Immune Netw.  2017 Dec;17(6):424-436. 10.4110/in.2017.17.6.424.

Species Specific Antiviral Activity of Porcine Interferon-α8 (IFNα8)

• Kim E
• Jhun H
• Kim J
• Park U
• Jo S
• Kwak A
• Kim S
• Nguyen TT
• Kang Y
• Choi I
• Lee J
• Kim H
• Kim Y
• Lee S
• Kim S

• KMID: 2400628
• DOI: http://doi.org/10.4110/in.2017.17.6.424

Abstract

Interferons (IFNs) have been known as antiviral genes and they are classified by type 1, type 2, and type 3 IFN. The type 1 IFN consists of IFNα, IFNβ, IFNτ, and IFNω whereas the type 2 IFN consists of only IFNγ, which is a key cytokine driving T helper cell type 1 immunity. IFNλ belongs to the type 3 IFN, which is also known as IL-28 and IL-29 possessing antiviral activities. Type 1 IFN is produced by viral infection whereas type 2 IFN is induced by mitogenic or antigenic T-cell stimuli. The IFNτ of bovine was first discovered in an ungulate ruminant recognition hormone. IFNτ belongs to the type 1 IFN with the common feature of type 1 IFN such as antiviral activity. IFNs have been mostly studied for basic research and clinical usages therefore there was no effort to investigate IFNs in industrial animals. Here we cloned porcine IFNα8 from peripheral blood mononuclear cells of Korean domestic pig (Sus scrofa domestica). The newly cloned IFNα8 amino acid sequence from Korean domestic pig shares 98.4% identity with the known porcine IFNα8 in databank. The recombinant porcine IFNα8 showed potent antiviral activity and protected bovine Madin-Darby bovine kidney epithelial (MDBK) cells from the cytopathic effect of vesicular stomatitis virus, but it failed to protect human Wistar Institute Susan Hayflick (WISH) cells and canine Madin-Darby canine kidney epithelial-like (MDCK) cells. The present study demonstrates species specific antiviral activity of porcine IFNα8.

Keyword

Porcine IFNα8; Mx dynamin-like GTPase-1; 2′, 5′-oligoadenylate synthetase-1; Recombinant protein; Antiviral assay

MeSH Terms

Amino Acid Sequence
Animals
Clone Cells
Humans
Interferons
Kidney
Ruminants
Sus scrofa
T-Lymphocytes
T-Lymphocytes, Helper-Inducer
Vesicular Stomatitis
Interferons

Figure

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  Figure 1 The regulation of porcine IFNα8 transcript. RT-PCR was performed with the whole blood RNA from Korean domestic pig (Sus scrofa domestica) under stimulation of LPS, polyinosinic-polycytidylic acid (Poly I:C), and VSV at time point of 3 h. The control β-actin showed no difference in transcript. The data represents one of 4 independent experiments. Ctl, control; LPS, lipopolysaccharide.

• 

  Figure 2 Translated amino acid sequence of porcine IFNα. The nucleotide sequence was used to convert into amino acid sequence. The hydrophobic signal sequence of 69 base pairs nucleotide sequences were highlighted by green color with underlined 23 amino acid residues. This sequence was deposited in databank and obtained accession number as “KX275310”.

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  Figure 3 Comparison of porcine IFNα8 to the known porcine IFNα. The ORF of porcine IFNα8 mRNA from Korean domestic pig was analyzed and newly identified as porcine IFNα8 (accession No. KX275310). The amino acid sequence was deduced by newly obtained the DNA sequence of porcine IFNα8 and the different amino acid residue of Korean porcine IFNα8 compared to porcine IFNα8 (accession No. ACV42397). Three distinct amino acid residues are highlighted with yellow color. ORF, open reading frame.

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  Figure 4 The expression and purification of recombinant porcine IFNα8 with Talon affinity chromatography. The recombinant porcine IFNα8 mRNA from Korean domestic pig was expressed in E. coli and purified by a mini-Talon metal affinity column. (A) The IPTG-induced recombinant porcine IFNα8 detected with mouse anti-his6-tag monoclonal antibody. The arrow indicated porcine IFNα8 induced in the presence of IPTG for 3 h. (B) The a mini-Talon metal affinity chromatography purified recombinant porcine IFNα8 was visualized by Coomassie blue staining. The data represents one of 5 independent experiments.

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  Figure 5 The purification of porcine IFNα8 by anion exchange chromatography. (A) UV absorbance at 280 nm was recorded during the porcine IFNα8 the second step purification process with anion chromatography. (B) The purified recombinant porcine IFNα8 fractions were visualized by silver staining. (C) The bound fractions of recombinant porcine IFNα8 were pooled for quantification to compare with BSA in silver staining. The data represents one of 5 independent experiments. BSA, bovine serum albumin; UV, ultra-violet.

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  Figure 6 Recombinant porcine IFNα8 reduces the cytopathic effect caused by VSV. (A) Human epithelial WISH, (B) bovine MDBK, (C) canine MDCK cells were treated with recombinant porcine IFNα8 including positive control human IFNα2 then infected with VSV to analyze the reduction of cytopathic effect. The plate was visualized with crystal violet staining. The concentration of porcine IFNα8 (20 ng/ml) and human IFNα2 (2 ng/ml) were added by the 2-fold serial dilution. For control, the wells at bottom row were non-infected cells; the rows above non-infected wells were only VSV infected cells which shows the complete cytopathic effect to VSV challenge. The data represents one of 5 independent experiments. hIFN, human interferon; pIFN, porcine interferon.

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  Figure 7 Recombinant porcine IFNα8 induces antiviral genes. (A) Bovine MDBK, (B) human WISH, and (C) canine MDCK cells were treated with human porcine IFNα8 (20 ng/ml) and human IFNα2 (2 ng/ml) at indicated time points (3 and 6 h). Non-treated cells were prepared as negative control. After treatment of IFNs, total RNA was extracted for RT-PCR of Mx-1, OAS-1, and β-actin for normalization. The data represents one of 3 independent experiments. Ctl, control; hIFN, human interferon; pIFN, porcine interferon.

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