A cullin‐based ubiquitin ligase is involved in virus‐mediated IRF3 degradation

Objective The transcription factor Interferon Regulatory Factor (IRF) 3 is a key mediator of the innate immune system. Upon viral infection, IRF3 is degraded and the molecular mechanisms involved in this process are unknown. Thus, the principal objective of this research project is to characterize the signaling pathways involved in IRF3 degradation. Methods 293T cells were co‐transfected with different IRF3 and HA‐ubiquitin expression vectors and the cell extracts were submitted to coimmunoprecipitation experiments. Temperature‐sensitive (TS) cell lines, Ts20 and Ts41, were infected at 34°C or 39°C and Tet‐inducible 293 cells expressing a dominant negative Cullin1 (Cul1) were left untreated or inducted with doxycycline before infection. All experiments were analyzed by Western blots. Results We demonstrate that IRF3 is a polyubiquitinated protein and that its polyubiquitination level increases in response to virus infection. Using the Ts20 cell line, in which the ubiquitin‐activiting enzyme E1 is active at 34°C but inactive at 39°C, we demonstrate that ubiquitination is involved in virus‐induced degradation of IRF3. Using the Ts41 cell line, which harbors a TS mutation in the NEDD8‐activating E1 enzyme, we show that a NEDDylation pathway also plays a role in virus‐mediated IRF3 degradation. In agreement with these observations, overexpression of a dominant negative of Cul1 partly suppresses virus‐induced IRF3 degradation. Conclusion We suggest that virus‐induced proteasome degradation of IRF3 is dependent of its ubiquitination, a process accomplished in part by a Skp1‐Cul1‐F box complex. This research is supported by the CIHR.