Experimental design. Induction of TLR signaling by H. capsulatum Yps3p protein leads to the activation of IRAK2 and NF-κB. NF-κB is then released from its inhibitor IκB and translocates into the nucleus, where it binds to one of five NF-κB binding sites on the pNiFty2-Luc plasmid, and subsequently activates transcription to produce luciferase. Vaccinia virus [VV] protein A46R targets multiple TLR adaptors including MyD88, and A52R associates with IRAK-1 and TRAF6 to disrupt downstream signaling. VV N1L and K1L proteins prevent the release of NF-κB from IκB. Expression of these proteins from their corresponding pORF5-VV plasmid leads to the inhibition of NF-κB activation and decreased luciferase expression from pNiFty2-Luc.