Molecular Basis for Interferon-mediated Pathogen Restriction in Human Cells

To define novel mechanisms for cellular immunity to the intracellular pathogenToxoplasma gondii, we performed a genome-wide CRISPR loss-of-function screen to provide an unbiased assessment of genes important for IFN-γ-dependent growth restriction. We revealed a previously unknown role for the tumor suppressor NF2/Merlin for maximum induction of Interferon Stimulated Genes (ISG), which are positively regulated by the transcription factor IRF-1. We then performed an additional focused ISG-targeted CRISPR screen that identified the host E3 ubiquitin ligase RNF213 as essential for IFN-γ mediated control ofT. gondii. RNF213 mediated ubiquitination of targets on the parasite-containing vacuole and growth restriction in response to IFN-γ in a variety of cell types, thus identifying a conserved factor that plays a prominent role in human cells. Surprisingly, growth inhibition did not require the autophagy protein ATG5, indicating that RNF213 initiates restriction independent of a non-canonical autophagy pathway that has previously been implicated in control ofT. gondii. RNF213 was also important for control of unrelated intracellular pathogens in human cells treated with IFN, as shown here forMycobacterium tuberculosisand Vesicular Stomatitis Virus. Collectively, our findings establish RNF213 as a critical component of cell-autonomous immunity to a broad spectrum of intracellular pathogens in human cells.