Bacterial effector screening reveals RNF214 as a virus restriction factor in mammals

Arboviruses are arthropod-borne viruses that pose significant threats to human and animal health. Previously, we demonstrated that bacterial effector proteins can serve as molecular tools to identify host immunity factors in insect cells that restrict arbovirus replication. In this study, we apply our bacterial effector screening system to identify immunity factors in two mammalian hosts-bats and humans. Our screens identified three bacterial effectors (IpaH4, SopB and SidM) that enhanced the replication of unrelated arboviruses in bat and human cells. We also discovered several effectors that enhanced arbovirus replication in an arbovirus- or host-specific manner. Focusing on the Shigella flexneri-encoded E3 ubiquitin ligase IpaH4, we identified the uncharacterized mammalian really interesting new gene (RING)-domain-containing protein RNF214 as a direct target that is ubiquitinated and degraded by IpaH4. RNF214 belongs to a large family of RING finger (RNF) proteins that primarily function as E3 ubiquitin ligases and that have diverse roles in regulating and mediating innate immune responses to disparate pathogens. Phylogenetic analyses reveal that RNF214 is highly conserved across vertebrate species, suggesting a conserved role in host defense. Functional studies demonstrate that RNF214 overexpression suppresses arbovirus infection in a manner dependent on its putative E3 ubiquitin ligase activity, whereas RNF214 depletion enhances viral replication in both human and bat cells. Furthermore, knockout of RNF214 did not alter the upregulation of interferon (IFN)-stimulated gene expression during infection or upon treatment of cells with IFN. Screening of 11 RNA and DNA viruses, revealed that RNF214 specifically restricts single-stranded RNA (ssRNA) viruses. These findings establish RNF214 as a critical component of the innate immune response against ssRNA viruses that may function independently of the IFN response. More broadly, our work highlights the utility of bacterial effector proteins as powerful tools for uncovering novel antiviral machinery in mammals.