CLONING OF THE HOST FACTOR GENES REQUIRED FOR THE VIRUS INFECTION

为了确定病毒生长所需的宿主因子以及病毒诱导的细胞死亡，我们分离出了抵抗病毒感染的细胞突变体。突变体是利用H.E.Ruley博士亲切提供的U3Hygro启动子陷阱逆转录病毒(pt-逆转录病毒)通过插入突变产生的，其中无启动子的HygromycinB标记允许选择表达基因启动子附近的整合事件。从这些文库中分离到抗病毒突变体如下：1.从小麦胚芽凝集素(WGA)存在下存活的克隆中分离到18个抗流感病毒感染的突变体(WGAR)。它们可能在病毒受体上存在缺陷。2.从抗HA单克隆…处理后存活的克隆中分离到10个突变体(FluR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株部分抵抗水泡性口炎病毒感染的突变体。其中一个DTR突变体在新城疫病毒感染后没有引起合胞体形成，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。突变株的遗传分析测定了WGAR中的一个(7WR1-1)、两个FluR和一个梨中逆转录病毒整合位点附近的核苷酸序列。与GenBank数据库的序列同源性搜索显示，没有与这些序列有显着同源性的基因。7WR1-1的表型与CHO15B相似，但缺乏N-乙酰氨基葡萄糖转移酶I(GTI)。为了确定pt逆转录病毒是否能够敲除GTI基因，正在克隆7WR1-1、CHO22和CHO15B的GTI基因。较少

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