Type I and type III interferons (IFNs) are critical for controlling viral infections. However, the precise dynamics of the IFN response have been difficult to define in vivo. Signaling through type I IFN receptors leads to interferon-stimulated response element (ISRE)-dependent gene expression and an antiviral state. As an alternative to tracking IFN, we used an ISRE-dependent reporter mouse to define the cell types, localization, and kinetics of IFN responding cells during influenza virus infection. We find that measurable IFN responses are largely limited to hematopoietic cells, which show a high sensitivity to IFN. Inflammatory monocytes display high basal IFN responses, which are enhanced upon infection and correlate with infection of these cells. We find that inflammatory monocyte development is independent of IFN signaling; however, IFN is critical for chemokine production and recruitment following infection. The data reveal a role for inflammatory monocytes in both basal IFN responses and responses to infection.

The host tropism of viral infection is determined by a variety of factors, from cell surface receptors to innate immune signaling. Many viruses encode proteins that interfere with host innate immune recognition in order to promote infection. STAT2 is divergent between species and therefore has a role in species restriction of some viruses. To understand the role of STAT2 in human metapneumovirus (HMPV) infection of human and murine tissues, we first infected STAT2-/- mice and found that HMPV could be serially passaged in STAT2-/-, but not WT, mice. We then used in vitro methods to show that HMPV inhibits expression of both STAT1 and STAT2 in human and primate cells, but not in mouse cells. Transfection of the murine form of STAT2 into STAT2-deficient human cells conferred resistance to STAT2 inhibition. Finally, we sought to understand the in vivo role of STAT2 by infecting hSTAT2 knock-in mice with HMPV, and found that mice had increased weight loss, inhibition of type I interferon signaling, and a Th2-polarized cytokine profile compared to WT mice. These results indicate that STAT2 is a target of HMPV in human infection, while the murine version of STAT2 restricts tropism of HMPV for murine cells and tissue.

RIG-I-like receptors (RLRs: RIG-I, MDA5 and LGP2) play a major role in the innate immune response against viral infections and detect patterns on viral RNA molecules that are typically absent from host RNA. Upon RNA binding, RLRs trigger a complex downstream signaling cascade resulting in the expression of type I interferons and proinflammatory cytokines. In the past decade extensive efforts were made to elucidate the nature of putative RLR ligands. In vitro and transfection studies identified 5'-triphosphate containing blunt-ended double-strand RNAs as potent RIG-I inducers and these findings were confirmed by next-generation sequencing of RIG-I associated RNAs from virus-infected cells. The nature of RNA ligands of MDA5 is less clear. Several studies suggest that double-stranded RNAs are the preferred agonists for the protein. However, the exact nature of physiological MDA5 ligands from virus-infected cells needs to be elucidated. In this work, we combine a crosslinking technique with next-generation sequencing in order to shed light on MDA5-associated RNAs from human cells infected with measles virus. Our findings suggest that RIG-I and MDA5 associate with AU-rich RNA species originating from the mRNA of the measles virus L gene. Corresponding sequences are poorer activators of ATP-hydrolysis by MDA5 in vitro, suggesting that they result in more stable MDA5 filaments. These data provide a possible model of how AU-rich sequences could activate type I interferon signaling.

An exacerbated immune response is one of the main causes of influenza-induced lung damage during infection. The molecular mechanisms regulating the fate of the initial immune response to infection, either as a protective response or as detrimental immunopathology, are not well understood. The purinergic receptor P2X7 is an ionotropic nucleotide-gated ion channel receptor expressed on immune cells that has been implicated in induction and maintenance of excessive inflammation. Here, we analyze the role of this receptor in a mouse model of influenza virus infection using a receptor knockout (KO) mouse strain. Our results demonstrate that the absence of the P2X7 receptor results in a better outcome to influenza virus infection characterized by reduced weight loss and increased survival upon experimental influenza challenge compared to wild-type mice. This effect was not virus strain specific. Overall lung pathology and apoptosis were reduced in virus-infected KO mice. Production of proinflammatory cytokines and chemokines such as interleukin-10 (IL-10), gamma interferon (IFN-γ), and CC chemokine ligand 2 (CCL2) was also reduced in the lungs of the infected KO mice. Infiltration of neutrophils and depletion of CD11b+ macrophages, characteristic of severe influenza virus infection in mice, were lower in the KO animals. Together, these results demonstrate that activation of the P2X7 receptor is involved in the exacerbated immune response observed during influenza virus infection.IMPORTANCE A hallmark of influenza virus infection is the development of lung pathology induced by an exacerbated immune response. The mechanisms shared by the antiviral host defense required for viral clearance and those required for development of immunopathology are not clearly understood. Purinergic receptors, and in particular the purinergic receptor P2X7 (P2X7r), are involved in activation of the immune response. We used mice lacking the P2X7r (P2X7r KO mice) to better understand the mechanisms that lead to development of lung pathology during influenza virus infection. In our studies, we observed that P2X7r KO mice developed less lung immunopathology and had better survival than the wild-type mice. These results implicate P2X7r in the induction of an exacerbated local immune response to influenza virus and help us to better understand the mechanisms leading to the lung immunopathology observed during severe viral infections.