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Identification of new genes involved in early steps of retroviral and lentiviral replication

鉴定参与逆转录病毒和慢病毒复制早期步骤的新基因

基本信息

批准号：
341815-2008
负责人：
Caruso, Manuel
金额：
$ 2.19万
依托单位：
Université Laval
依托单位国家：
加拿大
项目类别：
Discovery Grants Program - Individual
财政年份：
2008
资助国家：
加拿大
起止时间：
2008-01-01 至 2009-12-31
项目状态：
已结题

来源：
https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=399625
关键词：
Identification new genes involved early

项目摘要

The different steps of retroviral and lentiviral replications have been well studied. However, most of cellular proteins involved in the early steps of viral replication have yet to be discovered. We propose to generate a library of cells mutagenized with a retroviral vector. Retroviral vectors integrate into the host genomic DNA and can then inactivate genes. Compared to other mutagenic agents like chemicals or radiations, retroviral vectors will allow the easy identification of disrupted genes. We will then generate cell populations that are resistant to retroviral and lentiviral replication using 2 different strategies. Retroviral and lentiviral vectors containing CD19 and H21G, a diphtheria toxin mutant with less activity, will be used to enrich for cells that are resistant to viral infection. Cells expressing CD19 will be depleted after each infection cycle with an anti-CD19 antibody and magnetic beads. Cells infected with H21G vectors will be enriched due to the cell toxicity mediated by H21G. After several infections and cell sorting, cells will be cloned and disrupted genes responsible for the resistance to retroviral or lentiviral infection will be identified by PCR. Future studies will aim to pinpoint the exact role during viral replication of the genes identified from our screen.

逆转录病毒和慢病毒复制的不同步骤已经得到了很好的研究。然而，大多数参与病毒复制早期步骤的细胞蛋白质尚未被发现。我们建议用逆转录病毒载体诱变产生细胞库。逆转录病毒载体整合到宿主基因组DNA中，然后可以复制基因。与其他诱变剂如化学品或辐射相比，逆转录病毒载体将允许容易地识别被破坏的基因。然后，我们将使用2种不同的策略产生抗逆转录病毒和慢病毒复制的细胞群。含有CD 19和H21 G（一种活性较低的白喉毒素突变体）的逆转录病毒和慢病毒载体将用于富集对病毒感染具有抵抗力的细胞。每个感染周期后，表达CD 19的细胞将被抗CD 19抗体和磁珠耗尽。由于H21G介导的细胞毒性，用H21G载体感染的细胞将被富集。在几次感染和细胞分选后，将克隆细胞，并通过PCR鉴定负责抗逆转录病毒或慢病毒感染的破坏基因。未来的研究将旨在确定从我们的筛选中鉴定出的基因在病毒复制过程中的确切作用。