| Project/Area Number |
09307005
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Virology
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| Research Institution | The University of Tokyo |
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Principal Investigator |
NAGAI Yoshiyuki Institute of Medical Science, The University of Tokyo, Professor, 医科学研究所, 教授 (20022874)
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| Co-Investigator(Kenkyū-buntansha) |
TAGAWA Yuko Institute of Medical Science, The University of Tokyo, Associate Researcher, 医科学研究所, 教務職員 (40178538)
KATO Atsushi National institute of Infectionus Diseases, Laboratory Chief (researcher), (研究職)室長 (40152699)
SHIODA Tatsuo Institute of Medical Science, The University of Tokyo, Associate professor, 医科学研究所, 助教授 (00187329)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥38,800,000 (Direct Cost: ¥38,800,000)
Fiscal Year 1999: ¥8,400,000 (Direct Cost: ¥8,400,000)
Fiscal Year 1998: ¥8,000,000 (Direct Cost: ¥8,000,000)
Fiscal Year 1997: ¥22,400,000 (Direct Cost: ¥22,400,000)
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| Keywords | Sendai virus / genetic engineering / accessory genes / pathogenicity / transcription control / vector use / foreign gene expression / C蛋白 / アセンブリー / 抗インターフェロン / センダイウイルスベクター / 転写制御シグナル / lac Z / V蛋白 / HIV-1 gp120 |
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| Research Abstract |
In 1996, we established a system to recover Sendai virus (SeV), a nonsegmented negative strand RNA virus, entirely from cDNA. The purpose of this project has been to use this system to manipulate SeV genome at will and evaluate the contribution of viral cis- and trans-acting elements to viral replication and pathogenesis. An additional aim has been to learn the feasibility of using SeV as a novel expression vector.
By creating and characterizing knock-out viruses, we found that the accessory V and C proteins are essential for maintaining high viral load and counteracting the antiviral action of type I interferons, respectively, in the lung of mice, the natural host. Thus, these two proteins appeared to encode luxury functions required for viral pathogenesis. In addition, the C proteins were found to play a critical role in the assembly of viral proteins into mature virions, although they have been regarded basically as being nonstructural proteins. We were further able to identify trans
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cription START and STOP signals, which are present at the beginning and the end of each gene in the SeV genome. Further, the transcription was found to be down-regulated or attenuated before the envelope protein -encoding gene by subtle modification of the START signal. The SeV reverse genetics has settled several other outstanding questions including the significance of matrix protein phosphorylation.
The foreign genes inserted into SeV genome were maintained considerably stably. Their expression levels were extremely high. For instance, the gp 120 of HIV-1 expressed from recombinant SeV reached as high as 6 mg/L culture supernatant, the highest among available in mammalian cells. SeV genome of 15.3 kb could accommodate and express at least 3.2 kb additional sequence. Recombinant SeVs could transfer genes of interest into nondividing cells such as neurons. BY supplying the envelope protein in trans, it was possible to create recombinant SeV that lacks one of the envelope genes, hence undergoes no secondary transmission beyond the primary infection and obviously safer than the replication competent wild-type. These results suggest that SeV will serve as a novel class of viral vector in future. Less
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