Abstract
The production of cytokines such as type I interferon (IFN) is an essential component of innate immunity. Insufficient amounts of cytokines lead to host sensitivity to infection, whereas abundant cytokine production can lead to inflammation. A tight regulation of cytokine production is, thus, essential for homeostasis of the immune system. IFN-α production during RNA virus infection is mediated by the master transcription factor IRF7, which is activated upon ubiquitination by TRAF6 and phosphorylation by IKKε and TBK1 kinases. We found that Fas-associated death domain (FADD), first described as an apoptotic protein, is involved in regulating IFN-α production through a novel interaction with TRIM21. TRIM21 is a member of a large family of proteins that can impart ubiquitin modification onto its cellular targets. The interaction between FADD and TRIM21 enhances TRIM21 ubiquitin ligase activity, and together they cooperatively repress IFN-α activation in Sendai virus-infected cells. FADD and TRIM21 can directly ubiquitinate IRF7, affect its phosphorylation status, and interfere with the ubiquitin ligase activity of TRAF6. Conversely, a reduction of FADD and TRIM21 levels leads to higher IFN-α induction, IRF7 phosphorylation, and lower titers of RNA virus of infected cells. We conclude that FADD and TRIM21 together negatively regulate the late IFN-α pathway in response to viral infection.
Highlights
We show here that Fas-associated death domain (FADD) interacts with TRIM21 through its death-effector domain (DED) and enhances TRIM21 auto-ubiquitination and its ubiquitination activities on IRF7
FADD Interacts with TRIM 21 but Not the Other Closely Related TRIM Family Members—To understand how FADD might play a role in biological processes other than cell death, we attempted to identify any proteins that associated with FADD in non-stimulating conditions. 293T cells were transfected with a FLAG-tagged FADD expression plasmid or an empty control plasmid
We demonstrate here that FADD interacts with TRIM21, an E3 ubiquitin ligase that can ubiquitinate itself, IRF transcription factors, and p27 cell cycle inhibitor (22, 28 –31)
Summary
Subsequent immunoblotting with anti-HA antibodies to detect ubiquitinated proteins revealed a high molecular weight smear of more than 50 kDa in samples co-transfected with FADD, TRIM21, and ubiquitin (Fig. 3A, lower panel, lane 5). We observed a small, albeit reproducible increase in NF-B or IFN- activation with FADD overexpression in unstimulated or SeV-stimulated 293T cells (Fig. 4, A and B). Transient transfections of both TRIM21 and FADD only resulted in a modest increase in IFN- and NF-B activation (Fig. 4, A and B).
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