TRAF6 and the Three C-Terminal Lysine Sites on IRF7 Are Required for Its Ubiquitination-Mediated Activation by the Tumor Necrosis Factor Receptor Family Member Latent Membrane Protein 1

Abstract

We have recently shown that interferon regulatory factor 7 (IRF7) is activated by Epstein-Barr virus latent membrane protein 1 (LMP1), a member of the tumor necrosis factor receptor (TNFR) superfamily, through receptor-interacting protein-dependent K63-linked ubiquitination (L. E. Huye, S. Ning, M. Kelliher, and J. S. Pagano, Mol. Cell. Biol. 27:2910-2918, 2007). In this study, with the use of small interfering RNA and TNFR-associated factor 6 (TRAF6) knockout cells, we first show that TRAF6 and its E3 ligase activity are required for LMP1-stimulated IRF7 ubiquitination. In Raji cells which are latently infected and express high levels of LMP1 and IRF7 endogenously, expression of a TRAF6 small hairpin RNA construct reduces endogenous ubiquitination and endogenous activity of IRF7. In TRAF6 ؊/؊ mouse embryonic fibroblasts, reconstitution with TRAF6 expression, but not with TRAF6(C70A), which lacks the E3 ligase activity, recovers LMP1's ability to stimulate K63-linked ubiquitination of IRF7. Further, we identify IRF7 as a substrate for TRAF6 E3 ligase and show that IRF7 is ubiquitinated by TRAF6 at multiple sites both in vitro and in vivo. Most important, we determine that the last three C-terminal lysine sites (positions 444, 446, and 452) of human IRF7 variant A are essential for activation of IRF7; these are the first such sites identified. A ubiquitination-deficient mutant of IRF7 with these sites mutated to arginines completely loses transactivational ability in response not only to LMP1 but also to the IRF7 kinase IB kinase . In addition, we find that K63-linked ubiquitination of IRF7 occurs independently of its C-terminal functional phosphorylation sites. These data support our hypothesis that regulatory ubiquitination of IRF7 is a prerequisite for its phosphorylation. This is the first evidence to imply that ubiquitination is required for phosphorylation and activation of a transcription factor. Intracellular signaling initiated by latent membrane protein 1 (LMP1), the principal oncoprotein of the human gammaherpesvirus Epstein-Barr virus (EBV), is of interest not only for viral oncogenesis but also for the reason that LMP1 shares early steps in pathways used by CD40, interleukin-1 (IL-1) receptor-Toll-like receptor (TLR), and tumor necrosis factor receptor (TNFR) for activation of NFB (reviewed in references 16 and 35). As a member of the TNFR superfamily, LMP1 recruits TNFR-associated death domain protein, TNFRinteracting protein (RIP), and several TNFR-associated factors (TRAFs). Unlike CD40 and receptor activator of NF-B (RANK), which contain TRAF6-binding sites, LMP1 does not have a consensus TRAF6-binding sequence but associates with TRAF6 indirectly (reviewed in references 8, 16, 35, 51, and 61).Ubiquitination through K48-polyubiquitin linkage is well known as a process whereby proteins are targeted for proteasomal degradation. Recently, proteasome-independent functions for ubiquitination through K63 polyubiquitin or monoubiquitin linkages have been identified, and the importance of these ubiquitin...

Fas-associated Death Domain (FADD) and the E3 Ubiquitin-Protein Ligase TRIM21 Interact to Negatively Regulate Virus-induced Interferon Production

“…TRIM21 and FADD also additionally affect TRAF6, which is a positive regulator of IRF7 activation. TRAF6 can confer lysine 63 ubiquitination on IRF7, resulting in recruitment of kinases that phosphorylate IRF7 (53,58). How TRIM21 ubiquitination inhibits TRAF6 enzymatic function has yet to be determined.…”

Aryl Hydrocarbon Receptor Interacting Protein Targets IRF7 to Suppress Antiviral Signaling and the Induction of Type I Interferon

“…GFP-IRF7, Flag-IRF7, and deletion mutants (1-503, 1-475, 1-416, 1-340, 1-246, and 1-200) were previously described (42). IFN-␣4 luciferase reporter was provided by Dr. Shunbin Ning (East Tennessee State University) (43). IFN-␤ and NF-B reporters were described previously (22,44).…”

Guanylate Binding Protein 4 Negatively Regulates Virus-Induced Type I IFN and Antiviral Response by Targeting IFN Regulatory Factor 7

“…5B, 5C). Reduction of the ubiquitination of IRF7 led to impaired transactivation ability of IRF7 (34). Therefore, we assumed that overexpression of GBP4 could inhibit TRAF6-induced IRF7 transactivation.…”