記述言語：英語   掲載種別：研究論文（学術雑誌）  

Hepatitis B virus (HBV) infection is a major public health problem. The sodium taurocholate cotransporting polypeptide (NTCP) has been identified as an essential HBV receptor. Human hepatocytes are infected with HBV via binding between the preS1 region of the HBV large envelope protein and the NTCP. However, the role of preS2 in HBV entry is not well understood. In this study, we induced anti-preS2 serum in mice by DNA immunization, and showed that the resulting antiserum neutralized HBV infectivity. Competition assays using overlapping peptides suggested that the neutralizing epitope is located in the N-terminal region of preS2. In addition, monoclonal antibodies targeting the N-terminal region of preS2 neutralized HBV infectivity, indicating that these domains are critical epitopes for viral neutralization. These findings provide new insights into the HBV entry machinery while suggesting a novel modality for the prevention and treatment of HBV infection.

DOI： 10.1016/j.virusres.2022.199014

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Nature Springer  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

Abstract

Although the current hepatitis B (HB) vaccine comprising small-HBs antigen (Ag) is potent and safe, attenuated prophylaxis against hepatitis B virus (HBV) with vaccine-escape mutations (VEMs) has been reported. We investigate an HB vaccine consisting of large-HBsAg that overcomes the shortcomings of the current HB vaccine. Yeast-derived large-HBsAg is immunized into rhesus macaques, and the neutralizing activities of the induced antibodies are compared with those of the current HB vaccine. Although the antibodies induced by the current HB vaccine cannot prevent HBV infection with VEMs, the large-HBsAg vaccine-induced antibodies neutralize those infections. The HBV genotypes that exhibited attenuated neutralization via these vaccines are different. Here, we show that the HB vaccine consisting of large-HBsAg is useful to compensate for the shortcomings of the current HB vaccine. The combined use of these HB vaccines may induce antibodies that can neutralize HBV strains with VEMs or multiple HBV genotypes.

DOI： 10.1038/s41467-022-32910-z

その他リンク： https://www.nature.com/articles/s41467-022-32910-z

記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

In this study, we show that the coronavirus (CoV) genome may encode many functional hydrophobic alpha-helical peptides (HAHPs) in overlapping reading frames of major coronaviral proteins throughout the entire viral genome. These HAHPs can theoretically be expressed from non-canonical sub-genomic (sg)RNAs that are synthesized in substantial amounts in infected cells. We selected and analyzed five and six HAHPs encoded in the S gene regions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively. Two and three HAHPs derived from SARS-CoV-2 and MERS-CoV, respectively, specifically interacted with both the SARS-CoV-2 and MERS-CoV S proteins and inhibited their membrane fusion activity. Furthermore, one of the SARS-CoV-2 HAHPs specifically inhibited viral RNA synthesis by accumulating at the site of viral RNA synthesis. Our data show that a group of HAHPs in the coronaviral genome potentially has a regulatory role in viral propagation.

DOI： 10.3390/pathogens11080877

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ASM  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

Novel neplanocin A derivatives have been identified as potent and selective inhibitors of hepatitis B virus (HBV) replication in vitro . These include (1S,2R,5R)-5-(5-bromo-4-methyl-7H-pyrrolo[2,3-d]-pyrimidin-7-yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (AR-II-04-26) and (1S,2R,5R)-5-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3-(hydroxylmethyl)cyclopent-3-ene-1,2-diol (MK-III-02-03).

DOI： 10.1128/aac.02073-21

記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Nature Springer  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF6 is an antagonist of interferon (IFN)-mediated antiviral signaling, achieved through the prevention of STAT1 nuclear localization. However, the exact mechanism through which ORF6 prevents STAT1 nuclear trafficking remains unclear. Herein, we demonstrate that ORF6 directly binds to STAT1 with or without IFN stimulation, resulting in the nuclear exclusion of STAT1. ORF6 also recognizes importin α subtypes with different modes, in particular, high affinity to importin α1 but a low affinity to importin α5. Although ORF6 potentially disrupts the importin α/importin β1-mediated nuclear transport, thereby suppressing the nuclear translocation of the other classical nuclear localization signal-containing cargo proteins, the inhibitory effect of ORF6 is modest when compared with that of STAT1. The results indicate that the drastic nuclear exclusion of STAT1 is attributed to the specific binding with ORF6, which is a distinct strategy for the importin α1-mediated pathway. Combined with the results from a newly-produced replicon system and a hamster model, we conclude that SARS-CoV-2 ORF6 acts as a virulence factor via regulation of nucleocytoplasmic trafficking to accelerate viral replication, resulting in disease progression.

DOI： 10.1038/s42003-022-03427-4

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1111/1348-0421.12964

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier  

DOI： 10.1016/j.antiviral.2022.105268

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier  

DOI： 10.1016/j.antiviral.2021.105165

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jcmgh.2021.07.013

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/v13071310

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1073/pnas.2026184118.

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Proceedings of the National Academy of Sciences  

Immunoevasins are viral proteins that prevent antigen presentation on major histocompatibility complex (MHC) class I, thus evading host immune recognition. Hepatitis C virus (HCV) evades immune surveillance to induce chronic infection; however, how HCV-infected hepatocytes affect immune cells and evade immune recognition remains unclear. Herein, we demonstrate that HCV core protein functions as an immunoevasin. Its expression interfered with the maturation of MHC class I molecules catalyzed by the signal peptide peptidase (SPP) and induced their degradation via HMG-CoA reductase degradation 1 homolog, thereby impairing antigen presentation to CD8⁺ T cells. The expression of MHC class I in the livers of HCV core transgenic mice and chronic hepatitis C patients was impaired but was restored in patients achieving sustained virological response. Finally, we show that the human cytomegalovirus US2 protein, possessing a transmembrane region structurally similar to the HCV core protein, targets SPP to impair MHC class I molecule expression. Thus, SPP represents a potential target for the impairment of MHC class I molecules by DNA and RNA viruses.

DOI： 10.1073/pnas.2026184118

PubMed

その他リンク： https://syndication.highwire.org/content/doi/10.1073/pnas.2026184118

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Weily  

DOI： 10.1002/hep4.1653

その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/hep4.1653

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

DOI： 10.1128/JVI.01784-20

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1002/hep.31308

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1111/hepr.13574

その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/hepr.13574

記述言語：日本語   掲載種別：研究論文（その他学術会議資料等）  

J-GLOBAL

記述言語：日本語   掲載種別：研究論文（その他学術会議資料等）  

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Recent development of hepatitis B virus (HBV) culture systems has made it possible to analyze the almost all steps of the viral life cycle. However, the reproducibility of interaction between HBV and host cells seemed inaccurate in those systems because of utilization of cancer cell lines with a difference from hepatocytes in the majority of cases. In this study, in order to resolve this point, a novel HBV culture system using non-cancer-derived immortalized human hepatocytes derived cell lines, producing exogenous human sodium taurocholate cotransporting polypeptide, was developed. One of the cell clones, E/NtG8 cells, was permissive to both blood-borne HBV (HBVbb) and culture-derived recombinant HBV when cultured in the three-dimensional condition. Furthermore, the production of infectious HBV particles, which showed the similar physicochemical properties to HBVbb, was observed for about a month after HBVbb infection in this system, suggesting that it may reproduce whole steps of the HBV lifecycle under the condition analogous to human liver cells infected with HBV. This system seemed to contribute not only to find novel interactions between HBV and host cells but also to understand mechanism of HBV pathogenesis.

DOI： 10.1038/s41598-020-78655-x

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society of Microbiology  

DOI： 10.1128/JVI.01680-20

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1111/hepr.13529

その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/hepr.13529

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/hepr.13449

PubMed

その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/hepr.13449

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.neo.2019.03.010

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.3389/fpubh.2019.00121

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1128/JVI.01708-18

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1371/journal.pone.0212559

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/hepr.13277

PubMed

記述言語：英語   掲載種別：研究論文（その他学術会議資料等）  

J-GLOBAL

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

DOI： 10.1128/JVI.01997-17

Scopus

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Japanese Society of Veterinary Science  

DOI： 10.1292/jvms.17-0527

Scopus

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1073/pnas.1712484114

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PLOS  

DOI： 10.1371/journal.pone.0188344

Web of Science

PubMed

担当区分：筆頭著者,　最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Tayer & Francis  

DOI： 10.1080/14728222.2017.1369959

Web of Science

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier  

DOI： 10.1016/j.antiviral.2017.08.001

Web of Science

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier  

DOI： 10.1016/j.antiviral.2017.07.018

Web of Science

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1099/jgv.0.000813

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier  

DOI： 10.1016/j.jdermsci.2017.03.015.

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER IRELAND LTD  

Background: Langerhans cells (LCs) are one of the initial target cells for HIV following sexual exposure and they are productively infected by HIV. HIV-infected LCs migrate to the draining lymph nodes (dLNs) and transmit the virus to CD4+ T cells, leading to the dissemination of HIV. In contrast with the role of LCs in initial HIV acquisition, little is known about the modulation of immune responses by HIV-infected LCs.
Objective: We aimed to elucidate the induction of HIV-specific CD8+ T cells and regulatory T cells (Tregs), both of which play important roles in regulating the progression of HIV infection.
Methods: We examined the inducibility of HLA-A*0201 restricted HIV-specific CD8+ T cells and Tregs by HIV -primed LCs or HIV -primed dendritic cells (DCs) as a control.
Results: The number of HIV-specific CD8+ T cells induced by HIV -primed monocyte-derived LCs (mLCs) was significantly higher than that by HIV -primed monocyte-derived DCs (mDCs). Additionally, HIV-specific CD8+ T cells induced by HIV -primed mLCs produced more IFN-gamma than HIV-nonspecific CD8+ T cells. HIV -primed human epidermal LCs also induced IFN-gamma-producing HIV-specific CD8+ T cells. As for the induction of Tregs, HIV -primed mLCs and human epidermal LCs significantly impaired the induction of FoxP3(hi)CD45RA(-) effector Tregs than HIV-unprimed mLCs and human epidermal LCs.
Conclusions: HIV -primed LCs trigger beneficial immune responses against HIV infection through the increased induction of HIV-specific CD8+ T cells and the decreased induction of effector Tregs in the initial phase of HIV infection, thereby contributing to the prolonged onset of AIDS. (C) 2017 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.jdermsci.2017.03.015

Web of Science

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

Hepatitis C virus (HCV) core protein is responsible for the formation of infectious viral particles and induction of pathogenicity. The C-terminal transmembrane region of the immature core protein is cleaved by signal peptide peptidase (SPP) for maturation of the core protein. SPP belongs to the family of presenilin-like aspartic proteases. Some presenilin inhibitors are expected to suppress HCV infection and production; however, this anti-HCV effect has not been investigated in detail. In this study, presenilin inhibitors were screened to identify anti-HCV compounds. Of the 13 presenilin inhibitors tested, LY411575 was the most potent inhibitor of SPP-dependent cleavage of HCV core protein. Production of intracellular core protein and supernatant infectious viral particles from HCV-infected cells was significantly impaired by LY411575 in a dose-dependent manner (half maximum inhibitory concentration =0.27 mu M, cytotoxic concentration of the extracts to cause death to 50% of viable cells > 10 mu M). No effect of LY411575 on intracellular HCV RNA in the subgenomic replicon cells was detected. LY411575 synergistically promoted daclatasvir-dependent inhibition of viral production, but not that of viral replication. Furthermore, LY411575 inhibited HCV-related production of reactive oxygen species and expression of NADPH oxidases and vascular endothelial growth factor. Taken together, our data suggest that LY411575 suppresses HCV propagation through SPP inhibition and impairs host gene expressions related to HCV pathogenicity.

DOI： 10.1111/1348-0421.12448

Web of Science

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/1348-0421.12448.

記述言語：英語   掲載種別：研究論文（研究会，シンポジウム資料等）  

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.antiviral.2016.08.008

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jid.2016.01.030

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 11.329

DOI： 10.1038/ncomms11379

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1080/15384101.2016.1148837

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1128/JVI.02776-15

Web of Science

PubMed

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：論文集（書籍）内論文   出版者・発行元：Springer Japan  

Hepatitis C virus (HCV) is a major causative agent of liver disorders and a major risk factor for hepatocellular carcinoma. The induction of hepatocellular carcinoma by HCV is thought to involve not only chronic inflammation, but also the biological activity of HCV components. Structural proteins of HCV are composed of the core protein and two envelope proteins, E1 and E2. The HCV core protein has been reported to exhibit multiple biological functions involved in lipid synthesis, iron metabolism, insulin response, oxidative stress and cell growth, and to thereby contribute to the development of carcinogenesis and metabolic disorders. Moreover, several reports suggest that envelope proteins also play an important role in viral entry as well as HCV-related pathogenic events. However, the mechanism by which the structural proteins induce hepatitis C-related disorders has not been fully understood. This review focuses on the current status of biological responses mediated by HCV structural proteins.

DOI： 10.1007/978-4-431-56098-2_6

Scopus

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 5.228

DOI： 10.1038/srep17047

Web of Science

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 5.228

DOI： 10.1038/srep16699

Web of Science

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

The current treatments of chronic hepatitis B (CHB) face a limited choice of vaccine, antibody and antiviral agents. The development of additional antiviral agents is still needed for improvement of CHB therapy. In this study, we established a screening system in order to identify compounds inhibiting the core promoter activity of hepatitis B virus (HBV). We prepared 80 extracts of marine organisms from the coral reefs of Indonesia and screened them by using this system. Eventually, two extracts showed high inhibitory activity (>95%) and low cytotoxicity (66% to 77%). Solvent fractionation, column chromatography and NMR analysis revealed that 3,5-dibromo-2-(2,4-dibromophenoxy)-phenol (compound 1) and 3,4,5-tribromo-2-(2,4-dibromophenoxy)-phenol (compound 2), which are classified as polybrominated diphenyl ethers (PBDEs), were identified as anti-HBV agents in the extracts. Compounds 1 and 2 inhibited HBV core promoter activity as well as HBV production from HepG2.2.15.7 cells in a dose-dependent manner. The EC50 values of compounds 1 and 2 were 0.23 and 0.80 mu M, respectively, while selectivity indexes of compound 1 and 2 were 18.2 and 12.8, respectively. These results suggest that our cell-based HBV core promoter assay system is useful to determine anti-HBV compounds, and that two PBDE compounds are expected to be candidates of lead compounds for the development of anti-HBV drugs.

DOI： 10.3390/md13116759

Web of Science

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 3.345

記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

Hepatitis C virus (HCV) is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3) helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure-activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 mu M. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 mu M, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition.

DOI： 10.3390/ijms160818439

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 2.371

DOI： 10.1016/j.bbrc.2015.04.137

Web of Science

PubMed

記述言語：日本語   掲載種別：（MISC）総説・解説（商業誌）   出版者・発行元：(一社)日本医学教育学会  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 4.606

DOI： 10.1128/Jvi.03127-14

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 1.733

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Imipramine, a major antidepressant, is known to inhibit reuptake of serotonin and norepinephrine, which contributes to recovery from major depressive disorder. It has recently been reported that acute imipramine treatment inhibits N-methyl-D-aspartate (NMDA) receptor activity. However, the mechanisms underlying lung term effects of imipramine have nor been identified. We rested these distinct effects in mouse cortical neurons and found that acute (30 s) imipramine treatment decreased Ca2+ influx through NMDA receptors, whereas long term treatment (48 h) increased Ca2+ influx via the same receptors. Furthermore, long term treatment increased NMDA receptor 2B (NR2B) subunit expression via epigenetic changes, including increased acetylation of histones H3K9 and H3K27 in the NR2B promoter and decreased activity of histone deacetylase 3 (HDAC3) and HDAC4. These results suggest that the long term effects of imipramine on NMDA receptors are quire different from its acute effects. Furthermore, increased NR2B expression via epigenetic alterations might be a part of the mechanism responsible for this long-term effect. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ejphar.2015.02.010

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1017/S2040174415000100

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 3.257

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 2.730

記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）  

記述言語：英語   掲載種別：研究論文（その他学術会議資料等）  

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 4.606

DOI： 10.1128/Jvi.02280-14

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 6.915

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

IF = 2.465

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：INFORMA HEALTHCARE  

Hepatitis C virus nonstructural protein 3 (NS3) helicase is a promising target for developing new therapeutics. In this study, we identified cholesterol sulfate (CS) as a novel NS3 helicase inhibitor (IC50 = 1.7 +/- 0.2 mu M with a Hill coefficient of 3.9) by screening the extracts from marine organisms. The lack of the sulfate group, sterol structure or alkyl side chain of CS diminished the inhibition, suggesting that an anion binding and hydrophobic region in NS3 may be a target site of CS. It was further found that CS partly inhibits NS3-RNA binding activity, but exerted no or less inhibition against ATPase and serine protease activities. Moreover, we demonstrated that CS probably does not bind to RNA. Our findings suggest that CS may inhibit NS3 helicase not by abolishing the other NS3 activities but by inducing conformational changes via interaction with possible allosteric sites of NS3.

DOI： 10.3109/14756366.2013.766607

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

The helicase portion of the hepatitis C virus nonstructural protein 3 (NS3) is considered one of the most validated targets for developing direct acting antiviral agents. We isolated polybrominated diphenyl ether (PBDE) 1 from a marine sponge as an NS3 helicase inhibitor. In this study, we evaluated the inhibitory effects of PBDE (1) on the essential activities of NS3 protein such as RNA helicase, ATPase, and RNA binding activities. The structure-activity relationship analysis of PBDE (1) against the HCV ATPase revealed that the biphenyl ring, bromine, and phenolic hydroxyl group on the benzene backbone might be a basic scaffold for the inhibitory potency.

DOI： 10.3390/molecules19044006

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

DOI： 10.1038/jid.2013.467

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 3.345

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 3.057

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

Glycoprotein K (gK) is a virion envelope protein of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), which plays important roles in virion entry, morphogenesis and egress. Two-hybrid and pull-down assays were utilized to demonstrate that gK and no other HSV-1 genes specifically binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. SPP dominant negative mutants, shRNA against SPP significantly reduced HSV-1 replication in vitro. SPP also affected lysosomes and ER responses to HSV-1 infection. Thus, in this study we have shown for the first time that gK, despite its role in fusion and egress, is also involved in binding the cytoplasmic protein SPP. These results also suggest that SPP plays an important role in viral replication and possibly virus pathogenesis. This makes SPP unique in that its function appears to be required by the virus as no other protein can compensate its loss in terms of viral replication.

DOI： 10.1371/journal.pone.0085360

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

Hepatitis C virus (HCV) is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3) helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3) and suvanine as novel NS3 helicase inhibitors, with IC50 values of 4 and 3 mu M, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC50 values of 8, 8, and 14 mu M, and 7, 3, and 34 mu M, respectively. However, the dengue virus (DENV) NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites) with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 mu M. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.

DOI： 10.3390/md12010462

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 4.606

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 3.057

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Variation of core amino acid (aa) 70 of hepatitis C virus (HCV) has been shown recently to be closely correlated with liver disease progression, suggesting that the core region might be present as a quasispecies during persistent infection and that this quasispecies nature might have an influence on the progression of disease. In our investigation, the subjects were 79 patients infected with HCV genotype 1b (25 with chronic hepatitis [CH], 29 with liver cirrhosis [LC], and 25 with hepatocellular carcinoma [HCC]). Deep sequencing of the HCV core region was carried out on their sera by using a Roche 454 GS Junior pyrosequencer. Based on a plasmid containing a cloned HCV sequence (pCV-J4L6S), the background error rate associated with pyrosequencing, including the PCR procedure, was calculated as 0.092 ± 0.005/base. Deep sequencing of the core region in the clinical samples showed a mixture of "mutant-type" Q/H and "wild-type" R at the core aa 70 position in most cases (71/79 [89.9%]), and the ratio of mutant residues to R in the mixture increased as liver disease advanced to LC and HCC. Meanwhile, phylogenetic analysis of the almost-complete core region revealed that the HCV isolates differed genetically depending on the mutation status at core aa 70. We conclude that the core aa 70 mixture ratio, determined by deep sequencing, reflected the status of liver disease, demonstrating a significant association between core aa 70 and disease progression in CH patients infected with HCV genotype 1b. © 2013, American Society for Microbiology.

DOI： 10.1128/JVI.00826-13

Scopus

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

DOI： 10.1038/jid.2013.215

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PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

Caffeic acid phenethyl ester (CAPE) has been reported as a multifunctional compound. In this report, we tested the effect of CAPE and its derivatives on hepatitis C virus (HCV) replication in order to develop an effective anti-HCV compound. CAPE and CAPE derivatives exhibited anti-HCV activity against an HCV replicon cell line of genotype 1b with EC50 values in a range from 1.0 to 109.6 mu M. Analyses of chemical structure and antiviral activity suggested that the length of the n-alkyl side chain and catechol moiety are responsible for the anti-HCV activity of these compounds. Caffeic acid n-octyl ester exhibited the highest anti-HCV activity among the tested derivatives with an EC50 value of 1.0 mu M and an SI value of 63.1 by using the replicon cell line derived from genotype 1b strain Con1. Treatment with caffeic acid n-octyl ester inhibited HCV replication of genotype 2a at a similar level to that of genotype 1b irrespectively of interferon signaling. Caffeic acid n-octyl ester could synergistically enhance the anti-HCV activities of interferon-alpha 2b, daclatasvir, and VX-222, but neither telaprevir nor danoprevir. These results suggest that caffeic acid n-octyl ester is a potential candidate for novel anti-HCV chemotherapy drugs.

DOI： 10.1371/journal.pone.0082299

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Hepatitis C virus (HCV) is the causative agent of hepatitis C, a chronic infectious disease that can lead to development of hepatocellular carcinoma. The NS3 nucleoside triphosphatase (NTPase)/helicase has an essential role in HCV replication, and is therefore an attractive target for direct-acting antiviral strategies. In this study, we employed high-throughput screening using a photo-induced electron transfer (PET) system to identify an inhibitor of NS3 helicase from marine organism extracts. We successfully identified psammaplin A as a novel NS3 inhibitor. The dose-response relationship clearly demonstrates the inhibition of NS3 RNA helicase and ATPase activities by psammaplin A, with IC₅₀ values of 17 and 32 μM, respectively. Psammaplin A has no influence on the apparent Km value (0.4 mM) of NS3 ATPase activity, and acts as a non-competitive inhibitor. Additionally, it inhibits the binding of NS3 to single-stranded RNA in a dose-dependent manner. Furthermore, psammaplin A shows an inhibitory effect on viral replication, with EC₅₀ values of 6.1 and 6.3 μM in subgenomic replicon cells derived from genotypes 1b and 2a, respectively. We postulate that psammaplin A is a potential anti-viral agent through the inhibition of ATPase, RNA binding and helicase activities of NS3.

DOI： 10.1007/s11418-013-0742-7

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 6.915

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/jid.2013.150

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 5.997

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

Background & Aim: FKBP8/FKBP38 is a unique FK506-binding protein with a C-terminal membrane anchor and localizes at the outer membranes of mitochondria and the endoplasmic reticulum. Similar to some immunophilins, such as FKBP51, FKBP52 and Cyclophilin 40, FKBP8/FKBP38 contain a putative Calmodulin-binding domain and a tetratricopeptide-repeat (TPR) domain for the binding of Hsp90. Both Hsp90 and the non-structural protein 5A (NS5A) of the hepatitis C virus (HCV) interact specifically with FKBP8/FKBP38 through its TPR domain, and the ternary complex formation plays a critical role in HCV RNA replication. The goal of this study is to evaluate that the host factor inhibits the ternary complex formation and the replication of HCV in vitro and in vivo. Methods: S100 proteins, FKBP38, FKBP8, HCV NS5A, Hsp90, and calmodulin were expressed in E.coli and purified. In vitro binding studies were performed by GST pull-down, S-tag pull-down and surface plasmon resonance analyses. The effect of S100 proteins on HCV replication was analysed by Western blotting using an HCV NS3 antibody following transfection of S100 proteins into the HCV replicon harbouring cell line (sO cells). Results: In vitro binding studies showed that S100A1, S100A2, S100A6, S100B and S100P directly interacted with FKBP8/FKBP38 in a Ca2+-dependent manner and inhibited the FKBP8/FKBP38-Hsp90 and FKBP8/FKBP38-NS5A interactions. Furthermore, overexpression of S100A1, S100A2 and S100A6 in sO cells resulted in the efficient inhibition of HCV replication. Conclusion: The association of the S100 proteins with FKBP8/FKBP38 provides a novel Ca2+-dependent regulatory role in HCV replication through the NS5A-host protein interaction.

DOI： 10.1111/liv.12151

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

Aim Prediction of treatment responses to pegylated interferon (PEG IFN) plus ribavirin (RBV) therapy is uncertain for genotype 1b chronic hepatitis C.
Methods In this study, 96 patients were investigated for the correlation between 36 pretreatment serum chemokine/cytokine levels and PEG IFN/RBV treatment efficacy by a sandwich enzyme-linked immunoassay (ELISA) and a bead array.
Results First, chemokines/cytokines were measured semiquantitatively by sandwich ELISA in 31 randomly-selected patients and the serum regulated on activation normal T-cell expressed and secreted (RANTES) level was found to be significantly higher in the sustained virological response (SVR) group than the non-SVR group (P=0.048). Precise RANTES measurement in all 96 patients using a bead array confirmed this correlation (P=0.002). However, the genetic RANTES haplotype was not significantly related to the serum level. The serum RANTES level was extracted by multivariate analysis (odds ratio=4.09, 95% confidence interval=1.02-16.5, P=0.048) as an independent variable contributing to SVR.
Conclusion The serum RANTES level is an important determinant influencing the virological response to PEG IFN/RBV therapy in chronic hepatitis C.

DOI： 10.1111/hepr.12032

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

Microglia survey the brain environment by sensing several types of diffusible molecules, among which extracellular nucleotides released/leaked from damaged cells have central roles. Microglia sense ATP or other nucleotides by multiple P2 receptors, after which they change into several different phenotypes. However, so far, it is largely unknown whether microglia themselves release ATP and, if so, by what mechanism. Here we show that exocytosis is the mechanism by which microglia release ATP. When we stimulated microglia with ionomycin, they released ATP and the release was dependent on Ca2+, vesicular H+-ATPase, or SNAREs but independent of connexin/pannexin hemichannels. VNUT was found to be expressed in microglia and exhibited no colocalization with lysosome. We also visualized the exocytosis of ATP by a quinacrine-based fluorescent time-lapse imaging. Moreover, we found that lipopolysaccharide increased the ionomycin-induced release of ATP, which was dependent on the increase in VNUT. Taken together, our data suggested that exocytosis is the mechanism of ATP release from microglia. When activated, they would release ATP by increasing VNUT-dependent exocytotic mechanisms. GLIA 2013;61:1320-1330

DOI： 10.1002/glia.22517

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PubMed

記述言語：日本語   掲載種別：研究論文（その他学術会議資料等）   出版者・発行元：(公社)日本生物工学会  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 3.413

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 4.173

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCI LTD  

Background: Cytomegalovirus (CMV) is the most common cause of congenital virus infection. Infection of guinea pigs with guinea pig CMV (GPCMV) can provide a useful model for the analysis of its pathogenesis as well as for the evaluation of vaccines. Although glycoprotein B (gB) vaccines have been reported to reduce the incidence and mortality of congenital infection in human clinical trials and guinea pig animal models, the mechanisms of protection remain unclear.
Methods: To understand the gB vaccine protection mechanisms, we analyzed the spread of challenged viruses in the placentas and fetuses of guinea pig dams immunized with recombinant adenoviruses expressing GPCMV gB and beta-galactosidase, rAd-gB and rAd-LacZ, respectively.
Results: Mean body weight of the fetuses in the dams immunized with rAd-LacZ followed by GPCMV challenge 3 weeks after immunization was 78% of that observed for dams immunized with rAd-gB. Under conditions in which congenital infection occurred in 75% of fetuses in rAd-LacZ-immunized dams, only 13% of fetuses in rAd-gB-immunized dams were congenitally infected. The placentas were infected less frequently in the gB-immunized animals. In the placentas of the rAd-LacZ- and rAd-gB-immunized animals, CMV early antigens were detected mainly in the spongiotrophoblast layer. Focal localization of viral antigens in the spongiotrophoblast layer suggests cell-to-cell viral spread in the placenta. In spite of a similar level of antibodies against gB and avidity indices among fetuses in each gB-immunized dam, congenital infection was sometimes observed in a littermate fetus. In such infected fetuses, CMV spread to most organs.
Conclusions: Our results suggest that antibodies against gB protected against infection mainly at the interface of the placenta rather than from the placenta to the fetus. The development of strategies to block cell-to-cell viral spread in the placenta is, therefore, required for effective protection against congenital CMV infection. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.vaccine.2013.04.078

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

Hepatitis C virus (HCV) is a major cause of chronic liver disease. HCV NSSA protein plays an important role in HCV infection through its interactions with other HCV proteins and host factors. In an attempt to further our understanding of the biological context of protein interactions between NSSA and host factors in HCV pathogenesis, we generated an extensive physical interaction map between NSSA and cellular factors. By combining a yeast two-hybrid assay with comprehensive literature mining, we built the NSSA interactome composed of 132 human proteins that interact with NSSA These interactions were integrated into a high-confidence human protein interactome (HPI) with the help of the TargetMine data warehouse system to infer an overall protein interaction map linking NSSA with the components of the host cellular networks. The NSSA-host interactions that were integrated with the HPI were shown to participate in compact and well-connected cellular networks. Functional analysis of the NSSA "infection" network using TargetMine highlighted cellular pathways associated with immune system, cellular signaling, cell adhesion, cellular growth and death among others, which were significantly targeted by NSSA host interactions. In addition, cellular assays with in vitro HCV cell culture systems identified two ER-localized host proteins RTN1 and RTN3 as novel regulators of HCV propagation. Our analysis builds upon the present understanding of the role of NSSA protein in HCV pathogenesis and provides potential targets for more effective anti-HCV therapeutic intervention.

DOI： 10.1021/pr3011217

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/j.1365-2893.2012.01649.x

Scopus

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 4.179

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 4.470

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 12.552

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CELL PRESS  

Herpes simplex virus (HSV)-2 shedding is associated with increased risk for sexually acquiring HIV. Because Langerhans cells (LCs), the mucosal epithelium resident dendritic cells, are suspected to be one of the initial target cell types infected by HIV following sexual exposure, we examined whether and how HSV-2 affects HIV infection of LCs. Although relatively few HSV-2/HIV-coinfected LCs were detected, HSV-2 dramatically enhanced the HIV susceptibility of LCs within skin explants. HSV-2 stimulated epithelial cell production of antimicrobial peptides (AMPs), including human beta defensins and LL-37. LL-37 strongly upregulated the expression of HIV receptors in monocyte-derived LCs (mLCs), thereby enhancing their HIV susceptibility. Culture supernatants of epithelial cells infected with HSV-2 enhanced HIV susceptibility in mLCs, and this effect was abrogated by blocking LL-37 production. These data suggest that HSV-2 enhances sexual transmission of HIV by increasing HIV susceptibility of LCs via epithelial cell production of LL-37.

DOI： 10.1016/j.chom.2012.12.002

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PubMed

担当区分：責任著者   記述言語：日本語   掲載種別：研究論文（研究会，シンポジウム資料等）  

J-GLOBAL

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 3.057

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

Combination therapy with ribavirin, interferon, and viral protease inhibitors could be expected to elicit a high level of sustained virologic response in patients infected with hepatitis C virus (HCV). However, several severe side effects of this combination therapy have been encountered in clinical trials. In order to develop more effective and safer anti-HCV compounds, we employed the replicon systems derived from several strains of HCV to screen 84 extracts from 54 organisms that were gathered from the sea surrounding Okinawa Prefecture, Japan. The ethyl acetate-soluble extract that was prepared from marine sponge Amphimedon sp. showed the highest inhibitory effect on viral replication, with EC50 values of 1.5 and 24.9 mu g/ml in sub-genomic replicon cell lines derived from genotypes 1b and 2a, respectively. But the extract had no effect on interferon-inducing signaling or cytotoxicity. Treatment with the extract inhibited virus production by 30% relative to the control in the JFH1-Huh7 cell culture system. The in vitro enzymological assays revealed that treatment with the extract suppressed both helicase and protease activities of NS3 with IC50 values of 18.9 and 10.9 mu g/ml, respectively. Treatment with the extract of Amphimedon sp. inhibited RNA-binding ability but not ATPase activity. These results suggest that the novel compound(s) included in Amphimedon sp. can target the protease and helicase activities of HCV NS3.

DOI： 10.1371/journal.pone.0048685

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 1.280

記述言語：日本語   掲載種別：研究論文（その他学術会議資料等）   出版者・発行元：(公社)日本生物工学会  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/pr300121a

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 4.173

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

The mechanisms of induction of liver injury during chronic infection with hepatitis C virus (HCV) are not well understood. Gamma interferon (IFN-gamma)-inducible protein 10 (IP-10), a member of the CXC chemokine family, is expressed in the liver of chronic hepatitis C (CHC) patients and selectively recruits activated T cells to the sites of inflammation. Recently, it was shown that a low plasma concentration of IP-10 in CHC patients was closely associated with the outcome of antiviral therapy. In this study, we examined the role of the Toll-like receptor (TLR) pathway on IP-10 production in cells replicating HCV. Among the CXC chemokines, the expression of IP-10 was specifically increased in cells replicating HCV upon stimulation with conventional TLR2 ligands. The enhancement of IP-10 production upon stimulation with TLR2 ligands in cells replicating HCV induced CD44 expression. CD44 is a broadly distributed type I transmembrane glycoprotein and a receptor for the glycosaminoglycan hyaluronan (HA). In CHC patients, the expression of HA in serum has been shown to increase in accord with the progression of liver fibrosis, and HA also works as a ligand for TLR2. In the present study, IP-10 production upon HA stimulation was dependent on the expression of TLR2 and CD44, and a direct association between TLR2 and CD44 was observed. These results suggest that endogenous expression of HA in hepatocytes in CHC patients participates in IP-10 production through an engagement of TLR2 and CD44.

DOI： 10.1128/JVI.06872-11

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 3.662

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

The hepatitis C virus (HCV) causes one of the most prevalent chronic infectious diseases in the world hepatitis C, which ultimately develops into liver cancer through cirrhosis. The NS3 protein of HCV possesses nucleoside triphosphatase (NTPase) and RNA helicase activities. As both activities are essential for viral replication, NS3 is proposed as an ideal target for antiviral drug development. In this study, we identified manoalide (1) from marine sponge extracts as an RNA helicase inhibitor using a high-throughput screening photoinduced electron transfer (PET) system that we previously developed. Compound 1 inhibits the RNA helicase and ATPase activities of NS3 in a dose dependent manner, with IC50 values of 15 and 70 mu M, respectively. Biochemical kinetic analysis demonstrated that 1 does not affect the apparent K-m stranded RNA was inhibited by 1. Monoalide (1) also has the ability to inhibit the ATPase activity of human DHX36/RHAU, a putative RNA helicase. Taken together, we conclude that 1 inhibits the ATPase, RNA binding, and helicase activities of NS3 by targeting the helicase core domain conserved in both HCV NS3 and DHX36/RHAU.

DOI： 10.1021/np200883s

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PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

Hepatitis C virus (HCV) is a causative agent of acute and chronic hepatitis, leading to the development of hepatic cirrhosis and hepatocellular carcinoma. We prepared extracts from 61 marine organisms and screened them by an in vitro fluorescence assay targeting the viral helicase (NS3), which plays an important role in HCV replication, to identify effective candidates for anti-HCV agents. An ethyl acetate-soluble fraction of the feather star Alloeocomatella polycladia exhibited the strongest inhibition of NS3 helicase activity, with an IC50 of 11.7 mu g/mL. The extract of A. polycladia inhibited interaction between NS3 and RNA but not ATPase of NS3. Furthermore, the replication of the replicons derived from three HCV strains of genotype 1b in cultured cells was suppressed by the extract with an EC50 value of 23 to 44 mu g/mL, which is similar to the IC50 value of the NS3 helicase assay. The extract did not induce interferon or inhibit cell growth. These results suggest that the unknown compound(s) included in A. polycladia can inhibit HCV replication by suppressing the helicase activity of HCV NS3. This study may present a new approach toward the development of a novel therapy for chronic hepatitis C.

DOI： 10.3390/md10040744

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 4.606

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 3.345

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-beta-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.

DOI： 10.1128/JVI.06704-11

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.3892/etm.2011.415

Web of Science

PubMed

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 4.165

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：FRONTIERS RESEARCH FOUNDATION  

Hepatitis C virus (HCV), which is a major causative agent of blood-borne hepatitis, has chronically infected about 170 million individuals worldwide and leads to chronic infection, resulting in development of steatosis, cirrhosis, and eventually hepatocellular carcinoma. Hepatocellular carcinoma associated with HCV infection is not only caused by chronic inflammation, but also by the biological activity of HCV proteins. HCV core protein is known as a main component of the viral nucleocapsid. It cooperates with host factors and possesses biological activity causing lipid alteration, oxidative stress, and progression of cell growth, while other viral proteins also interact with host proteins including molecular chaperones, membrane-anchoring proteins, and enzymes associated with lipid metabolism to maintain the efficiency of viral replication and production. HCV core protein is localized on the surface of lipid droplets in infected cells. However, the role of lipid droplets in HCV infection has not yet been elucidated. Several groups recently reported that other viral proteins also support viral infection by regulation of lipid droplets and core localization in infected cells. Furthermore, lipid components are required for modification of host factors and the intracellular membrane to maintain or up-regulate viral replication. In this review, we summarize the current status of knowledge regarding the exploitation of lipid components by viral and host proteins in HCV infection.

DOI： 10.3389/fmicb.2012.00054

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Hepatitis C virus (HCV) is a major cause of chronic liver diseases. A high risk of chronicity is the major concern of HCV infection, since chronic HCV infection often leads to liver cirrhosis and hepatocellular carcinoma. Infection with the HCV genotype 1 in particular is considered a clinical risk factor for the development of hepatocellular carcinoma, although the molecular mechanisms of the pathogenesis are largely unknown. Autophagy is involved in the degradation of cellular organelles and the elimination of invasive microorganisms. In addition, disruption of autophagy often leads to several protein deposition diseases. Although recent reports suggest that HCV exploits the autophagy pathway for viral propagation, the biological significance of the autophagy to the life cycle of HCV is still uncertain. Here, we show that replication of HCV RNA induces autophagy to inhibit cell death. Cells harboring an HCV replicon RNA of genotype 1b strain Con1 but not of genotype 2a strain JFH1 exhibited an incomplete acidification of the autolysosome due to a lysosomal defect, leading to the enhanced secretion of immature cathepsin B. The suppression of autophagy in the Con1 HCV replicon cells induced severe cytoplasmic vacuolation and cell death. These results suggest that HCV harnesses autophagy to circumvent the harmful vacuole formation and to maintain a persistent infection. These findings reveal a unique survival strategy of HCV and provide new insights into the genotype-specific pathogenicity of HCV.

DOI： 10.1128/JVI.06099-11

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 4.509

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NEOPLASIA PRESS  

The human and Old World primate genomes possess conserved endogenous retrovirus sequences that have been implicated in evolution, reproduction, and carcinogenesis. Human endogenous retrovirus (HERV)-K with 5'LTR-gag-pro-pol-env-rec/np9-3'LTR sequences represents the newest retrovirus family that integrated into the human genome 1 to 5 million years ago. Although a high-level expression of HERV-K in melanomas, breast cancers, and teratocarcinomas has been demonstrated, the mechanism of the lineage-specific activation of the long terminal repeat (LTR) remains obscure. We studied chromosomal HERV-K expression in MeWo melanoma cells in comparison with the basal expression in human embryonic kidney 293 (HEK293) cells. Cloned LTR of HERV-K (HML-2.HOM) was also characterized by mutation and transactivation experiments. We detected multiple transcriptional initiator (Inr) sites in the LTR by rapid amplification of complementary DNA ends (5'RACE). HEK293 and MeWo showed different Inr usage. The most potent Inr was associated with a TATA box and three binding motifs of microphthalmia-associated transcription factor (MITF). Both chromosomal HERV-K expression and the cloned LTR function were strongly activated in HEK293 by transfection with MITF-M, a melanocyte/melanoma-specific isoform of MITF. Coexpression of MITF and the HERV-K core antigen was detected in retinal pigmented epithelium by an immunofluorescence analysis. Although malignant melanoma lines MeWo, G361, and SK-MEL-28 showed enhanced HERV-K transcription compared with normal melanocytes, the level of MITF-M messenger RNA persisted from normal to transformed melanocytes. Thus, MITF-M may be a prerequisite for the pigmented cell lineage-specific function of HERV-K LTR, leading to the high-level expression in malignant melanomas.

DOI： 10.1593/neo.11794

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is kept in a zoonotic transmission cycle between pigs and mosquitoes. JEV causes infection of the central nervous system with a high mortality rate in dead-end hosts, including humans. Many studies have suggested that the flavivirus core protein is not only a component of nucleocapsids but also an important pathogenic determinant. In this study, we identified heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) as a binding partner of the JEV core protein by pulldown purification and mass spectrometry. Reciprocal coimmunoprecipitation analyses in transfected and infected cells confirmed a specific interaction between the JEV core protein and hnRNP A2. Expression of the JEV core protein induced cytoplasmic retention of hnRNP A2 in JEV subgenomic replicon cells. Small interfering RNA (siRNA)-mediated knockdown of hnRNP A2 resulted in a 90% reduction of viral RNA replication in cells infected with JEV, and the reduction was cancelled by the expression of an siRNA-resistant hnRNP A2 mutant. In addition to the core protein, hnRNP A2 also associated with JEV nonstructural protein 5, which has both methyltransferase and RNA-dependent RNA polymerase activities, and with the 5'-untranslated region of the negative-sense JEV RNA. During one-step growth, synthesis of both positive-and negative-strand JEV RNAs was delayed by the knockdown of hnRNP A2. These results suggest that hnRNP A2 plays an important role in the replication of JEV RNA through the interaction with viral proteins and RNA.

DOI： 10.1128/JVI.00846-11

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 4.606

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 4.606

記述言語：日本語   掲載種別：研究論文（その他学術会議資料等）   出版者・発行元：(公社)日本生物工学会  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：W B SAUNDERS CO-ELSEVIER INC  

DOI： 10.1053/j.gastro.2011.05.027

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 18.187

記述言語：日本語  

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Japanese encephalitis virus (JEV) is a mosquito-borne RNA virus that belongs to the Flaviviridae family. In this study, we have examined the effect of cyclosporin A (CsA) on the propagation of JEV. CsA exhibited potent anti-JEV activity in various mammalian cell lines through the inhibition of CypB. The propagation of JEV was impaired in the CypB-knockdown cells and this reduction was cancelled by the expression of wild-type but not of peptidylprolyl cis-trans isomerase (PPlase)-deficient CypB, indicating that PPlase activity of CypB is critical for JEV propagation. Infection of pseudotype viruses bearing JEV envelope proteins was not impaired by the knockdown of CypB, suggesting that CypB participates in the replication but not in the entry of JEV. CypB was colocalized and immunoprecipitated with JEV NS4A in infected cells. These results suggest that CypB plays a crucial role in the replication of JEV through an interaction with NS4A. (c) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2011.01.011

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IF = 3.057

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

To eliminate hepatitis C virus (HCV) from infected hepatocytes, we generated two therapeutic molecules specifically activated in cells infected with HCV. A dominant active mutant of interferon (IFN) regulatory factor 7 (IRF7) and a negative regulator of HCV replication, VAP-C (Vesicle-associated membrane protein-associated protein subtype C), were fused with the C-terminal region of IPS-1 (IFN beta promoter stimulator-1), which includes an HCV protease cleavage site that was modified to be localized on the ER membrane, and designated cIRF7 and cVAP-C, respectively. In cells expressing the HCV protease, cIRF7 was cleaved and the processed fragment was migrated into the nucleus, where it activated various IFN promoters, including promoters of IFN alpha 6, IFN beta, and IFN stimulated response element. Activation of the IFN promoters and suppression of viral RNA replication were observed in the HCV replicon cells and in cells infected with the JFH1 strain of HCV (HCVcc) by expression of cIRF7. Suppression of viral RNA replication was observed even in the IFN-resistant replicon cells by the expression of cIRF7. Expression of the cVAP-C also resulted in suppression of HCV replication in both the replicon and HCVcc infected cells. These results suggest that delivery of the therapeutic molecules into the liver of hepatitis C patients, followed by selective activation of the molecules in HCV-infected hepatocytes, is a feasible method for eliminating HCV.

DOI： 10.1371/journal.pone.0015967

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1593/neo.101054

Scopus

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JOHN WILEY & SONS INC  

We have reported previously that the proteasome activator PA28 gamma participates not only in degradation of hepatitis C virus (HCV) core protein in the nucleus but also in the pathogenesis in transgenic mice expressing HCV core protein. However, the biological significance of PA28 gamma in the propagation of HCV has not been clarified. PA28 gamma is an activator of proteasome responsible for ubiquitin-independent degradation of substrates in the nucleus. In the present study, knockdown of PA28 gamma in cells preinfection or postinfection with the JFH-1 strain of HCV impaired viral particle production but exhibited no effect on viral RNA replication. The particle production of HCV in PA28 gamma knockdown cells was restored by the expression of an small interfering RNA (siRNA)-resistant PA28 gamma. Although viral proteins were detected in the cytoplasm of cells infected with HCV, suppression of PA28 gamma expression induced accumulation of HCV core protein in the nucleus. HCV core protein was also degraded in the cytoplasm after ubiquitination by an E3 ubiquitin ligase, E6AP. Knockdown of PA28 gamma enhanced ubiquitination of core protein and impaired virus production, whereas that of E6AP reduced ubiquitination of core protein and enhanced virus production. Furthermore, virus production in the PA28 gamma knockdown cells was restored through knockdown of E6AP or expression of the siRNA-resistant wild-type but not mutant PA28 gamma incapable of activating proteasome activity. Conclusion: Our results suggest that PA28 gamma participates not only in the pathogenesis but also in the propagation of HCV by regulating the degradation of the core protein in both a ubiquitin-dependent and ubiquitin-independent manner. (HEPATOLOGY 2010;52:411-420)

DOI： 10.1002/hep.23680

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

A major obstacle to gene transduction by viral vectors is inactivation by human complement in vivo. One way to overcome this is to incorporate complement regulatory proteins, such as CD55/decay accelerating factor (DAF), into viral particles. Lentivirus vectors pseudotyped with the baculovirus envelope protein GP64 have been shown to acquire more potent resistance to serum inactivation and longer transgene expression than those pseudotyped with the vesicular stomatitis virus (VSV) envelope protein G. However, the molecular mechanisms underlying resistance to serum inactivation in pseudotype particles bearing the GP64 have not been precisely elucidated. In this study, we generated pseudotype and recombinant VSVs bearing the GP64. Recombinant VSVs generated in human cell lines exhibited the incorporation of human DAF in viral particles and were resistant to serum inactivation, whereas those generated in insect cells exhibited no incorporation of human DAF and were sensitive to complement inactivation. The GP64 and human DAF were detected on the detergent-resistant membrane and were coprecipitated by immunoprecipitation analysis. A pseudotype VSV bearing GP64 produced in human DAF knockdown cells reduced resistance to serum inactivation. In contrast, recombinant baculoviruses generated in insect cells expressing human DAF or carrying the human DAF gene exhibited resistance to complement inactivation. These results suggest that the incorporation of human DAF into viral particles by interacting with baculovirus GP64 is involved in the acquisition of resistance to serum inactivation.

DOI： 10.1128/JVI.02519-09

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY  

Although a cell culture system for HCV JFH-1 strain has been developed, no robust cell culture system for serum-derived HCV is available. In this study, we have established systems capable of monitoring infection with JFH-1 virus based on specific reporter gene expression through proteolysis of chimeric transcription factors by HCV NS3/4A protease. We utilized a transcriptional factor Gal4-TBP that synergistically enhances transcription of the GAL4UAS and HIV-1 LTR tandem promoter with the Tat protein. We constructed chimeric Tat and Gal4-TBP transcription factors containing the HCV NS3/4A cleavage sequence of a mitochondria-resident IPS-1, but not those of the HCV polyprotein, and manipulated them to localize in the ER. Upon infection with JFH-1 virus, the transcription factors were efficiently cleaved by HCV protease, migrated into the nucleus and activated the reporter gene under the tandem promoter. Upon infection with JFH-1 virus, the Huh7OK1/TG-Luc cell line carrying the transcription factors and a luciferase gene under the promoter expressed luciferase in a dose-dependent manner in close correlation with HCV RNA replication. Huh7OK1/TG-LNGFR cells carrying the transcription factors and a cDNA of human low affinity nerve growth factor receptor under the promoter were selectively concentrated by immunomagnetic cell sorting upon infection with JFH-1 virus. These results indicate that the chimeric constructs bearing the ER-resident IPS-1 sequence are specifically recognized and efficiently cleaved by HCV protease and are harnessed for detection of HCV replication and for recovery of HCV-infected cells. This strategy may be applicable for the establishment of cell culture systems for the isolation of serum-derived HCV.

DOI： 10.1111/j.1348-0421.2010.00209.x

Web of Science

PubMed

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Japanese encephalitis virus (JEV) is a mosquito-borne RNA virus and one of the most important flaviviruses in the medical and veterinary fields. Although cholesterol has been shown to participate in both the entry and replication steps of JEV, the mechanisms of infection, including the cellular receptors of JEV, remain largely unknown. To clarify the infection mechanisms of JEV, we generated pseudotype (JEVpv) and recombinant (JEVrv) vesicular stomatitis viruses bearing the JEV envelope protein. Both JEVpv and JEVrv exhibited high infectivity for the target cells, and JEVrv was able to propagate and form foci as did authentic JEV. Anti-JEV envelope antibodies neutralized infection of the viruses. Treatment of cells with inhibitors for vacuolar ATPase and clathrin-mediated endocytosis reduced the infectivity of JEVpv, suggesting that JEVpv enters cells via pH-and clathrin-dependent endocytic pathways. Although treatment of the particles of JEVpv, JEVrv, and JEV with cholesterol drastically reduced the infectivity as previously reported, depletion of cholesterol from the particles by treatment with methyl beta-cyclodextrin enhanced infectivity. Furthermore, treatment of cells with sphingomyelinase (SMase), which hydrolyzes membrane-bound sphingomyelin to ceramide, drastically enhanced infection with JEVpv and propagation of JEVrv, and these enhancements were inhibited by treatment with an SMase inhibitor or C-6-ceramide. These results suggest that ceramide plays crucial roles in not only entry but also egress processes of JEV, and they should assist in the clarification of JEV propagation and the development of novel therapeutics against diseases caused by infection with flaviviruses.

DOI： 10.1128/JVI.02499-09

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROYAL SOC CHEMISTRY  

Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. Here we attempt to further our understanding of the biological context of protein interactions in HCV pathogenesis, by investigating interactions between HCV proteins Core and NS4B and human host proteins. Using the yeast two-hybrid (Y2H) membrane protein system, eleven human host proteins interacting with Core and 45 interacting with NS4B were identified, most of which are novel. These interactions were used to infer overall protein interaction maps linking the viral proteins with components of the host cellular networks. Core and NS4B proteins contribute to highly compact interaction networks that may enable the virus to respond rapidly to host physiological responses to HCV infection. Analysis of the interaction networks highlighted enriched biological pathways likely influenced in HCV infection. Inspection of individual interactions offered further insights into the possible mechanisms that permit HCV to evade the host immune response and appropriate host metabolic machinery. Follow-up cellular assays with cell lines infected with HCV genotype 1b and 2a strains validated Core interacting proteins ENO1 and SLC25A5 and host protein PXN as novel regulators of HCV replication and viral production. ENO1 siRNA knockdown was found to inhibit HCV replication in both the HCV genotypes and viral RNA release in genotype 2a. PXN siRNA inhibition was observed to inhibit replication specifically in genotype 1b but not in genotype 2a, while SLC25A5 siRNA facilitated a minor increase in the viral RNA release in genotype 2a. Thus, our analysis can provide potential targets for more effective anti-HCV therapeutic intervention.

DOI： 10.1039/c0mb00103a

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：KARGER  

A recent study by our group indicated that peripheral B cells in chronic hepatitis C (CHC) patients are infected with hepatitis C virus (HCV). This raised the logical question of how HCV circumvents the antiviral immune responses of B cells. Because type I interferon (IFN) plays a critical role in the innate antiviral immune response, IFN beta expression levels in peripheral B cells from CHC patients were analyzed, and these levels were found to be comparable to those in normal B cells, which suggested that HCV infection failed to trigger antiviral immune responses in B cells. Sensing mechanisms for invading viruses in host immune cells involve Toll-like receptor-mediated and retinoic acid-inducible gene-I (RIG-I)-mediated pathways. Both pathways culminate in IFN regulatory factor-3 (IRF-3) translocation into the nucleus for IFN beta gene transcription. Although the expression levels of RIG-I and its adaptor molecule, IFN promoter-stimulator-1, were substantially enhanced in CHC B cells, dimerization and subsequent nuclear translocation of IRF-3 were not detectable. TANK-binding kinase-1 (TBK1) and I kappa B kinase epsilon (IKK epsilon) are essential for IRF-3 phosphorylation. Constitutive expression of both kinases was markedly enhanced in CHC B cells. However, reduced expression of heat shock protein of 90 kDa, a TBK1 stabilizer, and enhanced expression of SIKE, an IKK epsilon suppressor, were observed in CHC B cells, which might suppress the kinase activity of TBK1/IKK epsilon for IRF-3 phosphorylation. In addition, the expression of vesicle-associated membrane protein-associated protein-C, a putative inhibitor of HCV replication, was negligible in B cells. These results strongly suggest that HCV utilizes B cells as a reservoir for persistent infection. Copyright (C) 2010 S. Karger AG, Basel

DOI： 10.1159/000317690

Web of Science

PubMed

記述言語：英語   掲載種別：（MISC）研究発表要旨（国際会議）   出版者・発行元：JOHN WILEY & SONS LTD  

Web of Science

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a component of the replication complex consisting of several host and viral proteins. We have previously reported that human butyrate-induced transcript 1 (hB-ind1) recruits heat shock protein 90 (Hsp90) and FK506-binding protein 8 (FKBP8) to the replication complex through interaction with NS5A. To gain more insights into the biological functions of hB-ind1 in HCV replication, we assessed the potential cochaperone-like activity of hB-ind1, because it has significant homology with cochaperone p23, which regulates Hsp90 chaperone activity. The chimeric p23 in which the cochaperone domain was replaced with the p23-like domain of hB-ind1 exhibited cochaperone activity comparable to that of the authentic p23, inhibiting the glucocorticoid receptor signaling in an Hsp90-dependent manner. Conversely, the chimeric hB-ind1 in which the p23-like domain was replaced with the cochaperone domain of p23 resulted in the same level of recovery of HCV propagation as seen in the authentic hB-ind1 in cells with knockdown of the endogenous hB-ind1. Immunofluorescence analyses revealed that hB-ind1 was colocalized with NS5A, FKBP8, and double-stranded RNA in the HCV replicon cells. HCV replicon cells exhibited a more potent unfolded-protein response (UPR) than the parental and the cured cells upon treatment with an inhibitor for Hsp90. These results suggest that an Hsp90-dependent chaperone pathway incorporating hB-ind1 is involved in protein folding in the membranous web for the circumvention of the UPR and that it facilitates HCV replication.

DOI： 10.1128/JVI.01035-09

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC INVESTIGATIVE PATHOLOGY, INC  

Hepatic steatosis and insulin resistance are factors that aggravate the progression of liver disease caused by hepatitis C virus (HCV) infection. In the pathogenesis of liver disease and metabolic disorders in HCV infection, oxidative stress due to mitochondrial respiratory chain dysfunction plays a pivotal role. Tacrolimus (FK506) is supposed to protect mitochondrial respiratory function. We studied whether tacrolimus affects the development of HCV-associated liver disease using HCV core gene transgenic mice, which develop hepatic steatosis, insulin resistance, and hepatocellular carcinoma. Administration of tacrolimus; to HCV core gene transgenic mice three times per week for 3 months led to a significant reduction in the amounts of lipid in the liver as well as in serum insulin. Tacrolimus treatment also ameliorated oxidative stress and DNA damage in the liver of the core gene transgenic mice. Tacrolimus administration reproduced these effects in a dose-dependent manner in HepG2 cells expressing the core protein. The intrahepatic level of tumor necrosis factor-alpha, which may be a key molecule for the pathogenesis in HCV infection, was significantly decreased in tacrolimus-treated core gene transgenic mice. Tacrolimus thus reversed the effect of the core protein in the patho genesis of HCV-associated liver disease. These results may provide new therapeutic tools for chronic hepatitis C, in which oxidative stress and abnormalities in lipid and glucose metabolism contribute to liver pathogenesis. (Am J Pathol 2009, 175:1515-1524; DOI: 10.2353/ajpath.2009.090102)

DOI： 10.2353/ajpath.2009.090102

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Autographa californica nuclear polyhedrosis virus (AcNPV) is a double-stranded-DNA virus that is pathogenic to insects. AcNPV was shown to induce an innate immune response in mammalian immune cells and to confer protection of mice from lethal viral infection. In this study, we have shown that production of type I interferon (IFN) by AcNPV in murine plasmacytoid dendritic cells (pDCs) and non-pDCs, such as peritoneal macrophages and splenic CD11c(+) DCs, was mediated by Toll-like receptor (TLR)-dependent and -independent pathways, respectively. IFN regulatory factor 7 (IRF7) was shown to play a crucial role in the production of type I IFN by AcNPV not only in immune cells in vitro but also in vivo. In mouse embryonic fibroblasts (MEFs), AcNPV produced IFN-beta and IFN-inducible chemokines through TLR-independent and IRF3-dependent pathways, in contrast to the TLR-dependent and IRF3/IRF7-independent production of proinflammatory cytokines. Although production of IFN-beta and IFN-inducible chemokines was severely impaired in IFN promoter-stimulator 1 (IPS-1)-deficient MEFs upon infection with vesicular stomatitis virus, AcNPV produced substantial amounts of the cytokines in IPS-1-deficient MEFs. These results suggest that a novel signaling pathway(s) other than TLR- and IPS-1-dependent pathways participates in the production of type I IFN in response to AcNPV infection.

DOI： 10.1128/JVI.00679-09

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Human vesicle-associated membrane protein-associated protein (VAP) subtype A (VAP-A) and subtype B (VAP-B) are involved in the regulation of membrane trafficking, lipid transport and metabolism, and the unfolded protein response. VAP-A and VAP-B consist of the major sperm protein (MSP) domain, the coiled-coil motif, and the C-terminal transmembrane anchor and form homo- and heterodimers through the transmembrane domain. VAP-A and VAP-B interact with NS5B and NS5A of hepatitis C virus (HCV) through the MSP domain and the coiled-coil motif, respectively, and participate in the replication of HCV. VAP-C is a splicing variant of VAP-B consisting of the N-terminal half of the MSP domain of VAP-B followed by the subtype-specific frameshift sequences, and its biological function has not been well characterized. In this study, we have examined the biological functions of VAP-C in the propagation of HCV. VAP-C interacted with NS5B but not with VAP-A, VAP-B, or NS5A in immunoprecipitation analyses, and the expression of VAP-C inhibited the interaction of NS5B with VAP-A or VAP-B. Overexpression of VAP-C impaired the RNA replication of the HCV replicon and the propagation of the HCV JFH1 strain, whereas overexpression of VAP-A and VAP-B enhanced the replication. Furthermore, the expression of VAP-C was observed in various tissues, whereas it was barely detected in the liver. These results suggest that VAP-C acts as a negative regulator of HCV propagation and that the expression of VAP-C may participate in the determination of tissue tropism of HCV propagation.

DOI： 10.1128/JVI.00889-09

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATL ACAD SCIENCES  

Hepatitis E virus (HEV) is a causative agent of acute hepatitis. The crystal structure of HEV-like particles (HEV-LP) consisting of capsid protein was determined at 3.5-angstrom resolution. The capsid protein exhibited a quite different folding at the protruding and middle domains from the members of the families of Caliciviridae and Tombusviridae, while the shell domain shared the common folding. Tyr-288 at the 5-fold axis plays key roles in the assembly of HEV-LP, and aromatic amino acid residues are well conserved among the structurally related viruses. Mutational analyses indicated that the protruding domain is involved in the binding to the cells susceptive to HEV infection and has some neutralization epitopes. These structural and biological findings are important for understanding the molecular mechanisms of assembly and entry of HEV and also provide clues in the development of preventive and prophylactic measures for hepatitis E.

DOI： 10.1073/pnas.0903699106

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

We have previously reported on the ubiquitylation and degradation of hepatitis C virus core protein. Here we demonstrate that proteasomal degradation of the core protein is mediated by two distinct mechanisms. One leads to polyubiquitylation, in which lysine residues in the N-terminal region are preferential ubiquitylation sites. The other is independent of the presence of ubiquitin. Gain- and loss-of-function analyses using lysineless mutants substantiate the hypothesis that the proteasome activator PA28 gamma, a binding partner of the core, is involved in the ubiquitin-independent degradation of the core protein. Our results suggest that turnover of this multifunctional viral protein can be tightly controlled via dual ubiquitin-dependent and -independent proteasomal pathways.

DOI： 10.1128/JVI.01690-08

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者,　責任著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）  

DOI： 10.2222/jsv.58.183

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PubMed

記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：秀潤社  

CiNii Books

その他リンク： http://search.jamas.or.jp/link/ui/2009042020

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Hepatitis C virus (HCV) core protein has shown to be localized in the detergent-resistant membrane (DRM), which is distinct from the classical raft fraction including caveolin, although the biological significance of the DRM localization of the core protein has not been determined. The HCV core protein is cleaved off from a precursor polyprotein at the lumen side of Ala(191) by signal peptidase and is then further processed by signal peptide peptidase (SPP) within the transmembrane region. In this study, we examined the role of SPP in the localization of the HCV core protein in the DRM and in viral propagation. The C terminus of the HCV core protein cleaved by SPP in 293T cells was identified as Phe(177) by mass spectrometry. Mutations introduced into two residues (Ile(176) and Phe(177)) upstream of the cleavage site of the core protein abrogated processing by SPP and localization in the DRM fraction. Expression of a dominant-negative SPP or treatment with an SPP inhibitor, L685,458, resulted in reductions in the levels of processed core protein localized in the DRM fraction. The production of HCV RNA in cells persistently infected with strain JFH-1 was impaired by treatment with the SPP inhibitor. Furthermore, mutant JFH-1 viruses bearing SPP-resistant mutations in the core protein failed to propagate in a permissive cell line. These results suggest that intramembrane processing of HCV core protein by SPP is required for the localization of the HCV core protein in the DRM and for viral propagation.

DOI： 10.1128/JVI.00306-08

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) regulates viral replication through its interaction with host and other viral proteins. We have previously shown that FK506-binding protein 8 (FKBP8) binds to NS5A and recruits Hsp90 to form a complex that participates in the replication of HCV. In this study, we examined the biochemical characteristics of the interaction and the intracellular localization of NS5A and FKBP8. Surface plasmon resonance analysis revealed that the dissociation constant of the interaction between the purified FKBP8 and NS5A expressed in bacteria was 82 nM. Mutational analyses of NS5A revealed that a single amino acid residue of Val or Ile at position 121, which is well conserved among all genotypes of HCV, is critical for the specific interaction with FKBP8. Substitution of the Val(121) to Ala drastically impaired the replication of HCV replicon cells, and the drug-resistant replicon cells emerging after drug selection were shown to have reverted to the original arrangement by replacing Ala(121) with Val. Examination of individual fields of the replicon cells by both fluorescence microscopy and electron microscopy (the correlative fluorescence microscopy-electron microscopy technique) revealed that FKBP8 is partially colocalized with NS5A in the cytoplasmic structure known as the membranous web. These results suggest that specific interaction of NS5A with FKBP8 in the cytoplasmic compartment plays a crucial role in the replication of HCV.

DOI： 10.1128/JVI.02253-07

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The NS3 protein of Japanese encephalitis virus (JEV) is a large multifunctional protein possessing protease, helicase, and nucleoside 5'-triphosphatase (NTPase) activities, and plays important roles in the processing of a viral polyprotein and replication. To clarify the enzymatic properties of NS3 protein from a structural point of view, an enzymatically active fragment of the JEV NTPase/helicase catalytic domain was expressed in bacteria and the crystal structure was determined at 1.8 angstrom resolution. JEV helicase is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Each of the motifs I (Walker A motif), 11 (Walker B motif) and VI was composed of an NTP-binding pocket. Mutation analyses revealed that all of the residues in the Walker A motif (Gly(199) , LYS200 and Thr(201)), in addition to the polar residues within the NTP-binding pocket (Gln(199), Arg(200) and Arg(464)), and also Arg(459) in the outside of the pocket in the motif IV were crucial for ATPase and helicase activities and virus replication. Lys(200) was particularly indispensable, and could not be exchanged for other amino acid residues without sacrificing these activities. The structure of the NTP-binding pocket of JEV is well conserved in dengue virus and yellow fever virus, while different from that of hepatitis C virus. The detailed structural comparison among the viruses of the family Flaviviridae should help in clarifying the molecular mechanism of viral replication and in providing rationale for the development of appropriate therapeutics. (C) 2008 Published by Elsevier Inc.

DOI： 10.1016/j.virol.2007.12.018

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is required for the replication of the viral genome and is involved in several host signaling pathways. To gain further insight into the functional role of NS5A in HCV replication, we screened human cDNA libraries by a yeast two-hybrid system using NS5A as the bait and identified human butyrate-induced transcript 1 (hB-ind1) as a novel NS5A-binding protein. Endogenously and exogenously expressed hB-ind1 was coimmunoprecipitated with NS5A of various genotypes through the coiled-coil domain of hB-ind1. The small interfering RNA (siRNA)-mediated knockdown of hB-ind1 in human hepatoma cell lines suppressed the replication of HCV RNA replicons and the production of infectious particles of HCV genotype 2a strain JFH1. Furthermore, these reductions were canceled by the expression of an siRNA-resistant hB-ind1 mutant. Among the NS5A-binding host proteins involved in HCV replication, hB-ind1 exhibited binding with FKBP8, and hB-ind1 interacted with Hsp90 through the FxxW motif in its N-terminal p23 homology domain. The impairment of the replication of HCV RNA replicons and of the production of infectious particles of JFH1 virus in the hB-ind1 knockdown cell lines was not reversed by the expression of an siRNA-resistant hB-ind1 mutant in which the FxxW motif was replaced by AxxA. These results suggest that hB-ind1 plays a crucial role in HCV RNA replication and the propagation of JFH1 virus through interaction with viral and host proteins.

DOI： 10.1128/JVI.02153-07

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記述言語：英語   掲載種別：論文集（書籍）内論文   出版者・発行元：World Scientific Publishing Co.  

Hepatitis C virus (HCV) infection is a major health problem worldwide for almost two decades. However, precise mechanisms including an infection of HCV to target cells or a cause of hepatocellular carcinoma are largely unknown. Study of HCV has been hampered by the lack of a robust and reliable cell culture system and a small animal model that supports replication of HCV. Understanding the mechanisms of HCV infection is essential for the development of new effective therapies for chronic hepatitis C. HCV presumably binds to specific receptor(s) and then enters into cells by endocytosis, as do other members of the Flaviviridae family and several host membrane proteins have been identified as receptor candidates for HCV. Recent advances of pseudotype virus systems based on vesicular stomatitis virus or retrovirus have provided further information surrounding the initial steps of HCV infection. In this Chapter, we will present current status of our knowledge on the mechanisms of HCV infection including candidate receptors for HCV. (161 words).

DOI： 10.1142/9789812790859_0006

Scopus

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The baculovirus Autographa californica multiple nucleopolyhedrovirus has been widely used not only to acheive a high level of foreign gene expression in insect cells, but also for efficient gene transduction into mammalian cells. Recombinant and pseudotyped baculoviruses possessing chimeric or foreign ligands have been constructed to improve the efficiency of gene transduction and to confer specificity for gene delivery into mammalian cells, respectively. Baculoviral DNA CpG motifs induce proinflammatory cytokines through a Toll-like receptor (TLR9)/ MyD88-dependent signaling pathway. Other baculovirus components produce type I interferons via a TLR-independent pathway-Baculovirus exhibits a strong adjuvant property and recombinant baculoviruses encoding microbial antigens elicit antibodies to the antigens and provide protective immunity in mice. This review deals with recent progress in the application of baculovirus vectors to gene delivery and vaccine development, and discusses the future prospects of baculovirus vectors. © 2008 Future Medicine Ltd.

DOI： 10.2217/17460794.3.1.35

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

Hepatitis C virus (HCV) is a major cause of chronic liver diseases, including steatosis, cirrhosis and hepatocellular carcinoma, and epidemiological studies indicate that HCV is also associated with insulin resistance and type 2 diabetes mellitus. The HCV core protein is not only a viral structural component but also a pathogenic factor, since its expression leads to the development of liver steatosis, insulin resistance and hepatocellular carcinoma in mice. The nuclear proteasome activator PA28 gamma/REG gamma, which specifically binds to the core protein, is required for the virulence of the core protein. Elucidation of the mechanisms by which HCV core protein participates in the above conditions may provide clues toward the development of novel therapeutic measures for chronic hepatitis C. (C) 2008 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biocel.2008.01.027

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担当区分：筆頭著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）  

DOI： 10.2222/jsv.57.141

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担当区分：筆頭著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Development of therapeutics for chronic hepatitis C has been hampered by the lack of an efficient cell culture system and a small animal model for the hepatitis C virus (HCV). An RNA replicon system, in which the HCV genome replicates autonomously in cells, and replication competent viruses derived from an HCV genotype 2a JFH1 strain efficiently propagating in Huh7 cells have been developed, and these systems have contributed to the evaluation of anti-HCV drugs targeted to viral and host proteins involved in the replication of HCV. Several compounds counteracting the viral enzymes, such as RNA polymerase and proteases, and host proteins involved in the lipid synthesis and protein folding are reported to have anti-HCV activities based on assessments using these in vitro systems. Furthermore, a mouse model transplanted with human liver fragments was shown to be capable of replicating HCV and has been used to evaluate the efficacy of antiviral drugs in vivo. In this review, we summarize information regarding systems for studying the HCV life cycle and potential new targets for therapeutic intervention for chronic hepatitis C.

DOI： 10.1016/j.addr.2007.04.015

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Hepatitis C virus (HCV) infection induces a wide range of chronic liver injuries; however, the mechanism through which HCV evades the immune surveillance system remains obscure. Blood dendritic cells (DCs) play a pivotal role in the recognition of viral infection and the induction of innate and adaptive immune responses. Several reports suggest that HCV infection induces the dysfunction of DCs in patients with chronic hepatitis C. Toll-like receptor (TLR) has been shown to play various roles in many viral infections; however, the involvement of HCV proteins in the TLR signaling pathway has not yet been precisely elucidated. In this study, we established mouse macrophage cell lines stably expressing HCV proteins and determined the effect of HCV proteins on the TLR signaling pathways. Immune cells expressing NS3, NS3/4A, NS4B, or NS5A were found to inhibit the activation of the TLR2, TLR4, TLR7, and TLR9 signaling pathways. Various genotypes of NS5A bound to MyD88, a major adaptor molecule in TLR, inhibited the recruitment of interleukin-1 receptor-associated kinase I to MyD88, and impaired cytokine production in response to TLR ligands. Amino acid residues 240 to 280, previously identified as the interferon sensitivity-determining region (ISDR) in NS5A, interacted with the death domain of MyD88, and the expression of a mutant NS5A lacking the ISDR partially restored cytokine production. These results suggest that the expression of HCV proteins modulates the TLR signaling pathway in immune cells.

DOI： 10.1128/JVI.00649-07

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JOHN WILEY & SONS LTD  

Hepatitis C virus (HCV) is the major causative agent of blood-borne hepatitis. The majority of HCV-infected individuals develop chronic hepatitis, which eventually progresses to liver cirrhosis, and hepatocellular carcinoma. Although the precise mechanisms of entry, replication, assembly, egress and pathogenesis of HCV are largely unknown, information about viral receptor candidates has accumulated by the development of pseudotype viruses and an in vitro replication system of the HCV JFH1 strain. Furthermore, the autonomous RNA replication system based on the artificial viral genome revealed that HCV replicates in the intracellular replication complex composed of viral and host proteins. Recently, an immunosuppressant, cyclosporin A and inhibitors for sphingolipid synthesis and chaperon were reported to inhibit the replication of HCV by counteracting the interplay between host and viral proteins. This review considers the current knowledge of the host proteins that participate in HCV replication and the possibility of developing novel therapeutics intervention for chronic hepatitis C. Copyright (c) 2007 John Wiley & Sons, Ltd.

DOI： 10.1002/rmv.542

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

The flavivirus capsid protein not only is a component of nucleocapsids but also plays a role in viral replication. In this study, we found a small capsid protein in cells infected with Japanese encephalitis virus (JEV) but not in the viral particles. The small capsid protein was shown to be generated by processing with host cysteine protease cathepsin L. An in vitro cleavage assay revealed that cathepsin L cleaves the capsid protein between amino acid residues Lys(18) and Arg(19), which are well conserved among the mosquito-borne flaviviruses. A mutant JEV resistant to the cleavage of the capsid protein by cathepsin L was generated from an infectious cDNA clone of JEV by introducing a substitution in the cleavage site. The mutant JEV exhibited growth kinetics similar to those of the wild-type JEV in monkey (Vero), mosquito (C6/36), and porcine (PK15) cell lines, whereas replication of the mutant JEV in mouse macrophage (RAW264.7) and neuroblastoma (N18) cells was impaired. Furthermore, the neurovirulence and neuroinvasiveness of the mutant JEV to mice were lower than those of the wild-type JEV. These results suggest that the processing of the JEV capsid protein by cathepsin L plays a crucial role in the replication of JEV in neural and macrophage cells, which leads to the pathogenesis of JEV infection.

DOI： 10.1128/JVI.00477-07

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Although in vitro replication of the hepatitis C virus (HCV) JFH1 clone of genotype 2a (HCVcc) has been developed, a robust cell culture system for the la and 1b genotypes, which are the most prevalent viruses in the world and resistant to interferon therapy, has not yet been established. As a surrogate virus system, pseudotype viruses transiently bearing HCV envelope proteins based on the vesicular stomatitis virus (VSV) and retrovirus have been developed. Here, we have developed a replication-competent recombinant VSV with a genome encoding unmodified HCV El and E2 proteins in place of the VSV envelope protein (HCVrv) in human cell lines. HCVrv and a pseudotype VSV bearing the unmodified HCV envelope proteins (HCVpv) generated in 293T or Huh7 cells exhibited high infectivity in Huh7 cells. Generation of infectious HCVrv was limited in some cell lines examined. Furthermore, HCVrv but not HCVpv was able to propagate and form foci in Huh7 cells. The infection of Huh7 cells with HCVpv and HCVrv was neutralized by anti-hCD81 and anti-E2 antibodies and by sera from chronic HCV patients. The infectivity of HCVrv was inhibited by an endoplasmic reticulum alpha-glucosidase inhibitor, N-(n-nonyl) deoxynojirimycin (Nn-DNJ), but not by a Golgi mannosidase inhibitor, deoxymannojirimycin. Focus formation of HCVrv in Huh7 cells was impaired by Nn-DNJ treatment. These results indicate that the HCVrv developed in this study can be used to study HCV envelope proteins with respect to not only the biological functions in the entry process but also their maturation step.

DOI： 10.1128/JVI.00608-07

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional Protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.

DOI： 10.1128/JVI.01684-06

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

The hepatitis C virus (HCV) core protein is a component of nucleocapsids and a pathogenic factor for hepatitis C. Several epidemiological and experimental studies have suggested that HCV infection is associated with insulin resistance, leading to type 2 diabetes. We have previously reported that HCV core gene-transgenic (PA28 gamma(+/+)CoreTg) mice develop marked insulin resistance and that the HCV core protein is degraded in the nucleus through a PA28 gamma-dependent pathway. In this study, we examined whether PA28 gamma is required for HCV core-induced insulin resistance in vivo. HCV core gene-transgenic mice lacking the PA28 gamma gene (PA28 gamma(-/-)CoreTg) were prepared by mating of PA28 gamma(+/+)CoreTg with PA28 gamma-knockout mice. Although there was no significant difference in the glucose tolerance test results among the mice, the insulin sensitivity in PA28 gamma(-/-)CoreTg mice was recovered to a normal level in the insulin tolerance test. Tyrosine phosphorylation of insulin receptor substrate 1 (IRS1), production of IRS2, and phosphorylation of Akt were suppressed in the livers of PA28 gamma(+/+)CoreTg mice in response to insulin stimulation, whereas they were restored in the livers of PA28 gamma(+/+)CoreTg mice. Furthermore, activation of the tumor necrosis factor alpha promoter in human liver cell lines or mice by the HCV core protein was suppressed by the knockdown or knockout of the PA28 gamma gene. These results suggest that the HCV core protein suppresses insulin signaling through a PA28 gamma-dependent pathway.

DOI： 10.1128/JVI.01683-06

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担当区分：筆頭著者   記述言語：英語  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATL ACAD SCIENCES  

Hepatitis C virus (HCV) is a major cause of chronic liver disease that frequently leads to steatosis, cirrhosis, and eventually hepatocellular carcinoma (HCC). HCV core protein is not only a component of viral particles but also a multifunctional protein because liver steatosis and HCC are developed in HCV core gene-transgenic (CoreTg) mice. Proteasome activator PA28 gamma/REG gamma regulates host and viral proteins such as nuclear hormone receptors and HCV core protein. Here we show that a knockout of the PA28 gamma gene induces the accumulation of HCV core protein in the nucleus of hepatocytes of CoreTg mice and disrupts development of both hepatic steatosis and HCC. Furthermore, the genes related to fatty acid biosynthesis and srebp-1c promoter activity were up-regulated by HCV core protein in the cell line and the mouse liver in a PA28y-dependent manner. Heterodimer composed of liver X receptor alpha (LXR alpha) and retinoid X receptor alpha (RXR alpha) is known to up-regulate srebp-1c promoter activity. Our data also show that HCV core protein enhances the binding of LXR alpha/RXR alpha to LXR-response element in the presence but not the absence of PA28 gamma. These findings suggest that PA28 gamma plays a crucial role in the development of liver pathology induced by HCV infection.

DOI： 10.1073/pnas.0607312104

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Hepatitis C virus (HCV) contains two membrane-associated envelope glycoproteins, E1 and E2, which assemble as a heterodimer in the endoplasmic reticulum (ER). In this study, predictive algorithms and genetic analyses of deletion mutants and glycosylation site variants of the E1 glycoprotein were used to suggest that the glycoprotein can adopt two topologies in the ER membrane: the conventional type I membrane topology and a polytopic topology in which the protein spans the ER membrane twice with an intervening cytoplasmic loop (amino acid residues 288 to 360). We also demonstrate that the E1 glycoprotein is able to associate with the HCV core protein, but only upon oligomerization of the core protein in the presence of tRNA to form capsid-like structures. Yeast two-hybrid and immunoprecipitation analyses reveal that oligomerization of the core protein is promoted by amino acid residues 72 to 91 in the core. Furthermore, the association between the E1 glycoprotein and the assembled core can be recapitulated using a fusion protein containing the putative cytoplasmic loop of the E1 glycoprotein. This fusion protein is also able to compete with the intact E1 glycoprotein for binding to the core. Mutagenesis of the cytoplasmic loop of E1 was used to define a region of four amino acids (residues 312 to 315) that is important for interaction with the assembled HCV core. Taken together, our studies suggest that interaction between the self-oligomerized HCV core and the E1 glycoprotein is mediated through the cytoplasmic loop present in a polytopic form of the E1 glycoprotein.

DOI： 10.1128/JVI.01203-06

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a component of viral replicase and is well known to modulate the functions of several host proteins. Here, we show that NS5A specifically interacts with FKBP8, a member of the FK506-binding protein family, but not with other homologous immunophilins. Three sets of tetratricopeptide repeats in FKBP8 are responsible for interactions with NS5A. The siRNA-mediated knockdown of FKBP8 in a human hepatoma cell line harboring an HCV RNA replicon suppressed HCV RNA replication, and this reduction was reversed by the expression of an siRNA-resistant FKBP8 mutant. Furthermore, immunoprecipitation analyses revealed that FKBP8 forms a complex with Hsp90 and NS5A. Treatment of HCV replicon cells with geldanamycin, an inhibitor of Hsp90, suppressed RNA replication in a dose-dependent manner. These results suggest that the complex consisting of NS5A, FKBP8, and Hsp90 plays an important role in HCV RNA replication.

DOI： 10.1038/sj.emboj.7601367

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Although processing of the hepatitis C virus (HCV) polyprotein and characterization of each of its viral proteins have been described in detail, analysis of the structure and assembly of HCV particles has been hampered by the lack of a robust cell culture system to support efficient replication of HCV. In this study, we generated HCV-like particles (HCV-LP) using a recombinant baculovirus encoding structural and a part of non-structural proteins in a human hepatoma cell line. The HCV-LP exhibited a buoyant density of 1.17 g/ml in CsCl equilibrium gradient and particles of 40 to 50 nm in diameter. Binding of the HCV-LP to human hepatoma cells was partially inhibited by the treatment with anti-hCD81 antibody, in contrast to the hCD81-independent binding of HCV-LP produced in insect cells. These results indicate that HCV-LP generated in different types of cells exhibit different cellular tropism for binding to target cells. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2005.12.001

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CENTER ACADEMIC PUBL JAPAN  

Japanese encephalitis virus (JEV) core protein is detected not only in the cytoplasm but also in the nucleoli of infected cells. We previously showed that a mutant JEV lacking the nucleolar localization of the core protein impaired viral replication in mammalian cells. In this study, we identified a nucleolar phosphoprotein B23 as a protein binding with the core protein of JEV but not with that of dengue virus. The region binding with JEV core protein was mapped to amino acid residues 38 to 77 of B23. Upon JEV infection, some fraction of B23 was translocated from the nucleoli to the cytoplasm, and cytoplasmic B23 was colocalized with the core protein of wild-type JEV but not with that of the mutant JEV. Furthermore, overexpression of dominant negatives of B23 reduced JEV replication. These results suggest that B23 plays an important role in the intracellular localization of the core protein and replication of JEV.

DOI： 10.1111/j.1348-0421.2006.tb03789.x

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PubMed

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

CiNii Books

記述言語：日本語   掲載種別：研究論文（その他学術会議資料等）  

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

The hepatitis C virus (HCV) nonstructural protein (NS) 5A is a phosphoprotein that associates with various cellular proteins and participates in the replication of the HCV genome. Human vesicle-associated membrane protein-associated protein (VAP) subtype A (VAP-A) is known to be a host factor essential for HCV replication by binding to both NS5A and NS5B. To obtain more information on the NS5A protein in HCV replication, we screened human brain and liver libraries by a yeast two-hybrid system using NS5A as bait and identified VAP-B as an WA-binding protein. Immunoprecipitation and mutation analyses revealed that VAP-B binds to both NS5A and NS5B in mammalian cells and forms homo- and heterodimers with VAP-A. VAP-A interacts with VAP-B through the transmembrane domain. NS5A interacts with the coiled-coil domain of VAP-B via 70 residues in the N-terminal and 341 to 344 amino acids in the C-terminal polyproline cluster region. NS5A was colocalized with VAP-B in the endoplasmic reticulum and Golgi apparatus. The specific antibody to VAP-B suppressed HCV RNA replication in a cell-free assay. Overexpression of VAP-B, but not of a mutant lacking its transmembrane domain, enhanced the expression of NS5A and NS5B and the replication of HCV RNA in Huh-7 cells harboring a subgenomic replicon. In the HCV replicon cells, the knockdown of endogenous VAP-B by small interfering RNA decreased expression of NS5B, but not of NS5A. These results suggest that VAP-B, in addition to VAP-A, plays an important role in the replication of the HCV genome.

DOI： 10.1128/JVI.79.21.13473-13482.2005

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PubMed

記述言語：日本語   掲載種別：（MISC）総説・解説（学術雑誌）   出版者・発行元：(有)科学評論社  

担当区分：筆頭著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）  

CiNii Books

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

We have previously shown that mice inoculated intranasally with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus [AcNPV]) are protected from a lethal challenge by influenza virus. However, the precise mechanism of induction of this protective immune response by the AcNPV treatment remained unclear. Here we show that AcNPV activates immune cells via the Toll-like receptor 9 (TLR9)/MyD88-dependent signaling pathway. The production of inflammatory cytokines was severely reduced in peritoneal macrophages (PECs) and splenic CD11c(+) dendritic cells (DCs) derived from mice deficient in MyD88 or TLR9 after cultivation with AcNPV. In contrast, a significant amount of alpha interferon (IFN-alpha.) was still detectable in the PECs and DCs of these mice after stimulation with AcNPV, suggesting that a TLR9/MyD88-independent signaling pathway might also participate in the production of IFN-alpha by AcNPV. Since previous work showed that TLR9 ligands include bacterial DNA and certain oligonucleotides containing unmethylated CpG dinucleotides, we also examined the effect of baculoviral DNA on the induction of innate immunity. Transfection of the murine macrophage cell line RAW264.7 with baculoviral DNA resulted in the production of the inflammatory cytokine, while the removal of envelope glycoproteins from viral particles, UV irradiation of the virus, and pretreatment with purified baculovirus envelope proteins or endosomal maturation inhibitors diminished the induction of the immune response by AcNPV. Together, these results indicate that the internalization of viral DNA via membrane fusion mediated by the viral envelope glycoprotein, as well as endosomal maturation, which releases the viral genome into TLR9-expressing cellular compartments, is necessary for the induction of the innate immune response by AcNPV.

DOI： 10.1128/JVI.79.5.2847-2858.2005

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Japanese encephalitis virus (JEV) core protein was detected in both the nucleoli and cytoplasm of mammalian and insect cell lines infected with JEV or transfected with the expression plasmid of the core protein. Mutation analysis revealed that Gly(42) and Pro(43) in the core protein are essential for the nuclear and nucleolar localization. A mutant M4243 virus in which both Gly(42) and Pro(43) were replaced by Ala was recovered by plasmid-based reverse genetics. In C6/36 mosquito cells, the M4243 virus exhibited RNA replication and protein synthesis comparable to wild-type JEV, whereas propagation in Vero cells was impaired. The mutant core protein was detected in the cytoplasm but not in the nucleus of either C6/36 or Vero cell lines infected with the M4243 virus. The impaired propagation of M4243 in mammalian cells was recovered by the expression of wild-type core protein in trans but not by that of the mutant core protein. Although M4243 mutant virus exhibited a high level of neurovirulence comparable to wild-type JEV in spite of the approximately 100-fold-lower viral propagation after intracerebral inoculation to 3-week-old mice of strain Jcl:ICR, no virus was recovered from the brain after intraperitoneal inoculation of the mutant. These results indicate that nuclear localization of JEV core protein plays crucial roles not only in the replication in mammalian cells in vitro but also in the pathogenesis of encephalitis induced by JEV in vivo.

DOI： 10.1128/JVI.79.6.3448-3458.2005

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can infect a variety of mammalian cells, as well as insect cells, facilitating its use as a viral vector for gene delivery into mammalian cells. Glycoprotein gp64, a major component of the budded AcMNPV envelope, is involved in viral entry into cells by receptor-mediated endocytosis and subsequent membrane fusion. We examined the potential production of pseudotype baculovirus particles transiently carrying ligands of interest in place of gp64 as a method of ligand-directed gene delivery into target cells. During amplification of a gp64-null pseudotype baculovirus carrying a green fluorescent protein gene in gp64-expressing insect cells, however, we observed the high-frequency appearance of a replication-competent virus incorporating the gp64 gene into the viral genome. To avoid generation of replication-competent revertants, we prepared pseudotype baculoviruses by transfection with recombinant bacmids without further amplification in the gp64-expressing cells. We constructed gp64-null recombinant bacmids carrying cDNAs encoding either vesicular stomatitis virus G protein (VSVG) or measles virus receptors (CD46 or SLAM). The VSVG pseudotype baculovirus efficiently transduced a reporter gene into a variety of mammalian cell lines, while CD46 and SLAM pseudotype baculoviruses allowed ligand-receptor-directed reporter gene transduction into target cells expressing measles virus envelope glycoproteins. Gene transduction mediated by the pseudotype baculoviruses could be inhibited by pretreatment with specific antibodies. These results indicate the possible application of pseudotype baculoviruses in ligand-directed gene delivery into target cells.

DOI： 10.1128/JVI.79.6.3639-3652.2005

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Hepatitis C virus (HCV) core protein is a putative nucleocapsid protein with a number of regulatory functions. In tissue culture cells, HCV core protein is mainly located at the endoplasmic reticulum as well as mitochondria and lipid droplets within the cytoplasm. However, it is also detected in the nucleus in some cells. To elucidate the mechanisms by which cellular trafficking of the protein is controlled, we performed subcellular fractionation experiments and used confocal microscopy to examine the distribution of heterologously expressed fusion proteins involving various deletions and point mutations of the HCV core combined with green fluorescent proteins. We demonstrated that a region spanning amino acids 112 to 152 can mediate association of the core protein not only with the ER but also with the mitochondrial outer membrane. This region contains an 18-amino-acid motif which is predicted to form an amphipathic alpha-helix structure. With regard to the nuclear targeting of the core protein, we identified a novel bipartite nuclear localization signal, which requires two out of three basic-residue clusters for efficient nuclear translocation, possibly by occupying binding sites on importin-a. Differences in the cellular trafficking of HCV core protein, achieved and maintained by multiple targeting functions as mentioned above, may in part regulate the diverse range of biological roles of the core protein.

DOI： 10.1128/JVI.79.2.1271-1281.2005

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PubMed

記述言語：日本語   掲載種別：研究論文（その他学術会議資料等）  

J-GLOBAL

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

Scopus

PubMed

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

Scopus

PubMed

担当区分：筆頭著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：(株)日本臨床社  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Hepatitis C virus (HCV) core protein is suggested to localize to the endoplasmic reticulum (ER) through a C-terminal hydrophobic region that acts as a membrane anchor for core protein and as a signal sequence for El protein. The signal sequence of core protein is further processed by signal peptide peptidase (SPP). We examined the regions of core protein responsible for ER retention and processing by SPP. Analysis of the intracellular localization of deletion mutants of HCV core protein revealed that not only the C-terminal signal-anchor sequence but also an upstream hydrophobic region from amino acid 128 to 151 is required for ER retention of core protein. Precise mutation analyses indicated that replacement of Leu(139), Val(140), and Lett(144) of core protein by Ala inhibited processing by SPP, but cleavage at the core-E1 junction by signal peptidase was maintained. Additionally, the processed El protein was translocated into the ER and glycosylated with high-mannose oligosaccharides. Core protein derived from the mutants was translocated into the nucleus in spite of the presence of the unprocessed C-terminal signal-anchor sequence. Although the direct association of core protein with a wild-type SPP was not observed, expression of a loss-of-function SPP mutant inhibited cleavage of the signal sequence by SPP and coimmunoprecipitation with unprocessed core protein. 139 140 144 These results indicate that Leu(139), Val(140), and Leu(144) in core protein play crucial roles in the ER retention and SPP cleavage of HCV core protein.

DOI： 10.1128/JVI.78.12.6370-6380.2004

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PubMed

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

DOI： 10.2222/jsv.53.185

Scopus

PubMed

記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：日本ウイルス学会  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Hepatitis C virus (HCV) core protein plays an important role in the formation of the viral nucleocapsid and a regulatory protein involved in hepatocarcinogenesis. In this study, we have identified proteasome activator PA28gamma (1 IS regulator gamma) as an HCV core binding protein by using yeast two-hybrid system. This interaction was demonstrated not only in cell culture but also in the livers of HCV core transgenic mice. These findings are extended to human HCV infection by the observation of this interaction in liver specimens from a patient with chronic HCV infection. Neither the interaction of HCV core protein with other PA28 subtypes nor that of PA28gamma with other Flavivirus core proteins was detected. Deletion of the PA28gamma-binding region from the HCV core protein or knockout of the PA28gamma gene led to the export of the HCV core protein from the nucleus to the cytoplasm. Overexpression of PA28gamma enhanced the proteolysis of the HCV core protein. Thus, the nuclear retention and stability of the HCV core protein is regulated via a PA28gamma-dependent pathway through which HCV pathogenesis may be exerted.

DOI： 10.1128/JVI.77.19.10237-10249.2003

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Although recombinant baculovirus vectors can be an efficient tool for gene transfer into mammalian cells in vitro, gene transduction in vivo has been hampered by the inactivation of baculoviruses by serum complement. Recombinant baculoviruses possessing excess envelope protein gp64 or other viral envelope proteins on the virion surface deliver foreign genes into a variety of mammalian cell lines more efficiently than the unmodified baculovirus. In this study, we examined the efficiency of gene transfer both in vitro and in vivo by recombinant baculoviruses possessing envelope proteins derived from either vesicular stomatitis virus (VSVG) or rabies virus. These recombinant viruses efficiently transferred reporter genes into neural cell lines, primary rat neural cells, and primary mouse osteal cells in vitro. The VSVG-modified bacullovirus exhibited greater resistance to inactivation by animal sera than the unmodified baculovirus. A synthetic inhibitor of the complement activation pathway circumvented the serum inactivation of the unmodified baculovirus. Furthermore, the VSVG-modified baculovirus could transduce a reporter gene into the cerebral cortex and testis of mice by direct inoculation in vivo. These results suggest the possible use of the recombinant bacullovirus vectors in combination with the administration of complement inhibitors for in vivo gene therapy.

DOI： 10.1128/JVI.77.18.9799-9808.2003

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PubMed

記述言語：日本語   掲載種別：（MISC）総説・解説（学術雑誌）   出版者・発行元：(株)最新医学社  

著者等はHCVの感染機構をin vitroで評価するため,HCVエンベロープタンパク質による細胞融合アッセイ系とエンベロープタンパク質を被ったシュードタイプウイルスを作製した.その結果,HCVの2つのエンベロープタンパク質が細胞表面のタンパク質受容体を認識し,エンドサイトーシスによって細胞内へ侵入することが示唆された.更に,HCVのエンベロープタンパク質のCD81との結合や細胞融合活性を阻害できるヒト型抗HCVエンベロープ抗体を得ることができた

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：International Medical Press Ltd  

Hepatitis C virus (HCV) is the major causative agent of chronic non-A, non-B hepatitis. The life cycle of HCV is largely unknown because a reliable culture system has not yet been established. HCV presumably binds to specific receptor(s) and enters cells through endocytosis, as do other members of Flaviviridae. The viral genome is translated into a precursor polyprotein after uncoating, and viral RNA is synthesized by a virus-encoded polymerase complex. Progeny viral particles are released into the luminal side of the endoplasmic reticulum and secreted from the cell after passage through the Golgi apparatus. Understanding the mechanisms of HCV infection is essential to the development of effective new therapies for chronic HCV infection. Several host membrane proteins have been identified as receptor candidates for HCV. Recent advances using pseudotype virus systems have provided information surrounding the initial steps of HCV infection. An HCV RNA replicon system has been useful for elucidating the replication mechanism of HCV. In this review, we summarize our current understanding of the mechanisms of HCV infection and discuss potential antiviral strategies against HCV infection.

DOI： 10.1177/095632020301400601

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PubMed

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

It is known that infection with Sindbis virus (SNV) induces apoptosis, which is inhibited by two pro-survival members of the Bcl-2 family, Bcl-2 and Bcl-xL. However, the mechanism of involvement of the other members of the Bcl-2 family in SNV-induced apoptosis remains unclear. In this study we report that Bad protein, one of the pro-apoptotic Bcl-2 family members, mediates apoptosis in the mammalian cells infected with SNV Expression of Bad was shown to promote SNV-induced apoptosis in human embryonic kidney 293T and baby hamster kidney cells. SNV infection also induced translocation of endogenous Bad into mitochondria and heterodimerization of Bad with Bcl-xL. On the other hand, the structurally most similar pro-survival members, Bcl-2, Bcl-xL, and Bcl-w, suppressed SNV-induced apoptosis in the absence of Bad, whereas Mcl-1 and A1 did not. Bcl-w could inhibit SNV-induced apoptosis in the presence of Bad, but Bcl-xL could not. Bad could be coimmunoprecipitated with Bcl-xL or Bcl-2, but not with Bcl-w. Two viral Bcl-2 homologs, E1B19K and BHRF1, also suppressed SNV-induced apoptosis irrespective of the presence of Bad and no physical association with Bad was observed. These results suggest that direct interaction of Bad with pro-survival members of the Bcl-2 family contributes to the progress of SNV-induced apoptosis and that nonbinding members restrain SNV-induced apoptosis irrespective of Bad expression. (C) 2002 Elsevier Science.

DOI： 10.1006/viro.2001.1206

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC  

The genome of hepatitis C virus (HCV) encodes two envelope glycoproteins (E1 and E2), which are thought to be responsible for receptor binding and membrane fusion resulting in virus penetration. To investigate cell surface determinants important for HCV infection, we used a recombinant vesicular stomatitis virus (VSV) in which the glycoprotein gene was replaced with a reporter gene encoding green fluorescent protein (GFP) and produced HCV-VSV pseudotypes possessing chimeric HCV E1 or E2 glycoproteins, either individually or together. The infectivity of the pseudotypes was determined by quantifying the number of cells expressing the GFP reporter gene. Pseudotypes that contained both of the chimeric E1 and E2 proteins exhibited 10-20 times higher infectivity on HepG2 cells than the viruses possessing either of the glycoproteins individually. These results indicated that both E1 and E2 envelope proteins are required for maximal infection by HCV. The infectivity of the pseudotype virus was not neutralized by anti-VSV polyclonal antibodies, Bovine lactoferrin specifically inhibited the infection of the pseudotype virus. Treatment of HepG2 cells with Pronase, heparinase, and heparitinase but not with phospholipase C and sodium periodate reduced the infectivity. Therefore, cell surface proteins and some glycosaminoglycans play an important role in binding or entry of HCV into susceptible cells, The pseudotype VSV possessing the chimeric HCV glycoproteins might offer an efficient tool for future research on cellular receptors for HCV and for the development of prophylactics and therapeutics for hepatitis C. (C) 2001 Academic Press.

DOI： 10.1006/viro.2001.0971

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Anti-apoptotic members of the Bcl-2 family, such as Bcl-w, maintain cell viability by preventing the activation of the cell death effectors, the caspases, Gene targeting experiments in mice have demonstrated that Bcl-w is required for spermatogenesis and for survival of damaged epithelial cells in the gut. Bcl-w is, however, dispensable for physiological cell death in other tissues. Here we report on the analysis of Bcl-w protein expression using a panel of novel monoclonal antibodies. Bcl-w is found In a diverse range of tissues including colon, brain and testes, A survey of transformed cell lines and purified hematopoietic cells demonstrated that Bcl-w is expressed in cells of myeloid, lymphoid and epithelial origin. Subcellular fractionation and confocal laser scanning microscopy demonstrated that Bcl-w protein is associated with intracellular membranes. The implications of these results are discussed in the context of the phenotype of Bcl-w-null mice and recent data that suggest that Bcl-w may play a role in colon carcinogenesis.

DOI： 10.1038/sj.cdd.4400835

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PubMed

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PHARMACEUTICAL SOC JAPAN  

Lactoferrin (LFR) plays an important role in the anti-microbial defense through iron binding, lipopolysaccharide binding and immunomodulation. In this study, we demonstrate that bovine LFR specifically inhibits the hemolytic activity of listeriolysin O (LLO) produced by Listeria monocytogenes. The hemolytic activity of LLO was completely inhibited in the presence of bovine LFR that was highly purified on two cation-exchange columns, whereas that of streptolysin O or perfringolysin O was not inhibited at all. A rabbit anti-LFR antibody canceled this inhibitory activity of bovine LFR. Although human transferrin exhibits 62% amino acid identity with bovine LFR, human apo-transferrin could not inhibit LLO-induced hemolysis. An increase in the concentration of FeCl3 or the Fe3+-saturation of bovine LFR, however, slightly reduced its inhibition of the hemolysis. The inhibitory activity of bovine LFR was dependent on pH, since it was observed under neutral and alkali conditions, but not under acidic conditions. These results suggest that the inhibition of LLO-induced hemolysis by bovine LFR is influenced by pH and iron ions, both of which may lead to conformational changes of LFR.

DOI： 10.1248/bpb.22.1167

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL SCIENCE ASIA  

Exoenzyme C3 (C3) produced by Clostridium botulinum type C and D strains specifically modifies the small GTP, binding protein Rho. It has been reported that C3 induces neuronal differentiation of the PC12 cell, an adrenergic cell line. In this study, the effects of C3 on a cholinergic cell line (NG108-15) and a glioma (CG) were examined for their differentiation. Treatment of NG108-15 cells and C6 glioma cells with C3 for 3 days induced neurite or process formation. Moreover, treatment by C3 increased the activities of acetylcholine esterase and choline acetyltransferase in NG108-15 cells, and 2',3'-cyclic nucleotide 3'-phosphohydrolase in C6 cells. These results suggest that C3 induces neuronal differentiation of the cholinergic cell as well as the adrenergic cell, and also glial differentiation of the glioma cell.

DOI： 10.1046/j.1440-1789.1999.00247.x

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATL ACAD SCIENCES  

The Bcl-2 family of proteins regulates apoptosis, the cell death program triggered by activation of certain proteases (caspases), An attractive model for how Bcl-2 and its closest relatives prevent caspase activation is that they bind to and inactivate an adaptor protein required for procaspase processing. That model has been supported by reports that mammalian prosurvival Bcl-2 relatives bind the adaptor Apaf-1, which activates procaspase-9, However, the itt vivo association studies reported here with both overexpressed and endogenous Apaf-1 challenge this notion. Apaf-1 could be immunoprecipitated together with procaspase-9, and the Apaf-1 caspase-recruitment domain was necessary and sufficient for their interaction, Apaf-1 did not bind, however, to any of the six known mammalian prosurvival family members (Bcl-2, Bcl-x(L), Bcl-w, A1, Mel-1, or Boo), or their viral homologs adenovirus E1B 19K and Epstein-Barr virus BHRF-1. Endogenous Apaf-1 also failed to coimmunoprecipitate with endogenous Bcl-2 or Bcl-xL, or with two proapoptotic relatives (Bax and Bim), Moreover, apoptotic stimuli did not induce Apaf-1 to bind to these family members. Thus, the prosurvival Bcl-2 homologs do not appear to act by sequestering Apaf-1 and probably instead constrain its activity indirectly.

DOI： 10.1073/pnas.96.17.9683

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PubMed

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：COLD SPRING HARBOR LAB PRESS  

DOI： 10.1101/sqb.1999.64.351

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

1 The aim of this study was to determine whether different signal transduction mechanisms underlie the Ca2+ sensitizing effects of guanosine 5'-O-(3-thiotriphosphate) (GTP(gamma)S) and receptor agonists on beta-escin-skinned smooth muscle of rabbit mesenteric artery.
2 In the homogenate of the beta-escin-skinned arterial strip, C3 exoenzyme of Clostridium botulinum catalyzed the [P-32]-ADP-ribosylation of only one protein that had the same molecular mass as the protein detected in Western blots with anti-rho p21 antibody. Pretreatment of preparations with C3 resulted in great inhibition of GTP(gamma)S-induced Ca2+ sensitization, although the effect of GTP(gamma)S at higher concentrations (greater than or equal to 30 mu M) was not completely blocked by this treatment. In contrast, the enhancement by phenylephrine and histamine, in the presence of guanosine 5'-triphosphate, of the Ca2+-induced contraction was not affected by C3 pretreatment.
3 The protein kinase C (PKC) inhibitors calphostin C and staurosporine completely eliminated the enhancement by phorbol ester 12,13-dibutyrate of the Ca2+-induced contraction. However, these PKC inhibitors had no effect on GTP(gamma)S- and receptor agonist-induced Ca2+ sensitization.
4 The tyrosine kinase inhibitors genistein and tyrphostin 25 caused an irreversible and complete block of the enhancement by GTP(gamma)S of the Ca2+-induced contraction without affecting this Ca2+ contraction. The inactive genistein analogue daidzein did not modify the effect of GTP(gamma)S. The Ca2+ sensitizing effects of phenylephrine and histamine were also blocked by these tyrosine kinase inhibitors.
5 These results suggest that rho p21 predominantly mediates GTP(gamma)S-induced Ca2+ sensitization of beta-escin-skinned smooth muscle of rabbit mesenteric artery, while the Ca2+ sensitizing actions of heterotrimeric G protein-coupled receptor agonists do not involve this small G protein. However, it seems that tyrosine phosphorylation, but not PKC activation, plays an important role in both of the rho p21 protein- and heterotrimeric G protein-mediated Ca2+ sensitization mechanisms.

DOI： 10.1038/sj.bjp.0702242

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：EATON PUBLISHING CO  

Screening assays are the most time-consuming and labor-intensive part of generating monoclonal antibodies (MAbs). Antibodies identified by enzyme-linked immunosorbent assay (ELISA) screening often are not suitable for their intended application such as immunofluorescence staining. We describe here a rapid and efficient flow cytometric screening procedure for the identification of MAbs directed against low-abundance cytoplasmic proteins, in our case, the pro-apoptotic molecule Bim. Cells from all equal mixture of a parental cell line and a subline expressing Bim were fixed, permeabilized and incubated with hybridoma supernatants. The supernatants were derived from a fusion of Sp2/0 plasmacytoma cells and spleen cells from a rat immunized with recombinant glutathione-S-transferase nse (GST)-Bim(L) fusion protein. Secondary staining with fluorochrome-labeled anti-rat Ig antibodies allowed detection of clones expressing Bim-specific antibodies. The screening procedure was rapid and efficient, nod most monoclonal antibodies identified were proven to De useful for immunofluorescence staining and other applications.

DOI： 10.2144/98255st03

Web of Science

PubMed

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CENTER ACADEMIC PUBL JAPAN  

The region encoding proline-rich units of actA genes was amplified from 24 strains of Listeria monocytogenes using polymerase chain reaction (PCR), PCR products of 13 strains showed the expected size of 623 bp, whereas those of 11 strains showed a short size of 518 bp, The shortening of these PCR products resulted from the deletion of one proline-rich unit, These results indicate that ActA proteins are divided into at least two different types which are unrelated to bacterial serotypes.

DOI： 10.1111/j.1348-0421.1998.tb02261.x

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Seventeen monoclonal antibodies (MAbs) were previously established against the heavy chain (He) of botulinum type E neurotoxin in BALB/c mice immunized with the type E toroid, Five MAbs (LE15-5, LE34-6, EK19-7, EK21-4, and AE27-9) showed toxin-neutralizing activity in mice, Two of the five MAbs, EK19-7 and EK21-4, recognized the regions located at amino acid positions 731 to 787 and 811 to 897, respectively, One of the remaining three antibodies (LE34-6) reacted with the amino acid sequence VIKAIN, at amino acid positions 663 to 668, closed by the ion channel-forming domain, It is suggested that the ion channel-forming domain may also be associated with the blocking of acetylcholine release, Furthermore, the amino acid sequence YLTHMRD within 30 residues of the C-terminal region of the He component seemed to be recognized by LE15-5, It has been reported that the binding domain of the type E toxin is located on the C-terminal half of the He component, Therefore, the neutralizing activity of LE15-5 antibody may be attributed to its ability to block the binding of neurotoxin to the receptor of target cells.

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Listeria monocytogenes replicates in a phagocytic cell following escape into the host cytoplasm. Listeriolysin O, secreted by L. monocytogenes, which belongs to the thiol-activated hemolysin family, is known to play an important role in the escape of the bacterium into the host cytoplasm. In this study, we demonstrated that expression of listeriolysin O by infecting L. monocytogenes was lightly induced in J774.1 macrophage-like cells pretreated with lipopolysaccharide, although the growth of the bacteria was suppressed. The number of viable L. monocytogenes decreased until 4 h post-infection and then increased between 3 and 8 h post-infection in untreated J774.1 host cells, but it decreased until 8 h post-infection in lipopolysaccharide-treated host cells. However, expression of listeriolysin O by L. monocytogenes was not induced in the untreated host cells, while it increased between 0 and 4 h post-infection in the lipopolysaccharide-treated host cells. Expression of listeriolysin O mRNA in the lipopolysaccharide-treated host cells was also induced at 2 h post-infection, suggesting that listeriolysin O was newly synthesized in the macrophage-like cells. These results suggest that macrophage activation induced with lipopolysaccharide could lead to the expression of the listeriolysin O gene and the synthesis of listeriolysin O protein under suppression of the intracellular growth of L. monocytogenes.

DOI： 10.1016/S0928-8244(96)00085-5

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PubMed

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Listeria monocytogenes replicates in a phagocytic cell following escape into the host cytoplasm. Listeriolysin O, secreted by L. monocytogenes, which belongs to the thiol-activated hemolysin family, is known to play an important role in the escape of the bacterium into the host cytoplasm. In this study, we demonstrated that expression of listeriolysin O by infecting L. monocytogenes was lightly induced in J774.1 macrophage-like cells pretreated with lipopolysaccharide, although the growth of the bacteria was suppressed. The number of viable L. monocytogenes decreased until 4 h post-infection and then increased between 3 and 8 h post-infection in untreated J774.1 host cells, but it decreased until 8 h post-infection in lipopolysaccharide-treated host cells. However, expression of listeriolysin O by L. monocytogenes was not induced in the untreated host cells, while it increased between 0 and 4 h post-infection in the lipopolysaccharide-treated host cells. Expression of listeriolysin O mRNA in the lipopolysaccharide-treated host cells was also induced at 2 h post-infection, suggesting that listeriolysin O was newly synthesized in the macrophage-like cells. These results suggest that macrophage activation induced with lipopolysaccharide could lead to the expression of the listeriolysin O gene and the synthesis of listeriolysin O protein under suppression of the intracellular growth of L. monocytogenes.

DOI： 10.1111/j.1574-695X.1996.tb00138.x

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PubMed

記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：NATL INST INFECTIOUS DISEASES  

Web of Science

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

We isolated the gene encoding a botulinum neurotoxin (BoNT) of 1285 amino acids with a molecular weight of 147 364 from the toxigenic bacteriophage d-sA of Clostridium botulinum type D strain South African (Dsa). The BoNT of Dsa (BoNT/Dsa) is composed of three regions on the basis of the homology to BoNT types Cl (BoNT/Cl) and D (BoNT/D). The N-terminal (Met-1 to Val-522) and the C-terminal regions (Trp-945 to Glu-1285) have high identity to corresponding regions of BoNT/D (96% identity) and BoNT/Cl (74% identity), respectively. The core region (Pro-523 to Lys-944) is common to three toxins (83% to 92% identity). These results suggest that neurotoxins produced from Clostridium botulinum types C and D an composed in a mosaic-like fashion.

Web of Science

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/0167-4781(96)00006-1

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

The molecular composition of progenitor toxins produced by a Clostridium botulinum type A strain (A-NIH) was analyzed. The strain produced three types of progenitor toxins (19 S, 16 S, and 12 S) as reported previously. Purified 19 S and 16 S toxins demonstrated the same banding profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that they consist of the same protein components. The nontoxic components of the 19 S and 16 S toxins are a nontoxic non-hemagglutinin (HA) (molecular mass, 120 kDa) and HA. HA could be fractionated into five subcomponents with molecular masses of 52, 35, 20, 19, and 15 kDa in the presence of 2-mercaptoethanol. The molar ratios of neurotoxins, nontoxic non-HAs, and each HA subcomponent of the 19 S and 16 S toxins showed that only HA-35 of the 19 S toxin was approximately twice the size of that of the 16 S toxin, suggesting that the 19 S toxin is a dimer of the 16 S toxin cross-linked by the 35-kDa subcomponent. The nontoxic non-HA of the 12 S toxin, but not those of the 19 S and 16 S toxins, demonstrated two bands with molecular masses of 106 and 13 kDa on SDS-PAGE with or without 2-mercaptoethanol. It was concluded from the N-terminal amino acid sequences that 106- and 13-kDa proteins were generated by a cleavage of whole nontoxic non-HA. This may explain why the 12 S and 16 S (and 19 S) toxins exist in the same culture. We also found that the HA and its 35-kDa subcomponent exist in a free state in the culture fluid along with the three types of progenitor toxins.

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

Botulinum C2 toxin is composed of two nonlinked protein components, component-I (light chain) and component-II (heavy chain). It is produced by Clostridium botulinum types C and D, and is thought to play a lethal pathogenic role, These biological activities of C2 toxin may be due to the ADP-ribosylation of non-muscle actin by component-I of the toxin. We were able to isolate two overlapping gene fragments encoding component-I room the chromosomal DNA of Clostridium botulinum type C strain (C)-203U28, and determine the complete nucleotide sequence of component-I gene. The gene for component-I, bc21, consists of one open reading frame (ORF) encoding 431 amino acid residues (1293 nucleotides) without signaling peptide sequence. The molecular mass calculated from the deduced amino acid sequence was 49400.37 Da. Mono-ADP-ribosyltransferase activity was demonstrated in the lysate from E.coli transformed by the recombinant plasmid, pGEM-C2 encompassing whole component-I gene with its own promotor. (C) 1996 Academic Press, Inc.

DOI： 10.1006/bbrc.1996.0409

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PubMed

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

The DNA fragment common to the genes encoding botulinum neurotoxin types C1 (BN/C1) and D (BN/D) was amplified by PCR from the culture supernatant of Clostridium botulinum type C strain 6813 (C6813) that was treated with either DNase I or proteinase K but not from the supernatant that was treated with both DNase I and proteinase K, suggesting the neurotoxin gene is located on a certain bacteriophage DNA. Thus, to isolate the neurotoxin gene, we performed PCR with the culture supernatant of C6813 and seven primer pairs designed from the genes encoding BN/C1 and BN/D. The coding region in the connected sequence encodes a neurotoxin composed of 1,280 amino acids with a molecular weight of 147,817. The neurotoxin from C6813 has 95% amino acid identity to BN/C1, except for its C-terminal one-third, which is quite similar to the C-terminal one-third of BN/D (95% identity). When we performed PCRs with four primer pairs designed from the 5'-terminal two-thirds of the BN/C1 gene and two primers from the 3'-terminal one-third of the BN/D gene, DNA fragments of the expected sizes (0.5 to 1.3 kbp) could be amplified from C. botulinum type C strains 6812 and 6814. These results suggest that some strains of C. botulinum type C contain the gene encoding the mosaic neurotoxin composed of parts of BN/C1 and BN/D.

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（研究会，シンポジウム資料等）   出版者・発行元：NATL INST HEALTH  

Web of Science

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

The genetic structure of neurotoxin genes in a Clostridium botulinum strain producing both type A and B neurotoxins (type AB) was investigated. Analyses by polymerase chain reaction (PCR) using type-specific primers corresponding to the coding regions for N-terminals of light-chains and C-terminals of heavy-chains of type A and type B neurotoxins, and Southern hybridization of total DNA showed that the type AB strain I.P.7212 carries at least one copy each of type A and B neurotoxin genes. Partial nucleotide sequences obtained by direct sequencing of the PCR products indicate that the type A and B genes carried by this strain are not classical type A and non-proteolytic type B genes, but are similar to the type A gene present in a strain which had caused infant botulism in Kyoto and the type B gene present in a proteolytic type B strain. (C) 1995 Academic Press, Inc.

DOI： 10.1006/bbrc.1995.2192

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

In beta-escin-permeabilized cultured pig aortic smooth muscle cells GTP gamma S dose-dependently enhances Ca2+-induced, wortmannin-sensitive phosphorylation of 20 kDa myosin light chain (MLC(20)), GTP gamma S does not potentiate thiophosphorylation of MLC(20), but does inhibit its dephosphorylation, Pretreatment with C, botulinum exotoxin C-3, which specifically ADP-ribosylates and inactivates the rho family of the small molecular weight G proteins, completely abolishes the effects of GTP gamma S, These results indicate that rite is involved in the GTP gamma S-induced enhancement of Ca2+-dependent MLC(20) phosphorylation in aortic smooth muscle cells, and strongly suggest that this effect of rho is due to inhibition of protein phosphatase activity toward MLC(20).

DOI： 10.1016/0014-5793(95)00573-R

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

In beta-escin-permeabilized cultured pig aortic smooth muscle cells GTP gamma S dose-dependently enhances Ca2+-induced, wortmannin-sensitive phosphorylation of 20 kDa myosin light chain (MLC(20)), GTP gamma S does not potentiate thiophosphorylation of MLC(20), but does inhibit its dephosphorylation, Pretreatment with C, botulinum exotoxin C-3, which specifically ADP-ribosylates and inactivates the rho family of the small molecular weight G proteins, completely abolishes the effects of GTP gamma S, These results indicate that rite is involved in the GTP gamma S-induced enhancement of Ca2+-dependent MLC(20) phosphorylation in aortic smooth muscle cells, and strongly suggest that this effect of rho is due to inhibition of protein phosphatase activity toward MLC(20).

DOI： 10.1016/0014-5793(95)00573-r

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We have previously reported the presence of an endogenous inhibitory activity in bovine brain for the ADP-ribosylation of GTP-binding proteins catalyzed by pertussis toxin (PT) (Hara-Yokoyama, M., and Furuyama, S, (1989) Biochem. Biophys. Res. Commun. 160, 67-71). In the present study, we identified the inhibitor as a ganglioside, The screening of various gangliosides revealed that G(Q1b alpha) most effectively inhibited the ADP-ribosyltransferase activities of both the holoenzyme and the catalytic subunit of PT. G(Q1b alpha) is a ganglioside newly identified as one of the antigens recognized by the cholinergic neuron-specific antibody, anti-Chol-1 alpha (Hirabayashi, Y,, Nakao, T,, Irie, F,, Whittaker, V,P., Ken, R,, and Ando, S. (1992) J. Biol. Chem. 267, 12973-12978), G(Q1b alpha) also inhibited the PT-catalyzed NAD(+) glycohydrolysis. Unlike PT activity, the ADP-ribosylation and the NAD(+) glycohydrolysis catalyzed by the C3 exoenzyme from Clostridium botulinum type C were inhibited by G(T1b) and G(Q1b). The ADP-ribosylation catalyzed by either PT or the C3 exoenzyme was not inhibited by ceramide, galactocerebroside, or sialic acid. In addition to the inhibitory action of gangliosides on ADP-ribosylation, the importance of gangliosides as regulators of NAD(+) metabolism is discussed.

DOI： 10.1074/jbc.270.14.8115

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

We examined production of ADP-ribosyltransferase C3 in 11 strains of Clostridium botulinum type C and D and their nontoxigenic derivatives. Antisera to C3 proteins of type C organisms divided C3 proteins roughly into at least two groups, bearing no relation to their bacterial types. The C3 gene of type D strain South African was isolated from a toxigenic phage library, and the complete sequence of the C3 gene was determined. The C3 protein of type D strain South African had 98% homology to the C3 protein of type C strain 003-9 and 66% homology to that of type D strain 1873. These results indicate that there are two types of C3 protein in type D organisms, as there are in type C organisms.

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：HOKKAIDO UNIV  

Web of Science

PubMed

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

Botulinum type D neurotoxin inhibited Ca2+-evoked norepinephrine secretion in digitonin permeabilized PCl2 cells. Inhibition by the toxin required prior incubation with dithiothreitol (DTT). The inhibition was dependent on both concentration and incubation times of the toxin, and was affected by Ca2+ concentration. With less than 0.7 muM Ca2+ almost complete inhibition was observed; however, above 0.7 muM, Ca2+ stimulated additional norepinephrine release in a dose-dependent manner.

DOI： 10.1016/0041-0101(92)90027-3

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PubMed

記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：NATL INST HEALTH  

Web of Science

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Epidermal cell differentiation inhibitor (EDIN) is a recently discovered protein which inhibits terminal differentiation of cultured keratinocytes (Sugai, M., Enomoto, T., Hashimoto, K., Matsumoto, K., Matsuo, Y., Ohgai, H., Hong, Y.-M., Inoue, S., Yoshikawa, K., and Suginaka, H. (1990) Biochem. Biophys. Res. Commun. 173, 92-98). The amino acid sequence deduced from the EDIN gene has revealed that EDIN shares high amino acid sequence homology with the exoenzyme C3 of Clostridium botulinum (Inoue, S., Sugai, M., Murooka, Y., Paik, S.-Y., Hong, Y.-M., Ohgai, H., and Suginaka, H. (1991) Biochem. Biophys. Res. Commun. 174, 459-464), which has been shown to ADP-ribosylate the rho/rac proteins (members of the small GTP-binding protein family). We show here that EDIN ADP-ribosylates rhoB p21 in time- and dose-dependent manners in a cell-free system. Kinetic studies of the ADP-ribosylation and peptide mapping of the reaction products of rhoB p21 by EDIN and C3 suggest that the mode of action of the ADP-ribosylation by EDIN is quite similar to that by C3 and that the ADP-ribosylation site of rhoB p21 by EDIN is presumably the same as that by C3. Proteins in epidermal membranes and keratinocyte homogenate with M(r) values of about 22,000 are ADP-ribosylated by EDIN or C3. Treatment of cultured human keratinocytes by EDIN or C3 results in an inhibition of terminal differentiation and a stimulation of growth of the cells. Moreover, EDIN and C3 injected into adult mouse skin induce hyperplasia of epidermis. These results suggest that EDIN and C3 affect growth and differentiation of keratinocytes by ADP-ribosylation of protein(s) with a M(r) of about 22,000, which may be the rho/rac proteins or related proteins.

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CENTER ACADEMIC PUBL JAPAN  

Chromosomal DNAs were extracted from Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike. The 6.0 Kbp fragment coding for the entire light chain (L) component and the N-terminus of heavy chain (H) component of botulinum type E toxin was obtained from each extracted DNAs after digestion with HindIII. The entire nucleotide sequences for the light chain components of these cloned genes were determined, and the derived amino acid sequences were compared to each other, and with those of botulinum type A, Cl, D, and tetanus toxins reported previously. The cleavage site of L and H components of type E toxin was presumed to be Arg-422. In a total of 422 amino acid residues of L component, 17 residues were different between butyricum and type E toxins, and all these differences were found within 200 residues of N-terminus of L component. On the contrary, five regions showing highly homologous sequences were found in L components among these six toxins, and one more region between botulinum type E and tetanus toxins.

DOI： 10.1111/j.1348-0421.1992.tb01659.x

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PubMed

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

By cation-exchange column chromatography followed by gel filtration or hydroxylapatite column chromatography, ADP-ribosyltransferases (exoenzyme C3) were isolated from culture supernatants of Clostridium botulinum type C strains Stockholm (CST) and 6813 (C6813) and from type D strains South African (DSA) and 1873 (D1873), and their molecular properties were compared. The purified C3 enzymes were homogeneous in polyacrylamide gel electrophoresis. The C3 enzymes existed as single-chain polypeptides with molecular masses of 25.0 to 25.5 kDa and transferred ADP-riboses to the same substrates in rat brain membrane extract. The C3 enzymes could be roughly classified into two groups with respect to amino acid composition, amino-terminal sequence, and antigenicity. One group contains the C3 enzymes of strains C6813 and DSA, and the other contains those of strains CST and D1873. The specific activity of the C3 enzyme of strain C6813 was about 15 times higher than that of the C3 enzyme of strain CST. These results indicate that the classification of the C3 molecules differs from that of the neurotoxin molecules.

DOI： 10.1128/jb.173.19.6025-6029.1991

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PubMed

担当区分：筆頭著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPAN BIOCHEMICAL SOC  

DOI： 10.1093/oxfordjournals.jbchem.a123296

Web of Science

PubMed

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Web of Science

PubMed

担当区分：筆頭著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Web of Science

PubMed

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Web of Science

PubMed

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）  

Botulinum neurotoxins are classified into seven types, A to G, based on their antigenicities (1). Among the seven types, C1 and D toxins have complex antigenicities in that they can cross-react with antiserum for the other type (2-4). A partial explanation of the cross reaction is the recent finding that a subtype of C1 neurotoxin has antigenicities of C1 light chain and D heavy chain (2,3,5). On the other hand, specific antibodies against type D toxins from strains South African (D-SA) and 1873 (D-1873) were separated from anti-toxin sera by affinity chromatography (6). These observations suggest that within the same type, C1 and D neurotoxins differ in the molecular structure and antigenicity. In this paper, the antigenic and molecular characteristics of the purified South-African toxin are compared with those of the 1873 toxin. The molecular properties of D-SA toxin were compared to those of neurotoxins from type C strains, Stockholm (C-ST) and 6813 (C-6813), and a type D strain, 1873 (D-1873). D-SA toxin, purified 610-fold from the culture supernatant in an overall yield of 30%, consisted of an intact peptide chain with a molecular weight of 140,000. Limited proteolysis of the toxin by trypsin formed dichain structure consisting of a light chain (Mr 50,000) and a heavy chain (Mr 90,000) linked by disulfide bond(s). Anti D-SA light-chain IgG reacted with D-1873 toxin but not with C1 toxins. On the other hand, anti D-SA heavy chain ross-reacted with C-ST, D-1873 and C-6813 toxins in that order. Amino-acid sequences of the whole molecule, heavy and light chains of D-SA and D-1873 toxins were similar to each other but not identical. These results suggest the molecular diversity in botulinum D toxins.

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開催年月日： 2023年6月

記述言語：日本語   会議種別：口頭（一般）  

開催地：北九州市   国名：日本国  

開催年月日： 2022年11月

記述言語：日本語   会議種別：ポスター発表  

開催地：長崎  

開催年月日： 2022年11月

記述言語：日本語   会議種別：口頭（一般）  

開催地：長崎  

開催年月日： 2022年11月

記述言語：日本語   会議種別：口頭（一般）  

開催地：長崎  

開催年月日： 2022年11月

記述言語：日本語   会議種別：口頭（一般）  

開催地：長崎  

開催年月日： 2022年11月

記述言語：日本語   会議種別：口頭（招待・特別）  

開催地：長崎  

開催年月日： 2022年7月

記述言語：英語   会議種別：ポスター発表  

開催地：Ghent, Belgium