Establishment and Maintenance of the Innate Antiviral Response to West Nile Virus Involves both RIG-I and MDA5 Signaling through IPS-1▿ †

RIG-I and MDA5, two related pathogen recognition receptors (PRRs), are known to be required for sensing various RNA viruses. Here we investigated the roles that RIG-I and MDA5 play in eliciting the antiviral response to West Nile virus (WNV). Functional genomics analysis of WNV-infected fibroblasts from wild-type mice and RIG-I null mice revealed that the normal antiviral response to this virus occurs in two distinct waves. The initial response to WNV resulted in the expression of interferon (IFN) regulatory factor 3 target genes and IFN-stimulated genes, including several subtypes of alpha IFN. Subsequently, a second phase of IFN-dependent antiviral gene expression occurred very late in infection. In cells lacking RIG-I, both the initial and the secondary responses to WNV were delayed, indicating that RIG-I plays a critical role in initiating innate immunity against WNV. However, another PRR(s) was able to trigger a response to WNV in the absence of RIG-I. Disruption of both MDA5 and RIG-I pathways abrogated activation of the antiviral response to WNV, suggesting that MDA5 is involved in the host's defense against WNV infection. In addition, ablation of the function of IPS-1, an essential RIG-I and MDA5 adaptor molecule, completely disabled the innate antiviral response to WNV. Our data indicate that RIG-I and MDA5 are responsible for triggering downstream gene expression in response to WNV infection by signaling through IPS-1. We propose a model in which RIG-I and MDA5 operate cooperatively to establish an antiviral state and mediate an IFN amplification loop that supports immune effector gene expression during WNV infection

Critical role for Stat3 in T-dependent terminal differentiation of IgG B cells

Stat proteins are latent cytoplasmic transcription factors that are crucial in many aspects of mammalian development. In the immune system, Stat3 has distinct roles in T-cell, neutrophil, and macrophage function, but a role for Stat3 in B-cell development, particularly in the terminal differentiation of B cells into antibody-secreting plasma cells, has never been directly tested. In this study, we used the Cre/lox system to generate a mouse strain in which Stat3 was conditionally deleted in the B-cell lineage (Stat3fl/flCD19Cre/+). B-cell development, establishment of the peripheral B-cell compartment, and baseline serum antibody levels were unperturbed in Stat3fl/flCD19Cre/+ mice. Strikingly, Stat3fl/flCD19Cre/+ mice displayed profound defects in T-dependent (TD) IgG responses, but normal TD IgM, IgE, and IgA responses and T-independent (TI) IgM and IgG3 responses. In addition, germinal center (GC) formation, isotype switching, and generation of memory B cells, including IgG+ memory cells, were all intact in Stat3fl/flCD19Cre/+ mice, indicating that the requirement for Stat3 was limited to plasma cell differentiation. These results demonstrate a profound yet highly selective role for Stat3 in TD IgG plasma cell differentiation, and therefore represent a unique example of a transcription factor regulating isotype-specific terminal B-cell differentiation

Distinct RIG-I and MDA5 Signaling by RNA Viruses in Innate Immunity▿

Alpha/beta interferon immune defenses are essential for resistance to viruses and can be triggered through the actions of the cytoplasmic helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Signaling by each is initiated by the recognition of viral products such as RNA and occurs through downstream interaction with the IPS-1 adaptor protein. We directly compared the innate immune signaling requirements of representative viruses of the Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Reoviridae for RIG-I, MDA5, and interferon promoter-stimulating factor 1 (IPS-1). In cultured fibroblasts, IPS-1 was essential for innate immune signaling of downstream interferon regulatory factor 3 activation and interferon-stimulated gene expression, but the requirements for RIG-I and MDA5 were variable. Each was individually dispensable for signaling triggered by reovirus and dengue virus, whereas RIG-I was essential for signaling by influenza A virus, influenza B virus, and human respiratory syncytial virus. Functional genomics analyses identified cellular genes triggered during influenza A virus infection whose expression was strictly dependent on RIG-I and which are involved in processes of innate or adaptive immunity, apoptosis, cytokine signaling, and inflammation associated with the host response to contemporary and pandemic strains of influenza virus. These results define IPS-1-dependent signaling as an essential feature of host immunity to RNA virus infection. Our observations further demonstrate differential and redundant roles for RIG-I and MDA5 in pathogen recognition and innate immune signaling that may reflect unique and shared biologic properties of RNA viruses whose differential triggering and control of gene expression may impact pathogenesis and infection

P58IPK: A Novel “CIHD” Member of the Host Innate Defense Response against Pathogenic Virus Infection

To support their replication, viruses take advantage of numerous cellular factors and processes. Recent large-scale screens have identified hundreds of such factors, yet little is known about how viruses exploit any of these. Influenza virus infection post-translationally activates P58 IPK , a cellular inhibitor of the interferon-induced, dsRNA-activated eIF2α kinase, PKR. Here, we report that infection of P58 IPK knockout mice with influenza virus resulted in increased lung pathology, immune cell apoptosis, PKR activation, and mortality. Analysis of lung transcriptional profiles, including those induced by the reconstructed 1918 pandemic virus, revealed increased expression of genes associated with the cell death, immune, and inflammatory responses. These experiments represent the first use of a mammalian infection model to demonstrate the role of P58 IPK in the antiviral response. Our results suggest that P58 IPK represents a new class of molecule, a cellular inhibitor of the host defense (CIHD), as P58 IPK is activated during virus infection to inhibit virus-induced apoptosis and inflammation to prolong host survival, even while prolonging viral replication. Respiratory illness caused by influenza virus is the seventh leading cause of death in the United States, and there is considerable interest in determining how the virus and host interact during infection and how the virus causes disease. Because influenza virus encodes only eight genes, it is dependent upon cellular proteins and processes for the generation of new viruses. One of these proteins is P58 IPK , the activation of which has long been thought to be required for efficient viral replication. To study how P58 IPK affects the ability of influenza virus to replicate and cause disease, we infected a strain of mice that lacks the gene for P58 IPK . We found that viruses replicated just as efficiently in mice lacking the P58 IPK gene, but that these mice were more susceptible to a fatal infection. The increased mortality rate in these mice was due to greater lung pathology and a heightened inflammatory response. Our results show that P58 IPK represents a new type of protein that benefits the host by reducing pathology and prolonging survival, even though it may also benefit the virus by allowing it to replicate for a longer period of time