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 differentiationassociated 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.

The influenza pandemic of 1918-19 was responsible for about 50 million deaths worldwide 1 . Modern histopathological analysis of autopsy samples from human influenza cases from 1918 revealed significant damage to the lungs with acute, focal bronchitis and alveolitis associated with massive pulmonary oedema, haemorrhage and rapid destruction of the respiratory epithelium 2 . The contribution of the host immune response leading to this severe pathology remains largely unknown. Here we show, in a comprehensive analysis of the global host response induced by the 1918 influenza virus, that mice infected with the reconstructed 1918 influenza virus displayed an increased and accelerated activation of host immune response genes associated with severe pulmonary pathology. We found that mice infected with a virus containing all eight genes from the pandemic virus showed marked activation of pro-inflammatory and cell-death pathways by 24 h after infection that remained unabated until death on day 5. This was in contrast with smaller host immune responses as measured at the genomic level, accompanied by less severe disease pathology and delays in death in mice infected with influenza viruses containing only subsets of 1918 genes. The results indicate a cooperative interaction between the 1918 influenza genes and show that study of the virulence of the 1918 influenza virus requires the use of the fully reconstructed virus. With recent concerns about the introduction of highly pathogenic avian influenza viruses into humans and their potential to cause a worldwide pandemic with disastrous health and economic consequences, a comprehensive understanding of the global host response to the 1918 virus is crucial. Moreover, understanding the contribution of host immune responses to virulent influenza virus infections is an important starting point for the identification of prognostic indicators and the development of novel antiviral therapies.Correspondence and requests for materials should be addressed to J.K. (jkash@u.washington.edu). † Present address: Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, Maryland 20892, USA.Supplementary Information is linked to the online version of the paper at www.nature.com/nature.Author Information Raw data on expression microarrays are available at http://expression.microslu.washington.edu. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2009 January 9. showed that mice infected with r1918 had about tenfold higher levels of virus on days 1 and 3 after infection than those infected with 2:6 1918 or 5:3 1918. By day 5 after infection there was a slight decrease in the lung titres of r1918-infected mice, and mice infected with 2:6 1918, 5:3 1918 and r1918 showed similar lung titres of about 10 7 50% embryo-infective doses per ml. Our results were consistent with those in previous reports 3 : most mice infected with r1918 ...

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Influenza is a global health concern, causing death, morbidity, and economic losses. Chemotherapeutics that target influenza virus are available; however, rapid emergence of drug-resistant strains is common. Therapeutic targeting of host proteins hijacked by influenza virus to facilitate replication is an antiviral strategy to reduce the development of drug resistance. Nuclear export of influenza virus ribonucleoprotein (vRNP) from infected cells has been shown to be mediated by exportin 1 (XPO1) interaction with viral nuclear export protein tethered to vRNP. RNA interference screening has identified XPO1 as a host proinfluenza factor where XPO1 silencing results in reduced influenza virus replication. The Streptomyces metabolite XPO1 inhibitor leptomycin B (LMB) has been shown to limit influenza virus replication in vitro; however, LMB is toxic in vivo, which makes it unsuitable for therapeutic use. In this study, we tested the anti-influenza virus activity of a new class of orally available smallmolecule selective inhibitors of nuclear export, specifically, the XPO1 antagonist KPT-335 (verdinexor). Verdinexor was shown to potently and selectively inhibit vRNP export and effectively inhibited the replication of various influenza virus A and B strains in vitro, including pandemic H1N1 virus, highly pathogenic H5N1 avian influenza virus, and the recently emerged H7N9 strain. In vivo, prophylactic and therapeutic administration of verdinexor protected mice against disease pathology following a challenge with influenza virus A/California/04/09 or A/Philippines/2/82-X79, as well as reduced lung viral loads and proinflammatory cytokine expression, while having minimal toxicity. These studies show that verdinexor acts as a novel anti-influenza virus therapeutic agent. IMPORTANCEAntiviral drugs represent important means of influenza virus control. However, substantial resistance to currently approved influenza therapeutic drugs has developed. New antiviral approaches are required to address drug resistance and reduce the burden of influenza virus-related disease. This study addressed critical preclinical studies for the development of verdinexor (KPT-335) as a novel antiviral drug. Verdinexor blocks progeny influenza virus genome nuclear export, thus effectively inhibiting virus replication. Verdinexor was found to limit the replication of various strains of influenza A and B viruses, including a pandemic H1N1 influenza virus strain, a highly pathogenic H5N1 avian influenza virus strain, and a recently emerging H7N9 influenza virus strain. Importantly, oral verdinexor treatments, given prophylactically or therapeutically, were efficacious in limiting lung virus burdens in influenza virus-infected mice, in addition to limiting lung proinflammatory cytokine expression, pathology, and death. Thus, this study demonstrated that verdinexor is efficacious against influenza virus infection in vitro and in vivo.

We previously hypothesized that efficient translation of influenza virus mRNA requires the recruitment of P58 IPK , the cellular inhibitor of PKR, an interferon-induced kinase that targets the eukaryotic translation initiation factor eIF2␣. P58 IPK also inhibits PERK, an eIF2␣ kinase that is localized in the endoplasmic reticulum (ER) and induced during ER stress. The ability of P58 IPK to interact with and inhibit multiple eIF2␣ kinases suggests it is a critical regulator of both cellular and viral mRNA translation. In this study, we sought to definitively define the role of P58IPK during viral infection of mammalian cells. Using mouse embryo fibroblasts from P58 IPK؊/؊ mice, we demonstrated that the absence of P58 IPK led to an increase in eIF2␣ phosphorylation and decreased influenza virus mRNA translation. The absence of P58 IPK also resulted in decreased vesicular stomatitis virus replication but enhanced reovirus yields. In cells lacking the P58 IPK target, PKR, the trends were reversed-eIF2␣ phosphorylation was decreased, and influenza virus mRNA translation was increased. Although P58 IPK also inhibits PERK, the presence or absence of this kinase had little effect on influenza virus mRNA translation, despite reduced levels of eIF2␣ phosphorylation in cells lacking PERK. Finally, we showed that influenza virus protein synthesis and viral mRNA levels decrease in cells that express a constitutively active, nonphosphorylatable eIF2␣. Taken together, our results support a model in which P58 IPK regulates influenza virus mRNA translation and infection through a PKR-mediated mechanism which is independent of PERK.

Influenza A virus infection is a major global health concern causing significant mortality, morbidity, and economic loss. Antiviral chemotherapeutics that target influenza A virus are available; however, rapid emergence of drug-resistant strains has been reported. Consequently, there is a burgeoning need to identify novel anti-influenza A drugs, particularly those that target host gene products required for virus replication, to reduce the likelihood of drug resistance. In this study, a small interfering RNA (siRNA) screen was performed to identify host druggable gene targets for anti-influenza A virus therapy. The host organic anion transporter-3 gene (OAT3), a member of the SLC22 family of transporters, was validated as being required to support influenza A virus replication. Probenecid, a prototypical uricosuric agent and chemical inhibitor of organic anion transporters known to target OAT3, was shown to be effective in limiting influenza A virus infection in vitro (50% inhibitory concentration [IC 50 ] of 5.0 ؋ 10 ؊5 to 5.0 ؋ 10 ؊4 M; P < 0.005) and in vivo (P < 0.05). Probenecid is widely used for treatment of gout and related hyperuricemic disorders, has been extensively studied for pharmacokinetics and safety, and represents an excellent candidate for drug repositioning as a novel anti-influenza A chemotherapeutic.

Background: IFN activates JAK-STAT signaling, where STAT1 phosphorylation is crucial for ISG induction and expression of IFIT2 to limit West Nile virus infection. Results: IKK⑀ mediates STAT1 serine 708 phosphorylation exclusive of tyrosine phosphorylation but dependent on nuclear export and ISG synthesis. Conclusion: IKK⑀-mediated STAT1 S708 phosphorylation is crucial for IFIT2 expression to control WNV. Significance: We define a novel anti-WNV innate immune effector pathway.

Influenza A virus (IAV) causes seasonal epidemics of respiratory illness that can cause mild to severe illness and potentially death. Antiviral drugs are an important countermeasure against IAV; however, drug resistance has developed, thus new therapeutic approaches are being sought. Previously, we demonstrated the antiviral activity of a novel nuclear export inhibitor drug, verdinexor, to reduce influenza replication in vitro and pulmonary virus burden in mice. In this study, in vivo efficacy of verdinexor was further evaluated in two animal models or influenza virus infection, mice and ferrets. In mice, verdinexor was efficacious to limit virus shedding, reduce pulmonary pro-inflammatory cytokine expression, and moderate leukocyte infiltration into the bronchoalveolar space. Similarly, verdinexor-treated ferrets had reduced lung pathology, virus burden, and inflammatory cytokine expression in the nasal wash exudate. These findings support the anti-viral efficacy of verdinexor, and warrant its development as a novel antiviral therapeutic for influenza infection.