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ABSTRACT Induction of interferon beta (IFN-β), IFN-stimulated genes (ISGs), and inflammatory responses is critical for control of viral infection. We recently identified an essential linkage of stimulation of the inflammatory cytokine interleukin-1β (IL-1β) and induction of ISGs that function as host restriction pathways against the emerging flavivirus West Nile virus (WNV) in vivo. Here we utilized ex vivo global transcriptome analysis of primary dendritic cells, known targets of WNV replication, to define gene signatures required for this IL-1β-driven antiviral response. Dendritic cells that were deficient in IL-1 receptor signaling showed dysregulation of cell-intrinsic defense genes and loss of viral control during WNV infection. Surprisingly, we found that in wild-type cells, IL-1β treatment, in the absence of infection, drove the transcription of IFN-β and ISGs at late times following treatment. Expression of these antiviral innate immune genes was dependent on the transcription factor IFN regulatory factor 3 (IRF3) and appears to reflect a general shift in IL-1β signaling from an early inflammatory response to a late IFN-mediated response. These data demonstrate that inflammatory and antiviral signals integrate to control viral infection in myeloid cells through a process of IL-1β-to-IRF3 signaling crosstalk. Strategies to exploit these cytokines in the activation of host defense programs should be investigated as novel therapeutic approaches against individual pathogens. IMPORTANCE West Nile virus is an emerging mosquito-borne flavivirus that can result in serious illness, neuropathology, and death in infected individuals. Currently, there are no vaccines or therapies for human use against West Nile virus. Immune control of West Nile virus infection requires inflammatory and antiviral responses, though the effect that each arm of this response has on the other is unclear. The significance of our research is in defining how virus-induced inflammatory responses regulate critical antiviral immune programs for effective control of West Nile virus infection. These data identify essential mechanisms of immune control that can inform therapeutic efforts against West Nile virus, with potential efficacy against other neuroinvasive viruses.

Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as "central" interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, "peripheral" interactome members (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation-induced assembly of RNA splicing interactomes that is important for understanding T cell activation.