Separating the molecular determinants that govern the evolution of Orthoflavivirus NS5-mediated IFN antagonism 

PROJECT SUMMARY Flaviviruses infect up to hundreds of millions of people worldwide annually. The spectrum of diseases caused by these viruses display a range of symptoms from mild illness to hemorrhagic fever, encephalitis, and congenital defects. To establish infection and cause pathogenesis, viruses need to suppress their vertebrate host's innate immune response. Flaviviruses use their non-structural protein 5 (NS5) to suppress type I interferon (IFN) signaling. While this function is highly conserved, there is diversity in the host factors that are targeted. ZIKV, DENV, and YFV NS5s target signal transducer and activator of transcription 2 (STAT2) for degradation/sequestration. These flaviviruses are transmitted by Aedes mosquitoes and circulate in a sylvatic cycle between non-human primates and humans. TBEV, LIV, and LGTV NS5s target tyrosine kinase 2 (TYK2) to inhibit its catalytic activity. These viruses are tick-borne and mainly circulate in rodents, with humans acting as dead-end hosts. TBEV, LGTV, and WNV NS5s target prolidase (PEPD) to block the expression of IFNAR1 on the cell surface. WNV, transmitted by Culex mosquitoes, circulates in birds with humans acting as dead-end hosts. These findings suggest that some flaviviruses evolved NS5s that encode multiple modes of IFN antagonism. While tick-borne flaviviruses and Culex-borne flaviviruses primarily amplify in rodents and birds respectively, their host ranges are much broader. Perhaps this extended host range applied variable evolutionary pressures that resulted in diverse modes of antagonism. My proposal will expand the knowledge of NS5- mediated IFN antagonism evolution and provide a framework for categorizing these mechanisms. I will achieve these goals by testing my central hypothesis that the evolution of flaviviruses is constrained by the structural and molecular determinants that govern their IFN antagonism functions in essential reservoir vertebrate hosts. In Aim 1, I will use structure-function analyses to identify determinants that define each mode of NS5-mediated IFN antagonism. Cryo-EM and extensive mutagenesis will serve to create a molecular "fingerprint" for each mechanism that will be used to screen a range of flavivirus NS5s to identify their mode of antagonism. I will explore the overlap in antagonism and replication determinants by conducting multicycle growth curves of various NS5 mutants in vitro, since not only is NS5 evolutionarily restricted by its function in innate immune suppression but also in the replication of the RNA genome. In Aim 2, I will examine the coevolution of type I IFN signaling in vertebrate hosts and flavivirus NS5. On the viral side, chimeric NS5s that swap regions from flaviviruses with divergent antagonism mechanisms will be tested for both host factor interaction and antagonism capabilities to. On the host side, I will replace endogenous PEPD and TYK2 with orthologs of various vertebrate species and study the ability of various flavivirus NS5s to antagonize these proteins. Together, the proposed work will provide a broader understanding of flavivirus NS5-mediated antagonism both in distinct structural, molecular terms, as well as provide clues about the evolutionary history as well as potential of these viruses.