RIP Mediates LMP1 Ubiquitination and Activation of IRF7

Background: As a key mediator of type-I interferon (IFNalpha/beta) responses, Interferon Regulatory Factor 7 (IRF7) is essential for host immune defenses. Activation of IRF7 generally requires virus-induced C-terminal phosphorylation. Lys63-linked ubiquitination regulates functions of target proteins rather than their proteasomal degradation through Lys48-linked ubiquitination.

Methods: For luciferase assays, 293 or 3T3 cells were transfected by use of Fugene 6 or Lipofectamine reagent. ISRE-Luc was used as reporter, and Renilla as internal control. For immunoprecipitation, 3T3 cells were transfected with FlagIRF7, LMP1, RIP and HA-Ub by use of Lipofectamine reagent. Cell lysates were first immunoprecipitated with Flag M2 antibody. After washes, protein complexes on the beads were dissociated in 1% SDS, diluted 1:10, and a second immunoprecipitation was performed with IRF7 antibody. Western Blots were performed with HA antibody. For RNA interference, siRIP was transfected by use of Lipofectamine 2000 reagent, and cells were cultured for 36 h before transfection for promoter-reporter assay

Results: Transient expression of LMP1 enhances IRF7 transcriptional activity. Moreover, LMP1 promotes ubiquitination of IRF7. At least a portion of this ubiquitination involves Lys63-linked ubiquitin, which has protein regulatory functions. Overexpression of ubiquitin or Lys63-only ubiquitin, which can only participate in Lys63-linked ubiquitination, activates IRF7 in a dose-dependent manner and increases LMP1-stimulated IRF7 transcriptional activity. It suggests that LMP1-promoted ubiquitination enhances IRF7 activity. In addition, LMP1 promotes the interaction of IRF7 with RIP. Finally, repression of expression of RIP in 293 cells by RNA interference results in significant decrease in LMP1-stimulated IRF7 activity, and in RIP knockout (RIP-/-) 3T3 cells, LMP1 can neither promote ubiquitination of IRF7 nor increase its transcriptional activity. 

Conclusions: IRF7 can be activated by the EBV oncoprotein, LMP1, through the Lys63-linked ubiquitination pathway, and RIP is required for this activation. These findings may also have more general significance for LMP1 signalling.