Summary Type-I interferons (IFN-I) are essential antiviral cytokines produced upon microbial infection. IFN-I elicits this activity through the upregulation of hundreds of IFN-I stimulated genes (ISGs). The full breadth of ISG induction demands activation of a number of cellular factors including the IκB kinase epsilon (IKKε). However, the mechanism of IKKε activation upon IFN receptor signaling has remained elusive. Here we show that TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family of proteins, interacts with IKKε and promotes induction of IKKε-dependent ISGs. TRIM6 and the E2-ubiquitin conjugase UbE2K cooperate in the synthesis of unanchored K48-linked poly-ubiquitin chains, which activate IKKε for subsequent STAT1 phosphorylation. Our work attributes a previously unrecognized activating role of K48-linked unanchored poly-ubiquitin chains in kinase activation and identifies the UbE2K-TRIM6-ubiquitin axis as critical for IFN signaling and antiviral response.

Virus infection induces the production of type I and type II interferons (IFN-I and IFN-II), cytokines that mediate the antiviral response. IFN-I (IFN-α and IFN-β) induces the assembly of IFN-stimulated gene factor 3 (ISGF3), a multimeric transcriptional activation complex composed of STAT1, STAT2, and IFN regulatory factor 9. IFN-II (IFN-γ) induces the homodimerization of STAT1 to form the gamma-activated factor (GAF) complex. ISGF3 and GAF bind specifically to unique regulatory DNA sequences located upstream of IFN-I-and IFN-II-inducible genes, respectively, and activate the expression of distinct sets of antiviral genes. The balance between type I and type II IFN pathways plays a critical role in orchestrating the innate and adaptive immune systems. Here, we show that the phosphorylation of STAT1 by IκB kinase epsilon (IKKε) inhibits STAT1 homodimerization, and thus assembly of GAF, but does not disrupt ISGF3 formation. Therefore, virus and/or IFN-I activation of IKKε suppresses GAF-dependent transcription and promotes ISGF3-dependent transcription. In the absence of IKKε, GAF-dependent transcription is enhanced at the expense of ISGF3-mediated transcription, rendering cells less resistant to infection. We conclude that IKKε plays a critical role in regulating the balance between the IFN-I and IFN-II signaling pathways.H ost immune defenses counteract virus infection by coordinating an intracellular innate immune response with the adaptive immune response. Systemically, the antiviral defense is generally cell-mediated, involving the recruitment and activation of dendritic cells, macrophages, neutrophils, and natural killer cells to the site of infection (1). This initial response is followed by a second wave of specific antiviral defenses involving cytotoxic T cells and antibodies generated from plasma B cells (2). The success of the adaptive immune response is intricately linked to the intracellular innate defenses initiated at the site of infection (1). The signaling required to coordinate the successful induction of the intracellular immune response to virus infection relies on the activation of interferon (IFN) genes, which encode cytokines with antiviral and immunomodulatory activity (3).The vertebrate type I IFN (IFN-I) genes are arranged in a large gene cluster consisting of a single IFNβ gene and several tandemly arranged IFNα genes encoding distinct isotypes (4, 5). In contrast to type I IFNs, type II IFN (IFN-II) is encoded by a single IFNγ gene. Although IFN-I mediates cellular resistance to virus infection, IFN-II confers limited cellular protection through the induction of a subset of genes that are shared between the IFN-I and IFN-II transcriptomes (6, 7). Virus infection leads to the activation of IFN-I genes and the secretion of IFNα and -β, which bind to IFN receptors at the cell surface. This, in turn, leads to the transcriptional activation of a group of IFN-stimulated genes (ISGs), and these ISGs collectively establish a nonpermissive environment for virus replication (6).The transcription...

The transcriptional response to virus infection is thought to be predominantly induced by interferon (IFN) signaling. Here we demonstrate that, in the absence of IFN signaling, an IFNlike transcriptome is still maintained. This transcriptional activity is mediated from IFN-stimulated response elements (ISREs) that bind to both the IFN-stimulated gene factor 3 (ISGF3) as well as to IFN response factor 7 (IRF7). Through a combination of both in vitro biochemistry and in vivo transcriptional profiling, we have dissected what constitutes IRFspecific, ISGF3-specific, or universal ISREs. Taken together, the data presented here suggest that IRF7 can induce an IFNlike transcriptome in the absence of type-I or -III signaling and therefore provides a level of redundancy to cells to ensure the induction of the antiviral state.

Summary Influenza A virus is unique as an RNA virus in that it replicates in the nucleus and undergoes splicing. With only ten major proteins, the virus must gain nuclear access, replicate, assemble progeny virions in the cytoplasm and then egress. In an effort to elucidate the coordination of these events, we manipulated the transcript levels from the bicistronic NS segment that encodes the spliced virus product responsible for genomic nuclear export. We find that utilization of an erroneous splice site ensures the slow accumulation of the viral Nuclear Export Protein (NEP) while generating excessive levels of an antagonist designed to inhibit the cellular response to infection. Modulation of this simple transcriptional event results in improperly timed export and loss of virus infection. Together, these data demonstrate that coordination of the influenza A virus life cycle is set by a ‘molecular timer’ that operates on the inefficient splicing of a virus transcript.

Summary A successful cellular response to virus infection is essential for evolutionary survival. In plants, arthropods, and nematodes, cellular antiviral defenses rely on RNA interference (RNAi). Interestingly, the mammalian response to virus is predominantly orchestrated through interferon (IFN)-mediated induction of antiviral proteins. Despite the potency of the IFN system, it remains unclear whether mammals also have the capacity to employ antiviral RNAi. Here we investigate this by disabling either IFN, small RNA function or both activities in the context of virus infection. We find that loss of small RNAs in the context of an in vivo RNA virus infection lowers titers due to reduced transcriptional repression of the host antiviral response. In contrast, enabling a virus with the capacity to inhibit the IFN system results in increased titers. Taken together, we conclude that small RNA silencing is not a physiological contributor to the IFN-mediated cellular response to virus infection.