SUMMARY TRIM25 mediates Lys 63-linked ubiquitination of the N-terminal CARDs of the viral RNA sensor RIG-I, leading to type I interferon (IFN) production. Here, we report that the influenza A virus non-structural protein 1 (NS1) specifically inhibits TRIM25-mediated RIG-I CARD ubiquitination, thereby suppressing RIG-I signal transduction. A novel domain in NS1 comprising E96/E97 residues mediates its interaction with the coiled-coil domain of TRIM25, thus blocking TRIM25 multimerization and RIG-I CARD ubiquitination. Furthermore, a recombinant influenza A virus expressing an E96A/E97A NS1 mutant is defective in blocking TRIM25-mediated anti-viral IFN response and loses virulence in mice. Our findings reveal a novel mechanism of influenza virus to inhibit host IFN response and also emphasize the vital role of TRIM25 in modulating viral infections.

Long non-coding RNAs (lncRNAs) play critical roles in diverse cellular processes; however, their involvement in many critical aspects of the immune response including the interferon (IFN) response remains poorly understood. To address this gap, we compared the global gene expression pattern of primary human hepatocytes before and at three time points after treatment with IFN-α. Among ∼200 IFN-induced lncRNAs, one transcript showed ∼100-fold induction. This RNA, which we named lncRNA-CMPK2, was a spliced, polyadenylated nuclear transcript that was induced by IFN in diverse cell types from human and mouse. Similar to protein-coding IFN-stimulated genes (ISGs), its induction was dependent on JAK-STAT signaling. Intriguingly, knockdown of lncRNA-CMPK2 resulted in a marked reduction in HCV replication in IFN-stimulated hepatocytes, suggesting that it could affect the antiviral role of IFN. We could show that lncRNA-CMPK2 knockdown resulted in upregulation of several protein-coding antiviral ISGs. The observed upregulation was caused by an increase in both basal and IFN-stimulated transcription, consistent with loss of transcriptional inhibition in knockdown cells. These results indicate that the IFN response involves a lncRNA-mediated negative regulatory mechanism. lncRNA-CMPK2 was strongly upregulated in a subset of HCV-infected human livers, suggesting a role in modulation of the IFN response in vivo.

The availability of an influenza virus NS1 gene knockout virus (delNS1 virus) allowed us to establish the significance of the biological relationship between the influenza virus NS1 protein and double-stranded-RNAactivated protein kinase (PKR) in the life cycle and pathogenicity of influenza virus. Our results show that the lack of functional PKR permits the delNS1 virus to replicate in otherwise nonpermissive hosts, suggesting that the major function of the influenza virus NS1 protein is to counteract or prevent the PKR-mediated antiviral response.

Schizosaccharomyces pombe contains four putative (1,3)β-D-glucan synthase (GS) catalytic subunits, Bgs1p-4p. In this work, we cloned bgs4+ and show that Bgs4p is the only subunit found to be a part of the GS enzyme and essential for maintaining cell integrity during cytokinesis and polarized growth. Here we show that bgs4+, cwg1+ (cwg1-1 shows reduced cell-wall β-glucan and GS catalytic activity) and orb11+ (orb11-59 is defective in cell morphogenesis) are the same gene. bgs4+ is essential for spore germination and bgs4+ shut-off produces cell lysis at growing poles and mainly at the septum prior to cytokinesis, suggesting that Bgs4p is essential for cell wall growth and to compensate for an excess of cell wall degradation during cytokinesis. Shut-off and overexpression analysis suggest that Bgs4p forms part of a GS catalytic multiprotein complex and that Bgs4p-promoted cell-wall β-glucan alterations induce compensatory mechanisms from other Bgs subunits and (1,3)α-D-glucan synthase. Physiological localization studies showed that Bgs4p localizes to the growing ends, the medial ring and septum, and at each stage of wall synthesis or remodeling that occurs during sexual differentiation: mating, zygote and spore formation, and spore germination. Bgs4p timing and requirements for proper positioning during cytokinesis and its localization pattern during spore maturation differ from those of Bgs1p. Bgs4p localizes overlapping the contractile ring once Bgs1p is present and a Calcofluor white-stained septum material is detected, suggesting that Bgs4p is involved in a late process of secondary or general septum synthesis. Unlike Bgs1p, Bgs4p needs the medial ring but not the septation initiation network proteins to localize with the other septation components. Furthermore, Bgs4p localization depends on the polarity establishment proteins. Finally, F-actin is necessary for Bgs4p delocalization from and relocalization to the growing regions, but it is not needed for the stable maintenance of Bgs4p at the growing sites, poles and septum. All these data show for the first time an essential role for a Bgs subunit in the synthesis of a (1,3)β-D-glucan necessary to preserve cell integrity when cell wall synthesis or repair are needed.