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

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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.