Mapping the virus-host interactions that determine interferon resistance of Seoul orthohantavirus - Mammalian hantaviruses (order Bunyavirales, genus Orthohantavirus) are zoonotic RNA viruses that cause severe vascular inflammatory disease in humans, with case-fatality rates of up to 40%. These negative-sense, tri-segmented viruses are maintained, persistently and asymptomatically, within wild rodent and insectivore reservoir populations worldwide. As the first line of antiviral defense, innate immune signaling and type I interferon (IFN) act to limit viral replication and production of infectious progeny, preventing tissue dissemination and systemic infection. A dominant hypothesis posits that asymptomatic reservoir persistence is maintained through innate immune suppression, which allows for sustained viral replication without damaging inflammation. Evidence to support this hypothesis includes observations by our group and others that hantavirus infection of natural reservoir target endothelial cells (EC) results in dampened innate immune signaling and IFN-stimulated gene (ISG) expression. We now show that EC from the natural reservoir host (Rattus norvegicus) for Seoul virus (Orthohantavirus seoulense, SEOV) support sustained viral replication and infectious virion production in the presence of exogenous type I IFN and robust ISG expression. In contrast, the related Hantaan virus is highly susceptible to IFN-mediated restriction in human and rat EC, both of which represent non-reservoir hosts. Similarly, SEOV replication in human EC was reduced in the context of type I IFN signaling, either through exogenous treatment or induced by viral recognition receptors. Together, this led to our overall hypothesis that, through coevolution, SEOV has developed mechanisms to directly target rat antiviral effector proteins induced by type I IFN and inhibit their functionality. Our data further suggests that mechanisms of inhibition seen in rat cells are ineffective in the setting of human infection. The unique tractability of the natural rat host and the distinct divergence in immune modulation provides an opportunity to identify critical SEOV protein – host antiviral protein interfaces that prevent viral restriction in reservoir, but not human EC. Our team brings decades of experience in virology, innate immune signaling, and in identifying human host protein interactors for diverse families of RNA viruses, as well as all of the necessary reagents, assays, and expertise for success. Using novel monoclonal antibodies directed at each of the SEOV proteins to capture authentic viral protein complexes in the context of infection, we will undertake an innovative comparative proteomics approach to contrast the SEOV interactome in reservoir rat and non-reservoir human primary EC (Aim 1). In Aim 2, we will perform validation and functional analysis of selected IFN-induced antiviral host partners isolated in Aim 1 to test their significance for viral replication and progeny production in reservoir and non-reservoir host cells. This approach to identify unique virus-host interactors is the first described for a reservoir host of any hantavirus species and will yield insights into mechanisms of persistence and human disease.
