A novel antiviral function of AMOTL2 enhances the human type I interferon response against Zika virus

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 cause severe neurological complications, including developmental defects in infants 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. The type I interferon (IFN) response plays a particularly critical role in the innate immune response to viruses, including ZIKV. Type I IFN is released from virus-infected cells as a warning signal to surrounding cells, setting off a chain of signaling events in the target cells, including the phosphorylation of STAT1 and STAT2, their formation of a complex with IRF9, and the translocation of this complex to the nucleus, where it acts as a transcription factor driving the upregulation of hundreds of interferon-stimulated genes (ISGs). ISGs work collectively to potently restrict viral replication.While the type I IFN response is clearly important in restricting ZIKV, there has not been a systematic assessment of which host genes are responsible for this potent effect. This thesis describes a CRISPR knockout screen to identify genes that contribute to type I IFN restriction of ZIKV in A549 cells. Eleven gene hits were identified, including IFI6, a previously described ISG with described antiviral activity against the Orthoflavivirus genus of viruses, of which ZIKV is a member. I showed that inactivation of this gene led to increased ZIKV replication in the presence of IFN-β, confirming its role as a restriction factor against ZIKV. Because previous studies on IFI6 used different cell types than A549 cells, my results with IFI6 knockout in A549s support the idea that IFI6 is an important anti-ZIKV restriction factor in diverse cell types. The top hit in the screen was AMOTL2, a gene with no previously described role in innate immunity. Surprisingly, AMOTL2 is not upregulated by type I IFN in A549 cells, but nonetheless experiments in single-gene knockout cells confirmed its IFN-specific antiviral phenotype, meaning that knockout of AMOTL2 increased ZIKV replication in the presence, but not absence, of type I IFN. I found that inactivation of AMOTL2 caused inhibition of key steps in the IFN signaling pathway including STAT1 phosphorylation and nuclear translocation, which corresponded with blunting of ISG upregulation and increased ZIKV replication. Interestingly, inactivation of AMOTL2 constitutively increased the levels of unphosphorylated STAT1 (U-STAT1), which has been reported to prevent activation of the type I IFN signaling pathway. I propose a model in which the presence of AMOTL2 in the cell suppresses high constitutive levels of U-STAT1, enabling a potent antiviral response to type I IFN. Interestingly, a known binding partner of AMOTL2, TAZ, has been reported to antagonize multiple innate immune signaling pathways. I found that AMOTL2 and TAZ interact in A549s, and that inactivation of TAZ increases type I IFN restriction of ZIKV in A549 cells, adding to published work that reported a proviral function of TAZ towards other viruses in other cell types. However, an expression construct of AMOTL2 lacking the TAZ binding domain was still able to rescue AMOTL2's antiviral phenotype, suggesting that AMOTL2's antiviral mechanism does not depend on TAZ binding. In summary, I have defined a new antiviral factor, AMOTL2, that acts through a novel mechanism to enhance the type I IFN response. These studies define a new pathway of type I IFN restriction of ZIKV.