Figure 3.

CyHV-3 ORF12 Encodes a Soluble Decoy Receptor for TNF-α

(A) ORF12 homology modeling. (i) Human TNF-α (green) with human TNFR2 (c6 domains in yellow, orange, and pink, intervening sequences in blue; PDB: 3ALQ). (ii) Homology model (colored as in Ai) of CyHV-3 ORF12 on 3ALQ.R, with proposed interaction with TNF-α (green) also shown. Residues in ball-and-stick representation are those with poor bond angles as assessed by Ramachandran plot. (iii) Overlay of (i) and (ii), superposition at 1.771Å is achieved. (iv) Ramachandran plot. Bad angles and the position of the affected residues in (ii) are indicated in red.

(B) Silver staining of total proteins found in concentrated supernatants of CCB cells infected with CyHV-3 (WT, 12Del, or 12Rev) or mock-infected (Mock). Arrowheads indicate the band corresponding to the ORF12 protein.

(C) ELISA binding assay of carp TNF-α1 and TNF-α2 to CyHV-3 secreted proteins. The data are the mean + SD of duplicate measurements. Results for which a significant difference was observed between WT/12Rev and 12Del/Mock groups are marked by asterisks. See also Figure S2.

(D) Neutralization of carp TNF-α1 and TNF-α2 by CyHV-3 secreted proteins was tested by using a TNF-α bioluminescent reporter assay. The data are expressed as fold change of luminescence relative to the respective controls and represent the mean + SD of quadruplicate measurements. Results for which a significant difference was observed between pTNF-α1 or pTNF-α2 and pEmpty are marked by asterisks. These data are representative of three independent experiments.