A missense polymorphism in porcine interferon-γ cDNA affects antiviral activity of the protein variant

We determined the interferon-γ (IFN-γ) cDNA sequence from three porcine breeds, Duroc, Landrance/Duroc hybrid, and Landrance breeds. Five single nucleotide polymorphisms (SNPs) of porcine IFN-γ (PoIFN-γ) were identified, respectively, at positions 269 (A/G), 376 (C/T), 426 (T/C), and 465 (T/C) of the coding sequence in Landrance/Duroc hybrid, and at position 251 (A/G) in Landrance breed. Among them, A269G and A251G polymorphisms resulted in Q67R and K61R replacements in the mature protein. PoIFN-γ cDNAs of Duroc breed (PoIFN-γ-W) and Landrance/Duroc hybrid (PoIFN-γ-M), which, respectively, encoded Q67 and R67, were introduced into a prokaryotic expression vector pET32 to express recombinant PoIFN-γ-W (rPoIFN-γ-W) and rPoIFN-γ-M protein variants in Escherichia coli. The identity of both protein variants was further confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We then compared bioactivities of these two recombinant proteins. Although both recombinant protein variants exhibited comparable activities in antiproliferation of PK-15 cells and in nitric oxide (NO) induction of porcine peripheral monocytes, antiviral activity of rPoIFN-γ-W protein was significantly higher (P < 0.001) than that of rPoIFN-γ-M protein in a plaque inhibition assay using pseudorabies virus (PRV). IC50 values of rPoIFN-γ-W and rPoIFN-γ-M protein in anti-PRV assay were determined as 5.3 ± 1.3 and 9.3 ± 4.3 nM, respectively. In conclusion, we have identified five novel SNPs in PoIFN-γ cDNA, including two missense polymorphisms that result in Q67R and K61R replacements. Our results further demonstrate that Q67R can markedly reduce antiviral activity of the PoIFN-γ protein. This is the first report that shows the functional SNP in the coding region of IFN-γ. In the future, it is imperative to determine whether Q67R replacement in IFN-γ may have disease association.