NF-κB activation is a turn on for vaccinia virus phosphoprotein A49 to turn off NF-κB activation

Abstract

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.

The characteristics and roles of β‐TrCP1/2 in carcinogenesis

“…Substrates of SCF b-TrCP ubiquitin ligase in other cellular processes SCF b-TrCP ubiquitin ligase plays a fundamental role in regulating the activities of multiple biological processes by targeting a range of cellular substrates for ubiquitination and degradation: (a) regulating cellular apoptosis through targeting BimEL [84], MAP3K5/ASK1 [85], MCL1 [86,87], and Pro-caspase-3 [88] for degradation; (b) involvement in virus infection by degrading A49 [89], BST-2 [90,91] (Fig. 4).…”

Vaccinia Virus Activation and Antagonism of Cytosolic DNA Sensing

“…Using this mechanism A49 binds tightly to β-TrCP only when IKKβ is activated and canonical β-TrCP targets including IκBα and β-catenin accumulate in their phosphorylated forms and are not processed (33,150,151). A49 antagonism of NF-κB contributes to virulence (33,34), but mutant A49 viruses unable to bind β-TrCP retain some degree of virulence suggesting the existence of other β-TrCP-independent functions, perhaps mediated by a second product identified in the A49 ORF (34,152). In addition to these proteins, several VACV proteins including K1 and A55 have been shown to act at the level of the NF-κB heterodimer preventing their translocation or their normal processing (35,43,153).…”

Leaky scanning translation generates a second A49 protein that contributes to vaccinia virus virulence

“…Recently, further insight to the mechanism of action of A49 showed that phosphorylation of S7 was necessary for A49 to bind β-TrCP and inhibit activation of NF-κB, whereas S12 was dispensable for these functions [ 8 ]. Therefore, A49 is a conditional inhibitor of NF-κB and requires activation by phosphorylation to become an inhibitor of this pathway.…”

“…Consequently, A49 is activated to become an inhibitor of NF-κB when the pathway leading to NF-κB activation is switched on. In other words, NF-κB activation is a turn on for A49 to turn off NF-κB activation [ 8 ]. This report also showed that A49 has a second function that contributes to virulence, independent of NF-κB inhibition.…”

“…This report also showed that A49 has a second function that contributes to virulence, independent of NF-κB inhibition. VACV strains expressing a mutant A49 protein that either cannot bind β-TrCP and so cannot inhibit NF-κB activation, or does these functions constitutively, both had virulence that was greater than that of vΔA49 and lower than that of wild-type (WT) virus [ 8 ].…”