LAUSR

Significance Vaccinia virus (VACV) encodes many proteins that inhibit innate immunity. For instance, protein A49 inhibits NF-κB activation by binding to β-TrCP. Here we show that A49 is phosphorylated on serine 7 and that this is necessary for binding β-TrCP and inhibition of NF-κB activation. Further, this phosphorylation occurs when the NF-κB pathway is stimulated and the kinase IKKβ is activated. Thus, A49 shows beautiful biological regulation, for activation of the pathway also activates the virus inhibitor of the pathway. The significance is seen in vivo, since VACVs expressing A49 S7A or S7E are less virulent than wild-type virus but more virulent than a virus lacking A49. Vaccinia virus protein A49 inhibits NF-κB activation by molecular mimicry and has a motif near the N terminus that is conserved in IκBα, β-catenin, HIV Vpu, and some other proteins. This motif contains two serines, and for IκBα and β-catenin, phosphorylation of these serines enables recognition by the E3 ubiquitin ligase β-TrCP. Binding of IκBα and β-catenin by β-TrCP causes their ubiquitylation and thereafter proteasome-mediated degradation. In contrast, HIV Vpu and VACV A49 are not degraded. This paper shows that A49 is phosphorylated at serine 7 but not serine 12 and that this is necessary and sufficient for binding β-TrCP and antagonism of NF-κB. Phosphorylation of A49 S7 occurs when NF-κB signaling is activated by addition of IL-1β or overexpression of TRAF6 or IKKβ, the kinase needed for IκBα phosphorylation. Thus, A49 shows beautiful biological regulation, for it becomes an NF-κB antagonist upon activation of NF-κB signaling. The virulence of viruses expressing mutant A49 proteins or lacking A49 (vΔA49) was tested. vΔA49 was attenuated compared with WT, but viruses expressing A49 that cannot bind β-TrCP or bind β-TrCP constitutively had intermediate virulence. So A49 promotes virulence by inhibiting NF-κB activation and by another mechanism independent of S7 phosphorylation and NF-κB antagonism. Last, a virus lacking A49 was more immunogenic than the WT virus.