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

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尚未被发现。我们建议建立一个逆转录病毒载体诱变细胞文库。逆转录病毒载体整合到宿主基因组DNA中，然后使基因失活。与化学物质或辐射等其他诱变剂相比，逆转录病毒载体可以很容易地识别出被破坏的基因。然后，我们将使用两种不同的策略产生对逆转录病毒和慢病毒复制具有抗性的细胞群。含有CD19和活性较低的白喉毒素突变体H21G的逆转录病毒和慢病毒载体将用于培养对病毒感染具有抗性的细胞。表达CD19的细胞将在每个感染周期后被抗CD19抗体和磁珠耗尽。由于H21G介导的细胞毒性，感染H21G载体的细胞会富集。经过几次感染和细胞分选后，细胞将被克隆，负责抵抗逆转录病毒或慢病毒感染的被破坏的基因将通过聚合酶链反应（PCR）鉴定出来。未来的研究将致力于查明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。

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.