|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1996 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1995 : ¥1,100,000 (Direct Cost : ¥1,100,000)
To identify host factors required for virus growth as well as virus induced cell death, we isolated cell mutants resistant to virus infection. The mutants were generated by insertional mutagenesis using the U3Hygro promoter trap retrovirus (pt-retrovirus) kindly provided by Dr.H.E.Ruley, in which a promoterless HygromycinB marker allows the selection of integration events adjacent to the promoter of expressed genes.
Isolation of virus resistant mutants
We made libraries consisting of a total of around 5x10^5 hygromycin resistant clones in which the pt-retrovirus was integrated into the downstream of the cellular promoter. The virus resistant mutants were isolated from these libraries as follows.
1, Eighteen mutants (WGAR) resistant to influenza virus infection were isolated from the clones surviving in the presence of wheat germ agglutinin (WGA). They may have defects in the virus receptors.
2, Ten mutants (FLUR) were isolated from the clones surviving after treatment with anti-HA monoclon
al antibody and complement following influenza virus infection.
3, Thirteen mutants partially resistant to the vesicular stomatitis virus infection were isolated from mutants resistant to Pseudomonas exotoxin A (PEAR) or Diphtheria toxin (DTR). One of the DTR mutants caused no syncytia formation after Newcastle disease virus infection, suggesting a mutant which has a defect in proteases activating the viral fusion glycoprotein.
Genetic analyzes of mutants
The nucleotide sequences adjacent to the retroviral integration sites in one (7WR1-1) of the WGAR,two of the FLUR and one of the PEAR were determined. A sequence homolgy search with the data bases of the GenBank revealed there were no genes significantly homologous to these sequences. The 7WR1-1 had the phenotype similar to that of CHO15B which has a defect in the Nacetylglucosaminyl transferase I (GTI). To determine whether the pt-retrovirus could knockout the GTI gene, cloning of the GTI genes from the 7WR1-1, the CHO22, and the CHO15B is in progress.
WGARの中の1株7WR1-1を含むいくつかの株について、pt-retrovirusの挿入部位に隣接した宿主遺伝子の塩基配列を決定したが、homology検索の結果では、該当する既知遺伝子は無かった。7WR1-1の表現型は、GlcNAc Transferase I (GTI)欠損であるCHO15B(あるいはlec1)に類似しており、現在、CHO細胞、15B細胞、7WR1-1からGTI遺伝子のクローニングをすすめ、当初期待したようにpt-retrovirusによるノックアウトによるものかを調べている。 Less