A CRISPR screen for Drosophila DNA viral immunity

About This Grant

Project Summary/Abstract DNA viruses are a difficult problem in health and disease. These viruses, including hepatitis B virus, poxviruses, adenovirus (respiratory diseases), and Epstein-Barr virus cause significant morbidity and mortality in humans. The lab has also used DNA viruses, including baculoviruses and nudiviruses, to naturally control insect pest populations and in biotechnology, for decades. Thus, there is much to be gained in developing a deeper understanding of how hosts and viruses co-evolve and the genetic mechanisms of their interactions. Researchers recently discovered and characterized the first DNA virus found to naturally infect Drosophila. This virus, a nudivirus, is common and causes significant mortality in hosts. The virus was recently adapted to infect D. melanogaster S2 cells, which are an extremely useful tool for studying host genetic factors involved in viral infection. This proposal employs a pooled CRISPR screenof nudivirus infection in S2 cells to identify host proviral factors. This involves transforming a pool of S2 cells with guide RNAs that each target a single gene in the genome and render that gene nonfunctional. Thus, each cell has a different gene knocked out. Cells are then infected with virus, incubated, and sorted based on the intensity of viral infection (measured by anti-capsid antibody in flow cytometry). These pools will then be amplicon sequenced and analyzed for genes that are enriched or depauperate in virus-infected or uninfected pools. Candidate genes will then be followed up by validating the roles for ten of these genes in nudivirus infection using RNAi and overexpression. This is the first such experiment for DNA viruses in Drosophila and promises to identify important pro-nudiviral factors.