CLONING OF THE HOST FACTOR GENES REQUIRED FOR THE VIRUS INFECTION

病毒感染所需宿主因子基因的克隆

• 批准号: 07670340
• 负责人: GOTOH Bin
• 金额: $ 1.47万
• 依托单位: FUKUI MEDICAL SCHOOL
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670340/
• 关键词: PROMOTER TRAP RETROVIRUS; CHO22; KNOCKOUT; INFLUENZA VIRUS; PSEUDOMONAS EXOTOXIN A; DIPHTHERIA TOXIN; VIRUS RESISTANT MUTANTS; TOXIN RESISTANT MUTANTS; promotertrap retrovirus; 毒素抵抗性変異株

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 mutantsWe 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 … More 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 mutantsThe 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. Less

为了鉴定病毒生长以及病毒诱导的细胞死亡所需的宿主因子，我们分离了抗病毒感染的细胞突变体。使用U3 Hygro启动子陷阱逆转录病毒通过插入诱变产生突变体（pt-retrovirus）由H.E.Ruley博士提供，其中无启动子的潮霉素B标记允许选择与表达基因的启动子相邻的整合事件。将逆转录病毒整合到细胞启动子的下游。从这些文库中分离到的病毒抗性突变体如下：1、从在小麦胚芽凝集素（WGA）存在下存活的克隆中分离到18个抗流感病毒的突变体（WGAR）。从用抗HA单克隆抗体处理后存活的克隆中分离出10个突变体（FLUR ...更多信息 3、从抗假单胞菌外毒素A（PEAR）和白喉毒素（DTR）的突变株中分离到13株对水疱性口炎病毒感染具有部分抗性的突变株。其中一个DTR突变体在感染纽卡斯尔病病毒后不引起合胞体的形成，表明该突变体在激活病毒融合糖蛋白的蛋白酶方面存在缺陷。与GenBank数据库进行同源性检索，没有发现与这些序列有显著同源性的基因。7 WR 1 -1具有与CHO 15 B相似的表型，CHO 15 B在N乙酰葡糖胺基转移酶I（GTI）中具有缺陷。为了确定pt-逆转录病毒是否可以敲除GTI基因，正在从7 WR 1 -1、CHO 22和CHO 15 B克隆GTI基因。少