The host innate immune response is triggered within hours of virus infection. As a whole, its function is to limit virus replication at local sites of infection and to orchestrate development of the adaptive immune response. Viruses are typically recognized by cellular pattern recognition receptors (PRRs), including toll-like receptors (TLRs) and the retinoic acid inducible gene (RIG)-like RNA receptors (RLRs). Ligation of these PRRs, often by viral nucleic acids, culminates in the activation of multiple transcription factors that cooperate in driving expression of cytokines and chemokines characteristic of the innate response. Nuclear factor-kappa B (NF-kappaB) and interferon (IFN) regulatory factors (IRFs) are particularly important transcription factors, responsible for induction of type I IFN (IFNalpha/beta), type III IFN, and other mediators of inflammation. IFNalpha/beta is central to the anti-viral response as it initiates its own transcriptional program resulting in expression of IFN-stimulated genes (ISGs) via the Janus kinase-signal transducer and activation of transcription (JAK-STAT) pathway. ISG expression influences many cellular processes including RNA processing, protein stability and cell viability that can directly affect virus replication. ISG expression in cells of the immune system such as dendritic cells (DCs) and macrophages is critical for antigen presentation and T- and B-cell activation, thus affecting the quality of the adaptive immune response and eventual virus clearance. To facilitate dissemination, pathogenic viruses have evolved mechanisms to suppress host innate immunity by antagonizing these signal transduction pathways. Hence, understanding the specific pathways by which viruses activate and evade innate immune responses is essential for understanding viral pathogenesis as well as for development of effective vaccines. To examine virus-host interactions that affect innate immunity, our laboratory utilizes flaviviruses as the primary model of infection. Flaviviruses have an essentially global distribution and represent a tremendous disease burden to humans, causing millions of infections annually. The success of flaviviruses as human pathogens is associated with the fact that they are arthropod-borne, transmitted by mosquitoes or ticks. Significant members of this group include dengue virus (DENV) and yellow fever virus (YFV) that cause hemorrhagic fevers, as well as Japanese encephalitis virus (JEV), West Nile virus (WNV), tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV) that cause infections of the central nervous system. These viruses are listed as NIAID category A, B and C pathogens for research into their basic biology and host response. The flavivirus single-stranded RNA genome is translated as one open reading frame; the resulting polyprotein is cleaved into at least ten proteins that include three structural (capsid C, membrane M, derived from the precursor preM and envelope E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Virus replication proceeds in association with modified membranes derived from the endoplasmic reticulum of host cells. NS5 is the largest and most conserved of the flavivirus proteins containing approximately 900 amino acids. It encodes a methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRP) and associates with NS3 (the viral protease) to form the functional unit of the viral replication complex. Despite the widespread and often severe infections caused by these pathogens, vaccines exist for only a few (YFV, JEV and TBEV) and no therapeutic exists to treat clinical infection caused by any flavivirus. One major area of research is determining how genes induced by type I interferon, called ISGs, confer virus-specific protection. We have found surprising roles for TRIM proteins in flavivirus-specific host protection. Tripartite motif-containing protein 5 (TRIM5) is a cellular antiviral restriction factor that prevents early events in retrovirus replication. The activity of TRIM5 is thought to be limited to retroviruses as a result of highly specific interactions with capsid lattices. In contrast to this current understanding, we demonstrated that both human and rhesus macaque TRIM5 suppress replication of specific flaviviruses. Multiple viruses in the tick-borne encephalitis complex are sensitive to TRIM5-dependent restriction, but mosquito-borne flaviviruses, including yellow fever, dengue, and Zika viruses, are resistant. In the past FY, we have identified the genetic basis for flavivirus resistance to TRIM5 within the viral NS3 protein, and shown that this confers a replication advantage in primary human dendritic cells. We have also used this information to develop a new animal model to study the pathogenesis of highly virulent tick-borne flaviviruses, Kyasanur forest disease virus and Alkhurma hemorrhagic fever virus. This work identifies human TRIM5 as an important barrier to infection and helps define the specific effector functions of the type I interferon response to emerging flaviviruses. A second emphasis in the lab is defining how innate immunity is induced to the yellow fever virus live-attentuated vaccine, YFV-17D. Administered to more than 500 million people worldwide, YFV-17D elicits strong innate and adaptive immune responses, and confers lifelong immunity to yellow fever in more than 95% of vaccinees. YFV-17D robustly induces cytotoxic T cells, TH1 and TH2 CD4 T cells, and neutralizing antibodies that persist for 40 or more years. The strength and quality of the adaptive immune response is strongly influenced by detectable viremia and a robust innate immune signature virus-host interactions that modulate host innate immune responses. However, whether signaling pathways uniquely engaged by YFV-17D drive heightened IFN and inflammatory responses compared to the parental strain or other orthoflaviviruses is not known. This FY, we determined that YFV-17D uniquely induces mitochondrial respiration and major metabolic perturbations, including hyperactivation of electron transport to fuel ATP synthase. Mitochondrial hyperactivity generates reactive oxygen species (mROS), blocking of which abrogated IFN expression in non-immune cells without reducing YFV-17D replication. Scavenging ROS in YFV-17D-infected human dendritic cells increased cell viability yet globally prevented expression of IFN signaling pathways. Thus, adaptation of YFV-17D for high growth uniquely imparts mitochondrial hyperactivity generating mROS as the critical messenger that converts a blunted IFN response into maximal activation of innate immunity essential for vaccine effectiveness.