Poxviral protein A46 antagonizes Toll-like receptor 4 signaling by targeting BB loop motifs in Toll-IL-1 receptor adaptor proteins to disrupt receptor:adaptor interactions.

Toll-like receptors (TLRs) have an anti-viral role in that they detect viruses, leading to cytokine and IFN induction, and as such are targeted by viruses for immune evasion. TLR4, although best known for its role in recognizing bacterial LPS, is also strongly implicated in the immune response to viruses. We previously showed that the poxviral protein A46 inhibits TLR4 signaling and interacts with Toll-IL-1 receptor (TIR) domain-containing proteins of the receptor complex. However the exact molecular mechanism whereby A46 disrupts TLR4 signaling remains to be established, and may yield insight into how the TLR4 complex functions, since viruses often optimally target key residues and motifs on host proteins for maximal efficiency. Here we show that A46 targets the BB loop motif of TIR proteins and thereby disrupts receptor:adaptor (TLR4:Mal and TLR4:TRAM), but not receptor:receptor (TLR4:TLR4) nor adaptor:adaptor (Mal:MyD88, TRAM:TRIF, and Mal:Mal) TIR interactions. The requirement for an intact BB loop for TIR adaptor interactions correlated with the protein:protein interfaces antagonized by A46. We previously discovered a peptide fragment derived from A46 termed VIPER (Viral Inhibitory Peptide of TLR4), which specifically inhibits TLR4 responses. Here we demonstrate that the region of A46 from which VIPER is derived represents the TLR4-specific inhibitory motif of the intact protein, and is essential for A46:TRAM interactions. This study provides the molecular basis for pathogen subversion of TLR4 signaling and clarifies the importance of TIR motif BB loops, which have been selected for viral antagonism, in the formation of the TLR4 complex.