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Analysis of functions of Epstein-Barr virus-encoded small RNA EBER

Research Project

Project/Area Number 13470062
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Virology
Research InstitutionHokkaido University

Principal Investigator

TAKADA Kenzo  Hokkaido Univ., Institute for Genetic Medicine, Prof., 遺伝子病制御研究所, 教授 (30133721)

Co-Investigator(Kenkyū-buntansha) IWAKIRI Dai  Hokkaido Univ., Institute for Genetic Medicine, Lec., 遺伝子病制御研究所, 助手 (10307853)
KANNNO Hiroyuki  Hokkaido Univ., Institute for Genetic Medicine, Asso. Prof., 遺伝子病制御研究所, 助教授 (40252663)
Project Period (FY) 2001 – 2002
Project Status Completed (Fiscal Year 2002)
Budget Amount *help
¥12,500,000 (Direct Cost: ¥12,500,000)
Fiscal Year 2002: ¥4,400,000 (Direct Cost: ¥4,400,000)
Fiscal Year 2001: ¥8,100,000 (Direct Cost: ¥8,100,000)
KeywordsEpstein-Barr virus / EBER / recombinant virus / Cre / loxP system / transformation / 小RNA
Research Abstract

Transgenesis of EBV genomes are usually accomplished via homologous recombination after transfection of targeting constructs into EBV-infected cells. However, the marker genes introduced into recombinant EBV genomes may affect their virological features. Low efficiency of homologous recombination is another problem. We utilized Cre/loxP-mediated site-specific recombination for removing marker genes from recombinant viruses, as well as for introducing transgenes into EBV genomes. We aimed to make recombinant EBV lacking viral genes encoding small, non-transcribed RNAs, EBERs (EBER-KO EBV), which appear to be responsible for Burkitt lymphoma tumorigenesis. We took advantage of a mutated loxP site, loxP2272, which has two base-pair mutations in its spacer region (Gene 216: 55, 1998). Since loxP2272 cannot recombine with wild-type loxP site, the region flanked with wild-type loxP and loxP2272 can be replaced with DNA sequences flanked with the same combination of loxP sites by means of Cre … More -mediated gene replacement. Recombinant Akata EBV carrying a neomycin-resistant gene inserted into the BamHI X region (XneoEBV) was subject to EBER targeting. First, EBER genes of XneoEBV were replaced with hygromycin-resistant (hygR) gene flanked with wild-type loxP and loxP2272 by means of homologous recombination in Akata cell [EBER(-)hygREBV]. Second, the hygromycin cassette of EBER(-)hygREBV was replaced with a short non-coding DNA sequence by means of Cre-mediated gene replacement in the host cells. Subsequently, virus production was induced, and the produced viruses were used for infecting EBV-negative Akata cells. Infected cells were selected by G418, and drug-resistant cell clones were examined for the presence of recombinant EBV lacking the hygR gene. Cre-mediated gene replacement occurred in 2 of out 76 clones, and those two cell clones produced a large quantity of EBER-KO virus lacking the hygR gene. B lymphocytes purified from cord blood were infected with equivalent amounts of XneoEBV and EBER-KO EBV, respectively. Although these viruses exhibited comparable infectivity, transforming titer of EBER-KO EBV was approximately 100 fold less than XneoEBV, demonstrating the contribution of EBERs for efficient transformation. Less

Report

(3 results)
  • 2002 Annual Research Report   Final Research Report Summary
  • 2001 Annual Research Report
  • Research Products

    (24 results)

All Other

All Publications (24 results)

  • [Publications] Nanbo, A.: "Epstein-Barr virus RNA confers resistance to interferon-a-induced apoptosis in Burkitt's lymphoma"EMBO J.. 21. 954-965 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nakayama, T.: "Human B cells immortalized with Epstein-Barr virus upregulate CCR6 and CCR10 and downregulate CXCR4 and CXCR5"J. Virol.. 76. 3072-3077 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Kassis J.: "EBV-expressing AGS gastric carcinoma cell sublines present increased motility and invasiveness"Int. J. Cancer. 99. 644-651 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nanbo, A.: "The role of Epstein-Barr virus-encoded small RNAs (EBERs) in oncogenesis"Rev. Med. Virol.. 12・5. 321-326 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Kiss, C.: "Improved subtractive suppression hybridization combined with high density cDNA array screening identifies differentially expressed viral and cellular genes"J. Virol. Methods. 107. 195-203 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Yang, L.: "Epstein-Barr virus infection of rat lymphocytes expressing human CD21 results in restricted latent viral gene expression and not in immunoblastic transformation"J. Med. Virol.. 70. 126-130 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nanbo, A.: "Epstein-Barr virus RNA confers resistance to interferon a induced apoptosis in Burkitt's lymphoma"EMBO J.. 21. 954-965 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nakayama, T.: "Human B cells immortalized with Epstein-Barr virus upregulate CCR6 and CCR10 and downregulate CXCR4 and CXCR5"J. Virol.. 76. 3072-3077 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Kassis J.: "EBV-expressing AGS gastric carcinoma cell sublines present increased motility and invasiveness"Int. J. Cancer. 99. 644-651 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nanbo, A.: "The role of Epstein-Barr virus-encoded small RNAs (EBERs) in oncogenesis"Rev. Med. Virol.. 12,5. 321-326 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Kiss, C.: "Improved subtractive suppression hybridization combined with high density cDNA array screening identifies differentially expressed viral and cellular genes"J. Virol. Methods. 107. 195-203 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Yang, L.: "Epstein-Barr virus infection of rat lymphocytes expressing human CD21 results in restricted latent viral gene expression but not in immunoblastic transformation"J. Med. Virol.. 70. 126-130 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nanbo, A: "Epstein-Barr virus RNA confers resistance to interferon-a-induced apoptosis in Burkett's lymphoma"EMBO J.. 21. 954-965 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Nakayama, T: "Human B cells immortalized with Epstein-Barr virus upregulate CCR6 and CCR10 and downregulate CXCR4 and CXCR5"J. Virol.. 76. 3072-3077 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Kassis J: "EBV-expressing AGS gastric carcinoma cell sublines present increased motility and invasiveness"Int. J. Cancer. 99. 644-651 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Nanbo, A: "The role of Epstein-Barr virus-encoded small RNAs (EBERs) in oncogenesis"Rev. Med. Virol.. 12・5. 321-326 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Kiss, C: "Improved subtractive suppression hybridization combined with high density cDNA array screening identifies differentially expressed viral and cellular genes"J. Virol. Methods. 107. 195-203 (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Yang, L.: "Epstein-Barr virus infection of rat lymphocytes expressing human CD2 1 results in restricted latent viral gene expression but not in immunoblastic transformation"J. Med. Virol.. 70号. 126-130 (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Nanbo, A.: "Epstein-Barr virus RNA confers resistance to interferon-a-induced apoptosis in Burkitt's lymphoma"EMBO J.. (in press).

    • Related Report
      2001 Annual Research Report
  • [Publications] Takada, K.: "The role of EBERs in oncogenesis (Review)"Cancer Biol.. 11. 461-467 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Maruo, S.: "Replacement of Epstein-Barr virus (EBV) plasmid with EBV-encoded EBER plasmid in Burkitt's lymphoma cells"J. Virol.. 75. 9977-9982 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Komano, J.: "Role of bcl-2 in Epstein-Barr virus-induced malignant conversion of Burkitt's lymphoma cell line Akata"J. Virol.. 75. 1561-1564 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Konishi, K.: "Role of Epstein-Barr virus-encoded latent membrane protein 2A on virus-induced transformation and virus activation"J. Gen. Virol.. 82. 1457-1463 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Maruo, S.: "Roles of Epstein-Barr virus glycoprotein gp350 and gp25 in the infection of various human epithelial cells"J. Gen. Virol.. 82. 2373-2383 (2001)

    • Related Report
      2001 Annual Research Report

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Published: 2001-04-01   Modified: 2016-04-21  

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