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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.

Following isolation of human IFN-␥ cDNA and gene in 1982 (Devos et al., 1982;Gray et al., 1982;Ullrich et al., 1982), cDNA of porcine IFN-␥ (PoIFN-␥) was isolated in 1988 (Charley et al., 1988). Nevertheless, the cDNA sequence has not been formally published. Although sequence of PoIFN-␥ gene was reported and aligned with human IFN-␥ gene to deduce the exon and intron structure (Dijkmans et al., 1990), little is known about the polymorphism of PoIFN-␥ gene or cDNA. Human population studies have revealed several polymorphic sites in both intron and exon regions of human IFN-␥ gene (Iwasaki et al., 2001;Tiroch et al., 2005). Two polymorphisms located in intron 1 of human IFN-␥ gene were further shown to affect the production of IFN-␥ (Pravica et al., 1999(Pravica et al., , 2000, and which might influence either disease manifestation of IgA nephropathy (Schena et al., 2006), or the infection susceptibility to parvovirus B19 (Kerr et al., 2003) and severe acute respiratory syndrome coronavirus (SARS-CoV) (Chong et al., 2006). Nonetheless, it remains to be explored whether any polymorphic sites in the coding region of IFN-␥ gene may affect its protein activity.
In this study, our specific aim was to explore functional polymorphism in the coding region of PoIFN-␥ gene. By employing reverse transcription-polymerase chain reaction (RT-PCR), three cDNA clones of PoIFN-␥ were isolated, respectively, from Duroc, Landrance/Duroc hybrid, and Landrance breeds. Nucleotide and encoded amino acid sequences of these three clones were aligned and compared with that of PoIFN-␥ gene (Dijkmans et al., 1990) to identify any polymorphic sites. Two PoIFN-␥ cDNA variants were cloned, respectively, into pET32 vector for expressing rPoIFN-␥ proteins. By performing a pseudorabies virus (PRV)-based plaque inhibition assay, we evaluated the antiviral activity of both rPoIFN-␥ protein variants. Other activities of IFN-␥, including antiproliferative effect on cells and stimulating nitric oxide (NO) production in porcine monocytes, were also assessed.

Isolation and stimulation of porcine leukocytes
Anticoagulant blood containing 2.5 mM EDTA was collected from Duroc, Landrance/Duroc hybrid, and Landrance breeds of Sus Scrofa. Leukocytes from each breed were isolated by Histopaque (Sigma, St. Louis, MO, USA) and cultured at 37 • C in RPMI 1640 supplemented with 10% fetal bovine serum (FBS). Cells were stimulated with 40 g/ml of concanavalin A (Con A, Sigma) for 18 and 46 h, respectively.

Cloning of PoIFN-γ cDNA variants and construction of rPoIFN-γ expression plasmids
Messenger RNA (mRNA) was extracted from Con Astimulated leukocytes by using a FastTrack 2.0 mRNA isolation kit (Invitrogen, Rockville, MD, USA). A total of 0.5 g of mRNAs were annealed with 0.5 g of oligo dT primer and reverse transcribed into cDNAs by AMV reverse transcriptase. PCR was performed in a 50 l reaction volume containing PCR buffer, dNTPs (0.2 mM each), cDNA template (10 ng), sense primer (20 pmole), antisense primer (20 pmole), and Taq DNA polymerase (1 U). After a denaturation step at 94 • C for 3 min, 35 PCR cycles were performed (denaturation at 94 • C for 1 min, primer annealing at 55 • C for 1.5 min, extension at 72 • C for 1.5 min), followed by a final extension at 72 • C for 10 min. Based on the sequence of PoIFN-␥ gene (Dijkmans et al., 1990), sense primer (5 -CAGAAGCTAACTCTCTCCG-3 ) and antisense primer (5 -GTGAAATAGTCACAGGATAC-3 ) were synthesized for the amplification of coding region. PCR-amplified PoIFN-␥ cDNA fragment was resolved by gel electrophoresis, cloned into pCR2.1 vector using a TA cloning kit (Invitrogen) and sequenced. DNA fragment encoding the mature PoIFN-␥ protein plus NcoI/XhoI restriction sites was further amplified by PCR, and subcloned into pET32a expression vector. The resultant pETpIFN-␥-series plasmids were transformed into Escherichia coli strain BL21(DE3).

Sequence alignment
Alignment of IFN-␥ cDNA sequences was performed by using DNAstar 5.0 software with clustal W program (DNAstar, Madison, WI, USA). Alignment of amino acid sequences of IFN-␥ among various animals was performed by using BioEdit software (Ibis Therapeutics, Carlsbad, CA, USA).

Expression and purification of rPoIFN-γ protein variants
E. coli strain carrying each pETpIFN-␥ variant was grown at 37 • C in Luria broth medium with ampicillin (50 g/ml). The recombinant protein expression was induced by 0.5 mM isopropyl ␤-d-thiogalactopyranoside (IPTG) when the OD 595 reached 0.8. Aliquot of the bacterial culture was harvested at different time points to determine the timing for optimal induction. Upon optimal induction, bacterial pellet was collected by centrifugation and resuspended in 1/25 volume (original culture) of binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). Cells were lysed with a sonicator and rPoIFN-␥ protein variants were purified by affinity chromatography on a nickelchelate column (Invitrogen).

SDS-PAGE and Western blot analysis
Bacterial pellets or purified recombinant proteins were analyzed by 13% SDS-PAGE. Following electrophoresis, proteins were transferred onto nitrocellulose (NC) membranes. Each membrane was incubated with blocking solution (5% non-fat dry milk and 1% normal serum in PBS) for 30 min before it was incubated with rabbit anti-PoIFN-␥ polyclonal antibody (1:2000 dilution in blocking solution) (R&D systems, Minneapolis, MN, USA) for 1 h at room temperature. After washing three times with phosphate-buffered saline containing 0.05% tween 20 (PBST), the membrane was further incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (1:3000 dilution). Finally, after PBST washings, NBT/BCIP was added for color detection.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis
MALDI-TOF MS analysis was performed as previously described (Lin et al., 2006). Briefly, gel plugs containing rPoIFN-␥ protein spots were excised from Coomassie blue stained gels to perform in-gel trypsin digestion. Using a Bruker ultraflex time-of-flight mass spectrometer equipped with a nitrogen laser and a 384 sample Scout source (Bruker Daltonics, Bremen, Germany), MALDI mass spectra were acquired by the reflectron mode. Peptide masses obtained were searched against a comprehensive nonredundant protein sequence database (NCBInr) using the Mascot program (Perkins et al., 1999) for protein identification.

Plaque inhibition assay for antiviral activity
PK-15 cells (porcine kidney cells) purchased from ATCC (Manassas, VA, USA) were seeded on 24-well plates (1 × 10 5 well −1 ) and grown in RPMI 1640 with 10% FBS. Upon adhesion, cells were treated either with each rPoIFN-␥ protein variant expressed in this study or commercial rPoIFN-␥ protein (R&D systems) for overnight. After three times of washes with PBS, cells were incubated with PRV (3.4 × 10 2 PFU/well) for 2 h at 37 • C. After removing unabsorbed virus, cells were covered with 1 ml of 1% methylcellulose in RPMI 1640. Three days later, cells were fixed by methanol for 10 min, stained with crystal violet, and plaque numbers were counted. Percentage of plaque formation inhibition was calculated as follows: [(mean number of plaques in the control) − (mean number of plaques in the treated sample)] × 100/(mean number of plaques in the control). IC50 was calculated as the concentration of rPoIFN-␥ that reached 50% of the inhibition.

Antiproliferation assay
PK-15 cells (1 × 10 5 well −1 ) were cultured in 24-well plates with RPMI 1640 plus 10% FBS. Cells were grown in medium (control) or treated with various concentrations of each rPoIFN-␥ protein variant for 72 h, washed with PBS and incubated at 37 • C for 3 h with p-nitrophenyl phosphate (pNpp) solution to measure acid phosphatase activity. The pNpp solution was prepared as described (Connolly et al., 1986). The reaction was stopped with 0.1 N NaOH (final concentration). The color development was determined at 405 nm using Quant spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA).

NO secretion assay
Adherent cells from porcine peripheral leukocytes (enriched monocytes) were cultured in 96-well plates (2 × 10 5 cells/well) and treated with various stimuli (in RPMI 1640/10% FBS) for 48 h. The supernatant of each stimulated culture was collected. By measuring nitrite and nitrate contents in the medium, NO production was determined with a nitric oxide colorimetric assay kit (BioVision, Mountain View, CA, USA).

Statistical analysis
All statistical analyses were performed using a GraphPad Prism version 4 software (GraphPad Software, San Diego, CA). Differences between groups were analyzed by two-way ANOVA.

Expression of rPoIFN-γ-W and rPoIFN-γ-M protein variants in E. coli
The open reading frame (ORF) of PoIFN-␥ gene encodes a polypeptide of 167 amino acid residues, containing a signal peptide (23 residues) coding sequence followed by the mature protein (144 residues) coding sequence (Dijkmans et al., 1990). The mature protein coding sequence of PoIFN-␥-W and PoIFN-␥-M cDNA was subcloned, respectively, into pET32 expression vector to generate bacterial expression constructs pETpIFN-␥-W and pETpIFN-␥-M. Upon induction with IPTG, E. coli strains carrying pETpIFN-␥-W and pETpIFN-␥-M unanimously overexpressed a recombinant protein around 33.7 kDa, which was the expected molecular mass of the mature PoIFN-␥ (15.8 kDa) fused with N-terminal tag (17.9 kDa). Five hours after IPTG induction, the recombinant protein reached a maximum amount and could be detected specifically by anti-PoIFN-␥ polyclonal antibody. Both recombinant protein variants were further purified by nickel-chelate affinity chromatography (Fig. 3).
Purified recombinant protein variants were treated with trypsin and subjected to MALDI-TOF MS analysis. Mass spectra of trypsin-digested rPoIFN-␥-W and rPoIFN-␥-M protein variants matched to that of PoIFN-␥ and thioredoxin fusion tag. Table 1 shows peptide mass and corresponding amino acid sequence of PoIFN-␥ following trypsin cleavage of both protein variants. The 1655.867 Da peptide mass corresponding to the amino acid sequence FFEIFKDNQAIQR (residues 56-68) was detected exclusively in the spectrum of rPoIFN-␥-W, which carries Q67 (Fig. 4A, Table 1). On the other hand, the peptide mass 1527.828 Da detected in rPoIFN-␥-M (with R67) spectrum matched to FFEIFKDNQAIR (residues 56-67) (Fig. 4B,  Table 1).

Antiviral activity of rPoIFN-γ protein variants
The antiviral activity of both rPoIFN-␥-W and rPoIFN-␥-M protein variants was determined by a PRV-based plaque inhibition assay on PK-15 cells. As shown in Fig. 5, both protein variants were able to inhibit the plaque formation of PRV in a dose-dependent manner (P < 0.001). However, antivi-    ral activity of rPoIFN-␥-M protein was significantly lower than that of rPoIFN-␥-W and commercial rPoIFN-␥ (P < 0.001).

NO induced in monocytes by rPoIFN-γ protein variants
IFN-␥ can stimulate macrophages and monocytes to secrete NO (Kim and Son, 1996;Malu et al., 2003). We thereby measured NO induction in monocytes by each rPoIFN-␥ protein variant. Untreated monocytes produced a low basal level of NO (Fig. 7). In contrast, NO secretion increased substantially in a  dose-dependent manner when monocytes were treated with various concentrations of rPoIFN-␥ (from 0.5 to 50 nM) (P < 0.001). However, levels of NO synthesis stimulated by rPoIFN-␥-W, rPoIFN-␥-M, and commercial rPoIFN-␥ were not significantly different.
Antiviral activity of PoIFN-␥ has been demonstrated in vitro on several swine viruses, including African swine fever (Esparza et al., 1988), transmissible gastroenteritis virus (Charley et al., 1988), influenza virus (Horisberger, 1992), porcine reproductive and respiratory syndrome virus (PRRSV) (Bautista and Molitor, 1999;Buddaert et al., 1998), and classical swine fever virus (CSFV) (Suradhat et al., 2001). All these viruses are RNA viruses that are highly sensitive to the antiviral mechanism of interferon. Instead of using RNA viruses, we evaluated the antiviral effect of PoIFN-␥ on pseudorabies virus, a DNA virus. PRV, a swine alphaherpesvirus, is distantly related to the human herpes simplex virus type 1 (HSV-1). Unlike HSV-1 that is well known for its anti-interferon mechanism, PRV lacks homologs of some anti-IFN genes (e.g. ␥34.5 and Us11) of HSV-1 and the viral growth could be inhibited in IFN-␤ treated PK-15 cells (Brukman and Enquist, 2006). The result of this study showed that PoIFN-␥ inhibited PRV in a dose-dependent manner, although the dosage of IFN-␥ used for PRV inhibition assay was higher than that for other RNA viruses. Therefore, we have established a PRV-based plaque inhibition assay to determine antiviral activity of PoIFN-␥. Except for carrying an extra fusion peptide encoded by pET32 vector at N-terminus, the amino acid sequence of rPoIFN-␥-W was identical to that of commercial rPoIFN-␥ protein (Vandenbroeck et al., 1991). The fact that antiviral activities of rPoIFN-␥-W and commercial rPoIFN-␥ proteins were not significantly different indicated that the fusion peptide did not interfere with antiviral activity of IFN-␥. Results from plaque inhibition assay showed that rPoIFN-␥-W presented a better antiviral activity than rPoIFN-␥-M protein (P < 0.001). The missense polymorphism, Q67R, indeed has an impact on the biological activity of PoIFN-␥-M protein, and this is the first report that identified a functional SNP in the coding region of IFN-␥.
In terms of antiproliferative effect (Friesel et al., 1987;Hansson et al., 1988;Maekawa et al., 1988), our data showed that both rPoIFN-␥-M and rPoIFN-␥-W expressed comparably lower antiproliferation activity than commercial rPoIFN-␥ (P < 0.001) on PK-15 cells. This could be resulted from the interference of fusion peptide carried in rPoIFN-␥-M and rPoIFN-␥-W proteins. In addition to growth inhibition, activation of macrophage/monocyte is another important immunoregulatory activity of IFN-␥. Measuring IFN-␥-induced NO secretion in macrophages is a common bioassay for evaluating the activity of murine IFN-␥ (Kim and Son, 1996;Malu et al., 2003). Instead of using macrophages, we measured PoIFN-␥-induced NO secretion in porcine monocytes because peripheral monocytes were more easily obtained from pigs. Our result showed that, in response to PoIFN-␥ stimulation, porcine peripheral monocytes produced NO in a dose-dependent manner. Nonetheless, induction levels of NO were not significantly different among monocytes treated with rPoIFN-␥-W, rPoIFN-␥-M, or commercial rPoIFN-␥. Taken altogether, Q67R and fusion peptide in the rPoIFN-␥ protein can, respectively, reduce antiviral activity and antiproliferation activity of IFN-␥, with little effect on NO induction. Our data indicate that these three activities reside in independent functional domains of PoIFN-␥.
In conclusion, we have identified five novel SNPs in PoIFN-␥ cDNA, including two missense polymorphisms that result in Q67R and K61R replacements. Interestingly, Q67R can markedly reduce antiviral activity of PoIFN-␥ protein. This in and of itself could warrant a potential association with a yet to be determined disease mechanism, of which the reduced immune response may lead to high susceptibility of virus infection.