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Intracellular innate immune responses are essential to protect host cells against pathogens. Upon infection, pattern recognition receptors detect incoming microbes and trigger downstream signaling pathways leading to production of antiviral type-I interferons (IFNs).These signaling pathways are regulated by different posttranslational modifications including the ubiquitin (Ub) system. Proteins covalently attached to K48-linked polyUb are targeted for degradation by the proteasome, whereas protein modification with K63-linked polyUb may have non-proteolytic activating functions. Unanchored Ub chains that are not covalently attached to any protein are also important for kinase activation. Tripartite motif (TRIM) proteins, which function as E3-ubiquitin ligases, have been implicated in antiviral activity. We found that TRIM6 is important in the synthesis of unanchored K48-linked polyUb chains, which activate the IKKε kinase for STAT1 phosphorylation and induction of an antiviral response. These polyUb chains and IKKε interact in vivo in an IFN-I signaling dependent manner upon virus infection in mice. To better understand the physiological role of unanchored polyUb during virus infection, we developed a system to isolate ubiquitin-interacting proteins from the lungs of influenza-infected mice. Using a proteomics approach we identified new cellular factors that interact with unanchored polyUb chains in vivo and may be involved in the innate immune antiviral response.

Type-I interferons (IFN-I) are essential antiviral cytokines produced upon microbial infection. IFN-I elicits this activity through the upregulation of 100s of IFN-I stimulated genes (ISGs), many of which have known antiviral activity. The full breadth of ISG induction requires activation of a number of cellular factors including the IκB kinase epsilon (IKKϵ). However, the mechanism of IKKϵ activation upon viral infection or IFN receptor signaling remains 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. Some studies have suggested a role for unanchored K63-linked polyubiquitin chains, which are not conjugated to any protein, in regulation of kinase activity. However, no role has yet been established for unanchored K48-linked polyubiquitin chains in kinase activation. We show that TRIM6 and the E2-ubiquitin conjugase UbE2K cooperate in the synthesis of unanchored K48-linked polyubiquitin chains, which activate IKKϵ for subsequent STAT1 phosphorylation. Our work defines a previously unrecognized activating role of K48-linked unanchored polyubiquitin chains in kinase activation and identifies the UbE2K-TRIM6-ubiquitin axis as critical for IFN signaling and antiviral response.