The C-Terminal Effector Domain of Non-Structural Protein 1 of Influenza A Virus Blocks IFN-ß Production by Targeting TNF Receptor-Associated Factor 3

Qian, Wei; Wei, Xiaoqin; Guo, Kelei; Li, Yongtao; Lin, Xian; Zou, Zhong; Zhou, Hongbo; Jin, Meilin

It is well-known that TRAF3 is modified with a polyubiquitin chain to provide a scaffold for complex formation. Thus, in order to study the effect of NS1/126-225 on TRAF3 ubiquitination, Co-IP experiments were conducted. Flag-TRAF3, pUb-HA, and NS1/126-225 along with RIG-I(N) were cotransfected into 293T cells and the ubiquitination level of TRAF3 was monitored. Compared to the control, NS1/126-225 reduced the RIG-I(N)-induced ubiquitination of TRAF3 (Figure 6, left) . To explore the type of TRAF3 ubiquitin chains, Flag-TRAF3 was transfected into 293T cells with ubiquitin mutants, including the pUb-K48-HA or pUb-K63-HA expression plasmid. The results showed that NS1/126-225 could decrease the K63-linked ubiquitination of TRAF3 but not the K48-linked ubiquitination of TRAF3 (Figure 6, right) . In summary, these results demonstrate that NS1/126-225 suppresses the K63-linked ubiquitination of TRAF3 that is important for the recruitment of the TBK1-IKKε kinase complex. expressing AA1 to 346 and containing the RING domain, the Zinc-fingers domain, the Isoleucine Zipper domain, and Flag-TRAF3-TD, expressing AA347 to 568 and containing the TRAF domain ( Figure 7A ). Co-IP assays showed that the TRAF domain of TRAF3 was found to be the crucial region responsible for the association of TRAF3 with NS1/126-225 ( Figure 7B ) as well as with wtNS1 (data not shown).