PROJECT SUMMARY / ABSTRACT Zika virus (ZIKV) re-emerged over the last several decades to cause large outbreaks in the Asian Pacific and Americas. ZIKV disease, though usually mild, can occasionally cause severe neurological complications, including developmental defects in babies born to a ZIKV-infected person. Much remains to be learned about ZIKV host-pathogen interactions, including how the innate immune system fights the early stages of infection in human cells. Given their role in inhibiting viral replication, innate immune factors could help explain the heterogeneity in ZIKV clinical outcome and could lead to the development of treatments for ZIKV. The type I interferon (IFN) response plays a particularly critical role in the innate immune response to viruses, including ZIKV. IFN is secreted from virus-infected cells, binds its receptor on nearby cells, and sets off a signaling pathway that culminates in a transcriptional program turning on hundreds of interferon-stimulated genes (ISGs), some of which encode antiviral proteins against a given virus. While the IFN response is clearly important in restricting ZIKV, systematic studies to define which host genes are responsible for this potent effect are lacking. While several specific antiviral ISGs have been identified, there have not been broader attempts to define all host genes that contribute to IFN restriction of ZIKV, including non-ISGs that may play a regulatory role in the pathway. Our lab performed a CRISPR knockout screen to identify genes that contribute to IFN restriction of ZIKV. The screen approach successfully identified genes involved in IFN signaling (positive controls). IFI6, a previously described ISG and flavivirus restriction factor, was also identified, as was AMOTL2, a non-ISG in our cell type and a gene with no described role in innate immunity. Despite the fact that AMOTL2 is not itself IFN-induced, AMOTL2 knockout increased ZIKV replication in the presence, but not absence, of IFN. These findings confirmed the IFN-specific antiviral phenotype of AMOTL2 and led to the hypothesis that AMOTL2 acts upstream of ISG transcription in the IFN response. My preliminary results support this model and are the basis for the proposed studies. In Aim 1, the mechanism of AMOTL2’s viral restriction will be better elucidated. This will be achieved by first validating the specificity of the observed phenotype through rescue experiments, then assessing the involvement of AMOTL2’s binding partners YAP and TAZ in IFN restriction of ZIKV through double knockouts and co-IP assays. In Aim 2, I will test the breadth of AMOTL2’s antiviral phenotype across diverse viruses (Aim 2a) and with ZIKV infections in a biologically-relevant primary cell type (Aim 2b). The complementary results of my two aims will enhance our understanding of the IFN response against ZIKV and possibly other viruses.