Zika virus nonstructural protein 5 inhibition of interferon signaling

PROJECT SUMMARY All vector-borne flavivirus NS5 proteins suppress host type I interferon (IFN) signaling, which is critical to successfully infecting humans and causing disease. Inhibitors of this function of NS5 or viruses engineered to lack this activity may be effective antiviral therapies and attenuated vaccines, respectively. However, a deeper understanding of how the flavivirus NS5 protein acts to suppress IFN is needed. Despite the importance of inhibiting IFN signaling, and the high degree of sequence conservation between NS5 proteins within this genus, flaviviruses have evolved numerous distinct ways to antagonize this innate immune response. Many flavivirus NS5 proteins, including those of dengue (DENV), Zika (ZIKV), and yellow fever (YFV) viruses, inhibit the Signal Transducer And Activator Of Transcription 2 (STAT2) protein, which is a transcription factor that mediates IFN signaling. In this application, we propose to fill critical gaps in our understanding of the ZIKV NS5-STAT2 interface by defining the essential viral and host genetic determinants for this interaction at the highest possible, single amino acid, resolution through deep mutational scanning (DMS). In Aim 1, we will screen libraries of all possible single amino acid ZIKV NS5 variants for the ability to suppress IFN signaling. We will examine these determinants in a range of flaviviruses to understand functional conservation and mechanisms. In Aim 2, we will identify how STAT2 genetics impact the ability of ZIKV NS5 to suppress IFN signaling and mediate infection. Finally, in Aim 3 we will define how ZIKV NS5 induces the degradation of human STAT2 by identifying the STAT2 sites where NS5 induces ubiquitination and the host factors ZIKV NS5 recruits to mediate this modification. The results of this project will provide in-depth insights into flavivirus host cell interactions and replication mechanisms that may aid in the development of therapies for ZIKV, and could perhaps be further applied in combating other future virus outbreaks.