転写因子FoxO1のリン酸化を抑制し、活性化状態を持続させること、2) 活性化されたFoxO1により、糖新生律速酵素遺伝子群の転写が促進され、肝細胞の糖新生が亢進することを報告した。HCVによるFoxO1の脱制御（強制持続的活性化）において、通常の糖新生制御経路インスリンレセプター–PI3キナーゼ–Akt/PKBシグナル経路の制御を受けないことから、それ以外のFoxO1リン酸化・脱リン酸化経路の関与が強く示唆された。近年、FoxO1の脱リン酸化の分子機序として、MAPK phosphatase-3 (MKP3)及びpro, Domestic conferenceＣ型肝炎ウイルス感染による転写因子FoxO1脱リン酸化の分子機序の解析Oral presentation
- 第65回日本細菌学会関西支部総会, Nov. 2012, Japanese, 日本細菌学会関西支部, 神戸, 【目的】Ｃ型肝炎ウイルス (HCV)は肝臓に慢性の炎症を起こし、高率に肝細胞癌を引き起こすが、その一方で、Ｃ型慢性肝炎患者において肝細胞のアポトーシスの亢進が認められている。我々は、Huh7.5細胞を用いたHCV J6/JFH1感染系で、HCV感染がBaxの活性化を引き起こし、ミトコンドリア障害を介したアポトーシスを誘導すること、及びreactive oxygen species (ROS)の産生を介してc-Jun N-terminal kinase (JNK)経路を活性化することを報告した。一方、アポトーシス促進因子であるBim は、Baxの活性化を促進することが知られている。そこで本研究では、HCV感染によるBax活性化の分子機構を明らかにすることを目的として、ROS/JNK経路及びBimの関与について検討を行った。【方法】HCV J6/JFH1, Domestic conferenceＣ型肝炎ウイルス感染によるBax活性化の分子機序の解析Oral presentation
- The 10th JSH Single Topic Conference, Nov. 2012, English, The Japan Society of Hepatology, 東京, Hepatitis C virus (HCV) infection causes not only intrahepatic diseases but also extrahepatic manifestations, including metabolic disorders. Chronic HCV infection is often associated with type 2 diabetes. However, the precise mechanism underlying this ass, Domestic conferenceMolecular mechanism of hepatitis C virus-induced glucose metabolism disorder[Invited]Invited oral presentation
- 第60回日本ウイルス学会学術集会, Nov. 2012, Japanese, 日本ウイルス学会, 大阪, 【目的と意義】Ｃ型肝炎ウイルス（HCV）慢性感染者は、日本で約200万人、全世界で約1億7,000万～2億人と推定されている。最近認可されたより治療効果の高い三者併用療法でも、30%近い症例で完全治癒が望めず、HCV 治療ワクチンと新たな感染者発生を防止する予防ワクチンの開発が強く求められている。我々は、HCVに対する予防ワクチンと治療ワクチンを開発する目的で、HCVのエンベロープタンパク質及び非構造タンパク質の一部をコードする遺伝子領域を組み込んだ発現プラスミドを数種類作製し、DNAワクチンとしてマウスに接種して、中和抗体産生及び細胞性免疫誘導を検討した。【材料と方法】1）DNAワクチン：HCV(1b型)のエンベロープタンパク質(E2)及び非構造タンパク質の一部(NS3)をコードする遺伝子領域を組み込んだ発現プラスミドを数種類作製した。2）マウス免, Domestic conferenceC型肝炎ウイルスに対する予防および治療ワクチン開発に関する研究Oral presentation
- 第60回日本ウイルス学会学術集会, Nov. 2012, Japanese, 日本ウイルス学会, 大阪, 【目的と意義】C型肝炎ウイルス(HCV)感染が高率にII型糖尿病を合併することが知られているが、詳しい発症機序はいまだ明らかではない。昨年、我々は本会において、HCV感染により細胞内の転写因子Hepatocyte nuclear factor-1α (HNF-1α)の転写が抑制されることが、GLUT2遺伝子転写抑制の原因の一つであることを報告した。その後の検討から、HCV感染によりHNF-1α蛋白質の分解誘導が引き起こされることを見出したので、その分子機序について報告する。【材料と方法】1) HCVによるHNF-1α蛋白質量の減少に蛋白質分解が関与するかを検討するために、各種阻害剤の影響を解析した。ヒト肝がん細胞株Huh-7.5細胞にHCV J6/JFH-1を感染させ、回収12時間前にライソソーム阻害剤またはプロテアソーム阻害剤を投与し、内在性HNF, Domestic conferenceC型肝炎ウイルスによるGLUT2遺伝子発現抑制の分子機構Oral presentation
- 第60回日本ウイルス学会学術集会, Nov. 2012, Japanese, 日本ウイルス学会, 大阪, 【目的と意義】C型肝炎ウイルス（HCV）は高率に持続感染し、慢性肝炎、肝硬変、肝細胞癌を引き起こす。HCVの非構造蛋白質の一つであるNS5Aはウイルスゲノム複製複合体の主構成因子であるだけでなく、HCVの病原性、ウイルス粒子産生、及びインターフェロン抵抗性に重要な役割を果たしている。しかしながら、その詳細な分子機序は未だ明らかにされていない。本研究では、NS5Aと結合する新規宿主因子を同定し、NS5Aによる新たな病原性発現機構を明らかにすることを目的とした。【材料と方法】 C-末端にMyc-His6タグを付加したNS5AをHuh-7細胞に発現させ、2段階タンデムタグ精製法と質量分析法により、NS5Aと結合する新規宿主因子を探索した。NS5A一過性発現Huh-7.5細胞およびHCV全長レプリコン細胞、HCV J6/JFH1感染細胞を用い、NS5Aと新規, Domestic conferenceC型肝炎ウイルスNS5A蛋白質の新規結合因子ヒストンメチル基転移酵素SMYD3の同定と機能解析Oral presentation
- 19th International Symposium on Hepatitis C Virus and Related Viruses., Oct. 2012, English, Organising Committee of the19th International Symposium on Hepatitis C Virus and Related Viruses, Venice, Italy, Chronic hepatitis C virus (HCV) infection is often associated with type 2 diabetes. We have recently reported that HCV infection promotes hepatic gluconeogenesis through a transcription factor forkhead box O1 (FoxO1)-dependent pathway. We demonstrated that HCV infection induced c-Jun N-terminal kinase (JNK) activation via increased mitochondrial reactive oxygen species (ROS) pr, International conferenceUp-regulation of MAPK phosphatase 3 is involved in HCV-induced suppression of FoxO1 phosphorylation.Oral presentation
- 19th International Symposium on Hepatitis C Virus and Related Viruses., Oct. 2012, English, Organising Committee of the19th International Symposium on Hepatitis C Virus and Related Viruses, Venice, Italy, Hepatitis C virus (HCV) infection is associated with the development of hepatocellular carcinoma (HCC). Increasing experimental evidence suggests that HCV contributes to HCC by directly modulating pathways that promote the malignant transformation of hepatocytes. HCV non-structural protein 5A (NS5A) is associated with a wide range of cellular proteins involved in cellular signa, International conferenceIdentification and characterization of a novel NS5A-interacting protein, SMYD3.Poster presentation
- 19th International Symposium on Hepatitis C Virus and Related Viruses., Oct. 2012, English, Organising Committee of the19th International Symposium on Hepatitis C Virus and Related Viruses, Venice, Italy, Hepatitis C virus (HCV) infection causes not only intrahepatic diseases but also extrahepatic manifestations, including metabolic disorders and autoimmune diseases. A number of studies have shown that HCV infection often predisposes the host towards type 2 diabetes. We previously reported that HCV replication suppresses cellular glucose uptake through down-regulation of cell su, International conferenceHCV infection induces lysosomal degradation of hepatocyte nuclear factor 1α via interaction with HCV NS5A protein.Poster presentation
- 19th International Symposium on Hepatitis C Virus and Related Viruses., Oct. 2012, English, Organising Committee of the19th International Symposium on Hepatitis C Virus and Related Viruses, Venice, Italy, An estimated 170 to 200 million individuals are chronically infected with Hepatitis C virus (HCV) worldwide and 3 to 4 million individuals are newly infected each year. There is currently no vaccine available to protect against HCV. Considering the limited efficacy and high cost of treatment for chronic Hepatitis C, preventive and therapeutic vaccines against HCV are thus much, International conferenceDevelopment of therapeutic and preventive vaccines against Hepatitis C virus.Poster presentation
- International Symposium on Hepatitis C Virus and Related Viruses, Oct. 2012, English, Organising Committee of the19th International Symposium on Hepatitis C Virus and Related Viruses, Venice, An estimated 170 to 200 million individuals are chronically infected with Hepatitis C virus (HCV) worldwide and 3 to 4 million individuals are newly infected each year. There is currently no vaccine available to protect against HCV. Considering the limited efficacy and high cost of treatment for chronic Hepatitis C, preventive and therapeutic vaccines against HCV are thus much, International conferenceDevelopment of therapeutic and preventive vaccines against hepatitis C virusPoster presentation
- 第34回日本分子生物学会年会, Dec. 2011, Japanese, 日本分子生物学会, 横浜, Domestic conferenceIdentification of an E3 ubiquitin ligase that mediates ubiquitylation of hepatitis C virus NS5A proteinPoster presentation
- 第64 回日本細菌学会関西支部総会, Nov. 2011, Japanese, 第64 回日本細菌学会関西支部, 大阪, Domestic conferenceC型肝炎ウイルスによるGLUT2遺伝子発現抑制の分子機構Oral presentation
- 第64 回日本細菌学会関西支部総会, Nov. 2011, Japanese, 第64 回日本細菌学会関西支部, 大阪, Domestic conferenceC型肝炎ウイルスNS5Aの新規結合タンパク質であるヒストンメチル基転移酵素SMYD3の同定Oral presentation
- 第15回日本肝臓学会大会, Oct. 2011, Japanese, 日本肝臓学会, 福岡, Domestic conferenceC型肝炎ウイルスのNS3変異Y56/Q86及びコア蛋白変異Q70は高発癌性ウイルス株の指標となるPoster presentation
- International Union of Microbiological Societies 2011 Congress, Sep. 2011, English, Federation of Microbiological Societies of Japan, 札幌, International conferencePolymorphisms of serine protease-domain of NS3 and Core protein of hepatitis C virus genotype 1b associate with hepatocellular carcinoma developmentPoster presentation
- International Union of Microbiological Societies 2011 Congress, Sep. 2011, English, Federation of Microbiological Societies of Japan, 札幌, International conferenceMolecular mechanisms involved in HCV infection-induced hepatic gluconeogenesisPoster presentation
- 18th International Symposium on Hepatitis C Virus and Related Viruses, Sep. 2011, English, Organising Committee of the18th International Symposium on Hepatitis C Virus and Related Viruses, Seattle, U.S.A., International conferenceIdentification of an E3 ubiquitinin ligase that targets hepatitis C virus NS5A protein for ubiquitylationPoster presentation
- International Union of Microbiological Societies 2011 Congress, Sep. 2011, English, Federation of Microbiological Societies of Japan, 札幌, International conferenceIdentification of an E3 ubiquitinin ligase that mediated ubiquitylation of hepatitis C virus NS5A proteinOral presentation
- International Union of Microbiological Societies 2011 Congress, Sep. 2011, English, Federation of Microbiological Societies of Japan, 札幌, International conferenceHepatitis C virus infection suppresses glucose transporter 2 gene expression by downregulation of hepatocyte nuclear factor 1α.Poster presentation
- 18th International Symposium on Hepatitis C Virus and Related Viruses, Sep. 2011, English, Organising Committee of the18th International Symposium on Hepatitis C Virus and Related Viruses, Seattle, U.S.A., International conferenceHepatitis C virus infection promotes hepatic gluconeogenesis through an NS5A-mediated, FoxO1-dependent pathwayPoster presentation
- 18th International Symposium on Hepatitis C Virus and Related Viruses, Sep. 2011, English, Organising Committee of the18th International Symposium on Hepatitis C Virus and Related Viruses, Seattle, U.S.A., International conferenceHCV-induced suppression of glucose transporter 2 gene expression via downregulation of hepatocyte nuclear factor 1α.Poster presentation
- 18th International Symposium on Hepatitis C Virus and Related Viruses, Sep. 2011, English, Organising Committee of the18th International Symposium on Hepatitis C Virus and Related Viruses, Seattle, U.S.A., International conferenceDevelopment of hepatocellular carcinoma in NS3 transgenic micePoster presentation
- 第47回日本肝臓学会総会, Jun. 2011, Japanese, 日本肝臓学会, 東京, Domestic conferenceC型肝炎ウイルスは酸化ストレスを介して糖新生を亢進し糖尿病発症に関与するOral presentation
- 第33回日本分子生物学会, Dec. 2010, Japanese, 日本分子生物学会, 神戸, Domestic conference型肝炎ウイルスNS5Aに結合するユビキチンリガーゼの同定Poster presentation
- 第58回日本ウイルス学会学術集会, Nov. 2010, Japanese, 日本ウイルス学会, 徳島, Domestic conference慢性C型肝炎患者血清および非感染者血清を用いたＣ型肝炎ウイルス中和感受性決定部位の検討Poster presentation
- 第58回日本ウイルス学会学術集会, Nov. 2010, Japanese, 日本ウイルス学会, 徳島, Domestic conference糖代謝に及ぼすC型肝炎ウイルスの影響及びその分子機序の解析Oral presentation
- 第58回日本ウイルス学会学術集会, Nov. 2010, Japanese, 日本ウイルス学会, 徳島, Domestic conferenceHCVによる糖代謝障害の分子機序Public symposium
- 第58回日本ウイルス学会学術集会, Nov. 2010, Japanese, 日本ウイルス学会, 徳島, Domestic conferenceHCV genotype 2aおよび2bのNS5A多様性はペグインターフェロン/リバビリン併用療法の治療効果と相関するOral presentation
- 第63回日本細菌学会関西支部総会, Nov. 2010, Japanese, 日本細菌学会関西支部, 大阪, Domestic conferenceC型肝炎ウイルス感染は転写因子FoxO1を介した糖新生を誘導するOral presentation
- 17th International meeting on hepatitis C virus and related viruses, Sep. 2010, English, Organising Committee of the17th International Meeting on Hepatitis C Virus and Related Viruses, Yokohama, Japan, International conferenceSequence heterogeneity of NS5A of HCV genotypes 2a and 2b affects RVR and SVR to PEG-IFN/RBV combination therapyPoster presentation
- 17th International meeting on hepatitis C virus and related viruses, Sep. 2010, English, Organising Committee of the17th International Meeting on Hepatitis C Virus and Related Viruses, Yokohama, Japan, International conferenceMolecular mechanism of HCV-induced suppression of glucose transporter (GLUT) 2 expressionPoster presentation
- 17th International meeting on hepatitis C virus and related viruses, Sep. 2010, English, Organising Committee of the17th International Meeting on Hepatitis C Virus and Related Viruses, Yokohama, Japan, International conferenceIdentification of an amino acid residue that determines sensitivity to virus neutralization by nonspecific inhibitors and specific neutralizing antibodies in human seraPoster presentation
- 17th International meeting on hepatitis C virus and related viruses, Sep. 2010, English, Organising Committee of the17th International Meeting on Hepatitis C Virus and Related Viruses, Yokohama, Japan, International conferenceHCV-induced generation of reactive oxygen species leads to Bax-mediated apoptosis through activation of the c-Jun NH2-terminal kinase signaling pathwayPoster presentation
- 第32回日本分子生物学会年会, Dec. 2009, Japanese, 日本分子生物学会, 横浜, Domestic conferenceC型肝炎ウイルスコア蛋白質の安定性調節因子E6AP及びPA28γの相互作用解析Poster presentation
- 第32回日本分子生物学会年会, Dec. 2009, Japanese, 日本分子生物学会, 横浜, Domestic conferenceC型肝炎ウイルスNS5A蛋白質と相互作用する細胞性キナーゼの解析Poster presentation
- 第57回日本ウイルス学会学術集会, Oct. 2009, Japanese, 日本ウイルス学会, 東京, Domestic conferenceグルコーストランスポーターGLUT2の転写制御に及ぼすC型肝炎ウイルスの影響.Poster presentation
- 16th International symposium on hepatitis C virus and related viruses, Oct. 2009, English, Organising Committee of the16th International Meeting on Hepatitis C Virus and Related Viruses, ニース, フランス, International conferenceMolecular mechanisms involved in HCV infection-associated predisposition of type 2 diabetes.Oral presentation
- 第57回日本ウイルス学会学術集会, Oct. 2009, Japanese, 日本ウイルス学会, 東京, Domestic conferenceC型肝炎ウイルス増殖に及ぼすエストラジオールの影響に関する検討.Poster presentation
- 第57回日本ウイルス学会学術集会, Oct. 2009, Japanese, 日本ウイルス学会, 東京, Domestic conferenceC型肝炎ウイルス感染によるインスリン抵抗性誘導の分子機序について.Oral presentation
- 第57回日本ウイルス学会学術集会, Oct. 2009, Japanese, 日本ウイルス学会, 東京, Domestic conferenceC型肝炎ウイルスによるBax活性化の分子機構の解析Oral presentation
- 16th International symposium on hepatitis C virus and related viruses, Oct. 2009, English, Organising Committee of the16th International Meeting on Hepatitis C Virus and Related Viruses, ニース, フランス, International conferenceActivation of JNK, but not p38 MAPK, is involved in HCV-induced, Bax-mediated apoptosis.Poster presentation
- 第60回日本細菌学会関西支部総会, Nov. 2007, Japanese, 日本細菌学会関西支部, 大阪, Domestic conference高感染力価のHCV J6/JFH-1株の産生とウイルス細胞変性効果の解析Oral presentation
- 第55回日本ウイルス学会学術集会, Oct. 2007, Japanese, 日本ウイルス学会, 札幌, Domestic conferenceＣ型肝炎ウイルス感染による細胞変性効果の分子機序の解析Oral presentation
- 第55回日本ウイルス学会学術集会, Oct. 2007, Japanese, 日本ウイルス学会, 札幌, Domestic conference高感染力価のHCV J6/JFH-1株の産生とウイルス細胞変性効果(CPE)の解析Poster presentation
- 14thInternationalMeetingonHepatitisCVirusandRelatedViruses, Sep. 2007, English, Organising Committee of the14th International Meeting on Hepatitis C Virus and Related Viruses, グラスゴー, イギリス, International conferenceHepatitis C virus infection induces apoptosis via a mitochondrial-related caspase pathwayPoster presentation
- 14thInternationalMeetingonHepatitisCVirusandRelatedViruses, Sep. 2007, English, Organising Committee of the14th International Meeting on Hepatitis C Virus and Related Viruses, グラスゴー, イギリス, International conferenceHCV with high replication/release capacity in Huh-7.5 cell cultures obtained by a simple, rapid method exhibits two distinct types of cytopathic effectPoster presentation
- 17th Asian Pacific Association for the study of the liver, Mar. 2007, English, Organising Committee of the 17th Asian Pacific Association for the study of the liver, 京都, International conferenceThe possible role for the NS5A and NS3/4A of hepatitis C virus in cell injury and hepatocarcinogenesis.Oral presentation
■ Research Themes
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, Apr. 2021 - Mar. 2024, Principal investigatorAnalysis of the effect of Nrf2-Prdx1 pathway on the degradation of hepatitis B virus RNAB型肝炎ウイルス(HBV)は、ヒト肝細胞に感染し、慢性肝炎、肝硬変、肝細胞癌などを含む肝疾患を引き起こすが、現時点において、根本的な治療法は確立されておらず、HBVを体内から完全に排除することは不可能とされている。本研究では、申請者が世界に先駆けて見出した、「HBVのXタンパク質 (HBx)の新規宿主結合因子Peroxiredoxin 1 (Prdx1)を介したHBV RNAの新規分解機構」のより詳細なメカニズムを明らかにすることにより、HBV関連疾患に対する新たな治療戦略シーズへの可能性を提示することを目的としている。 申請者は以前、Prdx1の発現を誘導する転写因子Nrf2の過剰発現がHBV増殖を有意に抑制することを見出している。そこで、令和3年度は、HBV感染によるNrf2-Prdx1経路の活性化の誘導機構について詳細に検討し、以下の成果を得た。(1) HBxタンパク質はNrf2タンパク質の安定化および核への移行を促進させることが明らかとなった。(2) 酸化ストレスセンサーであるKeap1はdouble-glycine repeat (DGR)ドメインを介してHBxと相互作用することが明らかとなった。(3) Nrf2はHBV core promoterに結合し、core promoter活性を阻害することが明らかとなった。以上のことから、HBV感染細胞において、Keap1はHBxと相互作用することにより、Nrf2の活性化を誘導し、活性化したNrf2はHBV core promoter活性を阻害することでHBV増殖を抑制すると考えられた。 これらの成果は、本研究の目的達成に向けた重要な知見であると考える。
- 学術研究助成基金助成金／基盤研究(C), Apr. 2012 - Mar. 2015Competitive research funding
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Kobe University, Apr. 2011 - Mar. 2014Hepatitis C Virus (HCV) NS5A is a multifunctional protein involved in HCV life cycle and HCV-induced liver pathologies. To elucidate the role of NS5A in HCV pathogenesis, we searched for host proteins interacting with NS5A by tandem affinity purification and identified a novel NS5A-interacting protein, SET and MYND domain-containing 3 (SMYD3), a histone methyltransferase involved in the proliferation of cancer cells. The interaction of NS5A with SMYD3 was confirmed by co-immunoprecipitation, proximity ligation assay, and confocal microscopy. NS5A predominately colocalized with SMYD3 in the cytoplasm. Moreover, the interaction between NS5A and SMYD3 transcriptionally activated AGR3, which is involved in breast, prostate and ovary tumorigenesis and belongs to the family of protein disulfide isomerases. Taken together, these data suggest that the interaction between NS5A and SMYD3 causes hepatocytic dysfunction through upregulation of AGR3 expression.Competitive research funding
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Young Scientists (B), Kobe University, Apr. 2009 - Mar. 2011In this study, by using the J6/JFH1 strain of HCV and Huh7.5 cells, I demonstrated that HCV-induced ROS production triggers JNK activation, which leads to Bax activation and apoptosis. I also found that HCV core plays a role in HCV-induced apoptosis.Competitive research funding
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Young Scientists (B), Kobe University, Apr. 2007 - Mar. 2009本研究では、HCV clone J6/JFH1株とHuh7.5細胞を用いて、HCV感染初期における肝細胞の細胞障害の分子機序について検討した。HCV感染により、ミトコンドリアにおける活性酸素種(ROS)の産生が増加し、アポトーシス促進タンパク質であるBaxの活性化及びBaxのミトコンドリアへの移行が亢進し、ミトコンドリア障害を介するcaspase-3依存的なアポトーシスが誘導されることを明らかにした。Competitive research funding