フルテキストURL K0005289_other1.pdf
著者 Takeda, Midori| Ikeda, Masanori| Ariumi, Yasuo| Wakita, Takaji| Kato, Nobuyuki|
抄録  A hepatitis C virus (HCV) infection system was developed previously using the HCV JFH-1 strain (genotype 2a) and HuH-7 cells, and this cell culture is so far the only robust production system for HCV. In patients with chronic hepatitis C, the virological effects of pegylated interferon and ribavirin therapy differ depending on the HCV strain and the genetic background of the host. Recently, we reported the hepatoma-derived Li23 cell line, in which the JFH-1 life cycle is reproduced at a level almost equal to that in HuH-7-derived RSc cells. To monitor the HCV life cycle more easily, we here developed JFH-1 reporter-assay systems using both HuH-7- and Li23-derived cell lines. To identify any genetic mutations by long-term cell culture, HCV RNAs in HuH-7 cells were amplified 130 days after infection and subjected to sequence analysis to find adaptive mutation(s) for robust virus replication. We identified two mutations, H2505Q and V2995L, in the NS5B region. V2995L but not H2505Q enhanced JFH-1 RNA replication. However, we found that H2505Q but not V2995L enhanced HCV RNA replication of strain O (genotype 1b). We also selected highly permissive D7 cells by serial subcloning of Li23 cells. The expression levels of claudin-1 and Niemann-Pick C1-like 1 in D7 cells are higher than those in parental Li23 cells. In this study, we developed HCV JFH-1 reporter-assay systems using two distinct hepatoma cell lines, HuH-7 and Li23. The mutations in NS5B resulted in different effects on strains O and JFH-1 HCV RNA replication.
備考 学位審査副論文
発行日 2012-07
出版物タイトル Journal of General Virology
93巻
7号
出版者 Cambridge Univ. Press for the Society for General Microbiology
開始ページ 1422
終了ページ 1431
ISSN 0022-1317
NCID AA00698722
資料タイプ 学術雑誌論文
言語 English
OAI-PMH Set 岡山大学
著作権者 https://creativecommons.org/licenses/by-nc-nd/4.0/deed.ja
論文のバージョン author
PubMed ID 22456614
DOI 10.1099/vir.0.040725-0
Web of Sience KeyUT 000306348900003
関連URL https://doi.org/10.1099/vir.0.040725-0 http://ousar.lib.okayama-u.ac.jp/54272
フルテキストURL K0005288_other.pdf
著者 Sejima, Hiroe| Mori, Kyoko| Ariumi, Yasuo| Ikeda, Masanori| Kato, Nobuyuki|
抄録  Persistent hepatitis C virus (HCV) infection frequently causes hepatocellular carcinoma. However, the mechanisms of HCV-associated hepatocarcinogenesis and disease progression are unclear. Although the human hepatoma cell line, HuH-7, has been widely used as the only cell culture system for robust HCV replication, we recently developed new human hepatoma Li23 cell line-derived OL, OL8, OL11, and OL14 cells, in which genome-length HCV RNA (O strain of genotype 1b) efficiently replicates. OL, OL8, OL11, and OL14 cells were cultured for more than 2 years. We prepared cured cells from OL8 and OL11 cells by interferon-γ treatment. The cured cells were also cultured for more than 2 years. cDNA microarray and RT-PCR analyses were performed using total RNAs prepared from these cells. We first selected several hundred highly or moderately expressed probes, the expression levels of which were upregulated or downregulated at ratios of more than 2 or less than 0.5 in each set of compared cells (e.g., parent OL8 cells versus OL8 cells cultured for 2 years). From among these probes, we next selected those whose expression levels commonly changed during a 2-year culture of genome-length HCV RNA-replicating cells, but which did not change during a 2-year culture period in cured cells. We further examined the expression levels of the selected candidate genes by RT-PCR analysis using additional specimens from the cells cultured for 3.5 years. Reproducibility of the RT-PCR analysis using specimens from recultured cells was also confirmed. Finally, we identified 5 upregulated genes and 4 downregulated genes, the expression levels of which were irreversibly altered during 3.5-year replication of HCV RNA. These genes may play roles in the optimization of the environment in HCV RNA replication, or may play key roles in the progression of HCV-associated hepatic diseases.
キーワード HCV HCV RNA replication system Li23 cells Long-term RNA replication Upregulated host genes Downregulated host genes
備考 学位審査副論文
発行日 2012-07
出版物タイトル Virus Research
167巻
1号
出版者 Elsevier Science
開始ページ 74
終了ページ 85
ISSN 0168-1702
NCID AA10642076
資料タイプ 学術雑誌論文
言語 English
OAI-PMH Set 岡山大学
著作権者 https://creativecommons.org/licenses/by-nc-nd/4.0/deed.ja
論文のバージョン author
PubMed ID 22579597
DOI 10.1016/j.virusres.2012.04.008
Web of Sience KeyUT 000305496700010
関連URL https://doi.org/10.1016/j.virusres.2012.04.008 http://ousar.lib.okayama-u.ac.jp/54271
著者 Kato, Nobuyuki| Sejima, Hiroe| Ueda, Youki| Mori, Kyoko| Satoh, Shinya| Dansako, Hiromichi| Ikeda, Masanori|
発行日 2014-03-13
出版物タイトル PLOS ONE
9巻
3号
資料タイプ 学術雑誌論文
著者 Ueda, Youki| Takeda, Midori| Mori, Kyoko| Dansako, Hiromichi| Wakita, Takaji| Kim, Hye-Sook| Sato, Akira| Wataya, Yusuke| Ikeda, Masanori| Kato, Nobuyuki|
発行日 2013-08-30
出版物タイトル PLOS ONE
8巻
8号
資料タイプ 学術雑誌論文
著者 Dansako, Hiromichi| Ueda, Youki| Okumura, Nobuaki| Satoh, Shinya| Sugiyama, Masaya| Mizokami, Masashi| Ikeda, Masanori| Kato, Nobuyuki|
発行日 2015
出版物タイトル The FEBS journal
資料タイプ 学術雑誌論文
JaLCDOI 10.18926/AMO/53335
フルテキストURL 69_2_71.pdf
著者 Hiramoto, Hiroki| Dansako, Hiromichi| Takeda, Midori| Satoh, Shinya| Wakita, Takaji| Ikeda, Masanori| Kato, Nobuyuki|
抄録 Persistent infection with hepatitis C virus (HCV) often causes chronic hepatitis, and then shows a high rate of progression to liver cirrhosis and hepatocellular carcinoma. To clarify the mechanism of the persistent HCV infection is considered to be important for the discovery of new target(s) for the development of anti-HCV strategies. In the present study, we found that the expression level of annexin A1 (ANXA1) in human hepatoma cell line Li23-derived D7 cells was remarkably lower than that in parental Li23 cells, whereas the susceptibility of D7 cells to HCV infection was much higher than that of Li23 cells. Therefore, we hypothesized that ANXA1 negatively regulates persistent HCV infection through the inhibition of viral RNA replication. The results revealed that HCV production was significantly inhibited without a concomitant reduction in the amount of lipid droplets in the D7 cells stably expressing exogenous ANXA1. Further, we demonstrated that ANXA1 negatively regulated the step of viral RNA replication rather than that of viral entry in human hepatocytes. These results suggest that ANXA1 would be a novel target for the development of anti-HCV strategies.
キーワード HCV annexin A1 Li23 cell line Li23-derived D7 cells HCV-JFH-1
Amo Type Original Article
発行日 2015-04
出版物タイトル Acta Medica Okayama
69巻
2号
出版者 Okayama University Medical School
開始ページ 71
終了ページ 78
ISSN 0386-300X
NCID AA00508441
資料タイプ 学術雑誌論文
言語 English
著作権者 CopyrightⒸ 2015 by Okayama University Medical School
論文のバージョン publisher
査読 有り
PubMed ID 25899628
Web of Sience KeyUT 000353181700001
著者 Kuroki, Misao| Ariumi, Yasuo| Hijikata, Makoto| Ikeda, Masanori| Dansako, Hiromichi| Wakita, Takaji| Shimotohno, Kunitada| Kato, Nobuyuki|
発行日 2013-01-11
出版物タイトル Biochemical and Biophysical Research Communications
430巻
2号
資料タイプ 学術雑誌論文
JaLCDOI 10.18926/AMO/49042
フルテキストURL 66_6_461.pdf
著者 Koike, Kazuko| Takaki, Akinobu| Kato, Nobuyuki| Ouchida, Mamoru| Kanzaki, Hirotaka| Yasunaka, Tetsuya| Shiraha, Hidenori| Miyake, Yasuhiro| Yamamoto, Kazuhide|
抄録 Hepatitis C virus (HCV) infection induces several changes in hepatocytes, such as oxidative stress, steatosis, and hepatocarcinogenesis. Although considerable progress has been made during recent years, the mechanisms underlying these functions remain unclear. We employed proteomic techniques in HCV replicon-harboring cells to determine the effects of HCV replication on host-cell protein expression. We examined two-dimensional electrophoresis (2-DE) and mass spectrometry to compare and identify differentially expressed proteins between HCV subgenomic replicon-harboring cells and their “cured” cells. One of the identified proteins was confirmed using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Full-length HCV genome RNA replicating and cured cells were also assessed using ELISA. Replicon-harboring cells showed higher expression of retinal dehydrogenase 1 (RALDH-1), which converts retinol to retinoic acid, and the cured cells showed higher expression of retinol-binding protein (RBP), which transports retinol from the liver to target tissues. The alteration in RBP expression was also confirmed by ELISA and Western blot analysis. We conclude that protein expression profiling demonstrated that HCV replicon eradication affected retinol-related protein expression.
キーワード hepatitis C virus retinol-binding protein
Amo Type Original Article
発行日 2012-12
出版物タイトル Acta Medica Okayama
66巻
6号
出版者 Okayama University Medical School
開始ページ 461
終了ページ 468
ISSN 0386-300X
NCID AA00508441
資料タイプ 学術雑誌論文
言語 English
著作権者 CopyrightⒸ 2012 by Okayama University Medical School
論文のバージョン publisher
査読 有り
PubMed ID 23254580
Web of Sience KeyUT 000312966100005
著者 Ueda, Youki| Mori, Kyoko| Ariumi, Yasuo| Ikeda, Masanori| Kato, Nobuyuki|
発行日 2011-06-17
出版物タイトル Biochemical and Biophysical Research Communications
409巻
4号
資料タイプ 学術雑誌論文
JaLCDOI 10.18926/AMO/32281
フルテキストURL fulltext.pdf
著者 Nozaki, Akito| Naganuma, Atsushi| Nakamura, Takashi| Tanaka, Katsuaki| Sekihara, Hisahiko| Kato, Nobuyuki|
抄録 <p>We have developed a reliable internally controlled RT-nested PCR method for the detection of hepatitis C virus (HCV) RNA using in vitro synthesized Renilla luciferase (Rluc) RNA as an internal control. Using this method, the 5'-noncoding region of HCV RNA (144 nucleotides) and Rluc RNA (276 nucleotides) were efficiently amplified in a single tube, and the sensitivity and specificity of this method were comparable to standard RT-nested PCR. This method was successfully performed on RNA specimens obtained from in vitro HCV-infected human hepatocyte PH5CH8 cells, which support HCV replication. In addition, we demonstrated that this method was useful for the evaluation of antiviral reagents by confirming the anti-HCV activity of bovine lactoferrin, which we previously found to be a new inhibitor of HCV infection. Therefore, this method may be useful for the studies of not only HCV but also of other viruses.</p>
キーワード Hepatitis C virus Reverse transcriptionnested PCR (RT-nested PCR) internal control
Amo Type Article
発行日 2000-12
出版物タイトル Acta Medica Okayama
54巻
6号
出版者 Okayama University Medical School
開始ページ 253
終了ページ 257
ISSN 0386-300X
NCID AA00508441
資料タイプ 学術雑誌論文
言語 English
論文のバージョン publisher
査読 有り
PubMed ID 11132918
Web of Sience KeyUT 000166042900003
JaLCDOI 10.18926/AMO/32025
フルテキストURL fulltext.pdf
著者 Kato, Nobuyuki|
抄録 <p>Hepatitis C virus (HCV), discovered in 1989, is the major causative agent of parenteral non-A, non-B hepatitis worldwide. Following the development of a method of diagnosing HCV infection, it became apparent that HCV frequently causes chronic hepatitis. Persistent infection with HCV is implicated in liver cirrhosis and hepatocellular carcinoma. Current worldwide estimations suggest that more than 170 million people have been infected with HCV, an enveloped positive single-stranded RNA (9.6-kilobases) virus belonging to the Flaviviridae. The HCV genome shows remarkable sequence variation, especially in the hypervariable region 1 of the E2 protein-encoding region, and globally, HCV appears to be distributed with more than 30 genotypes. Complicated &#34;quasispecies&#34; and frequent mutations of viral genomes have also emerged. The HCV genome encodes a large polyprotein precursor of about 3,000 amino acid residues, and this precursor protein is cleaved by the host and viral proteinases to generate at least 10 proteins in the following order: NH2-core-envelope (E1)-E2-p7-nonstructural protein 2 (NS2)-NS3-NS4A-NS4B-NS5A-NS5B-COOH. These viral proteins not only function in viral replication but also affect a variety of cellular functions. Although several explanations have been proposed, the mechanisms of HCV infection and replication in targeted cells, the mechanism of persistent viral infection, and the pathogenesis of hepatic diseases (hepatitis or hepatocellular carcinoma) are all poorly understood. A major reason why these mechanisms remain unclear is the lack of a good experimental HCV replication system. Although several classical trials using cultured cells have been reported, several new, more promising experimental strategies (generations of infectious cDNA clone, replicon, animal models, etc.) are currently being designed and tested, in order to resolve these problems. In addition, new therapies for chronic hepatitis have also been developed. The enormous body of information collected thus far in the field of HCV research is summarized below, and an overview of the current status of HCV molecular virology of HCV is provided.&#60;/P&#62;</p>
Amo Type Review
発行日 2001-06
出版物タイトル Acta Medica Okayama
55巻
3号
出版者 Okayama University Medical School
開始ページ 133
終了ページ 159
ISSN 0386-300X
NCID AA00508441
資料タイプ 学術雑誌論文
言語 English
論文のバージョン publisher
査読 有り
PubMed ID 11434427
Web of Sience KeyUT 000169512600001
JaLCDOI 10.18926/AMO/31716
フルテキストURL fulltext.pdf
著者 Alam, Shahjalal S.| Nakamura, Takashi| Naganuma, Atsushi| Nozaki, Akito| Nouso, Kazuhiro| Shimomura, Hiroyuki| Kato, Nobuyuki|
抄録 <p>We have shown that highly proofreading DNA polymerase is required for the polymerase chain reaction in the genetic analysis of hepatitis C virus (HCV). To clarify the status of HCV quasispecies in hepatic tissue using proofreading DNA polymerase, we performed a genetic analysis of the HCV core protein-encoding region in cancerous and noncancerous lesions derived from 4 patients with hepatocellular carcinoma. In contrast to the previously published data, we observed neither deletions nor stop codons in the analyzed region and no significant difference in the complexity of HCV quasispecies between cancerous and noncancerous lesions. This result suggests that the HCV core gene is never structurally defective in hepatic tissues, including cancerous lesions. However, in 3 of the patients, the consensus HCV species differed between cancerous and noncancerous lesions, suggesting that the predominant replicating HCV species differs between these 2 types of lesions. Moreover, during the course of the study, we obtained several interesting variants possessing a substitution at codon 9 of the core gene, whose substitution has been shown to induce the production of the F protein synthesized by a - 2/+1 ribosomal frameshift.</p>
キーワード hepatitis C virus core gene hepatocellular carcinoma quasispecies proofreading DNA polymerase
Amo Type Article
発行日 2002-06
出版物タイトル Acta Medica Okayama
56巻
3号
出版者 Okayama University Medical School
開始ページ 141
終了ページ 147
ISSN 0386-300X
NCID AA00508441
資料タイプ 学術雑誌論文
言語 English
論文のバージョン publisher
査読 有り
PubMed ID 12108585
Web of Sience KeyUT 000176521200004
JaLCDOI 10.18926/AMO/31696
フルテキストURL fulltext.pdf
著者 Nozaki, Akito| Kato, Nobuyuki|
抄録 <p>Based on recent LightCycler techniques developed for the quantitation of serum HCV RNA, we have developed a quantitative method for the intracellular hepatitis C virus (HCV) RNA using LightCycler PCR. A simple real-time PCR assay, based on the SYBR Green I dye and LightCycler fluorimeter and with no probe requirement, is described. In the presence of 0.5 microg of cellular RNA, it was demonstrated that as few as 25 copies of HCV RNA could be specifically detected with a set of primers that amplify a 144-base pair sequence unique to the 5'-noncoding region of HCV RNA. We demonstrated that this method was useful for the evaluation of antiviral reagents using HCV-infected human cultured cells.</p>
キーワード hepatitis C virus real-time PCR LightCycler
Amo Type Article
発行日 2002-04
出版物タイトル Acta Medica Okayama
56巻
2号
出版者 Okayama University Medical School
開始ページ 107
終了ページ 110
ISSN 0386-300X
NCID AA00508441
資料タイプ 学術雑誌論文
言語 English
論文のバージョン publisher
査読 有り
PubMed ID 12002616
Web of Sience KeyUT 000175176900007
著者 有海 康雄| 黒木 美沙緒| 團迫 浩方| 阿部 健一| 池田 正徳| 脇田 隆字| 加藤 宣之|
発行日 2010-04-01
出版物タイトル 岡山医学会雑誌
122巻
1号
資料タイプ 学術雑誌論文
著者 加藤 宣之| 小熊 惠二|
発行日 2001-04-28
出版物タイトル 岡山医学会雑誌
113巻
1号
資料タイプ 学術雑誌論文