A missense polymorphism in porcine interferon-gamma cDNA affects antiviral activity of the protein variant.
We determined the interferon-gamma (IFN-gamma) cDNA sequence from three porcine breeds, Duroc, Landrance/Duroc hybrid, and Landrance breeds. Five single nucleotide polymorphisms (SNPs) of porcine IFN-gamma (PoIFN-gamma) 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-gamma cDNAs of Duroc breed (PoIFN-gamma-W) and Landrance/Duroc hybrid (PoIFN-gamma-M), which, respectively, encoded Q67 and R67, were introduced into a prokaryotic expression vector pET32 to express recombinant PoIFN-gamma-W (rPoIFN-gamma-W) and rPoIFN-gamma-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-gamma-W protein was significantly higher (P<0.001) than that of rPoIFN-gamma-M protein in a plaque inhibition assay using pseudorabies virus (PRV). IC50 values of rPoIFN-gamma-W and rPoIFN-gamma-M protein in anti-PRV assay were determined as 5.3+/-1.3 and 9.3+/-4.3nM, respectively. In conclusion, we have identified five novel SNPs in PoIFN-gamma 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-gamma protein. This is the first report that shows the functional SNP in the coding region of IFN-gamma. In the future, it is imperative to determine whether Q67R replacement in IFN-gamma may have disease association.
Fan
Yi-Hsin
YH
Graduate Institute of Veterinary Microbiology, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC.
Chow
Kuan-Chih
KC
Huang
San-Yuan
SY
Chi
Lang-Ming
LM
Huang
Chienjin
C
Chiou
Shiow-Her
SH
D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000037:antagonists & inhibitors / Q000378:metabolism; D001483:Base Sequence; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000037:antagonists & inhibitors / Q000235:genetics*; D014644:Genetic Variation; D011558:Herpesvirus 1, Suid / Q000254:growth & development / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000235:genetics*; D011110:Polymorphism, Genetic; D013552:Swine; D010948:Viral Plaque Assay; D038301:Virus Inactivation
D000998:Antiviral Agents; D018076:DNA, Complementary; D007371:Interferon-gamma