Classic swine fever virus NS2 protein leads to the induction of cell cycle arrest at S-phase and endoplasmic reticulum stress

The pEGFP-C1 and pEGFP-N1 eukaryotic expression vector were purchased from Clontech (USA) and competent Escherichia coli DH5α used for cloning were purchased from Tiangen Biotech (China). Virulent CSFV (Shimen strain) was obtained from the Control Institute of Veterinary Bioproducts and Pharmaceuticals (China) and propagated in PK-15 cells. The established swine umbilical vein endothelial cell line, SUVEC, was cultured as previously described [13]. Briefly, SUVEC were cultured at 37°C and 5% CO₂ in M199 (Gibco, UK) medium containing 20% foetal calf serum (Hyclone, China), 50 μg/mL heparin (Sigma-Aldrich, USA), 20 μg/mL unrefined hypothalamus-pituitary supernatant (produced from sheep in this laboratory), and 100 μg/mL penicillin/streptomycin. The culture medium was replaced every 3 days. Porcine kidney cells (PK-15; ATCC CCL-33) were grown in Dulbecco's modified eagle medium (DMEM; Gibco, UK), supplemented with 10% foetal calf serum (Hyclone, China).

Mouse anti-GFP monoclonal antibody (mAb), horseradish peroxidase-conjugated goat anti-rabbit and horseradish peroxidase-conjugated goat anti-mouse antibodies were purchased from Millipore (USA). The anti-porcine cyclin A rabbit polyclonal antiserum was prepared by our laboratory. The mouse anti-porcine GAPDH antibody was obtained from LifeSpan Biosciences (USA). The MG132 proteasome inhibitor was purchased from Calbiochem (USA) and the nuclear staining dye Hoechst 33342 and ER-Tracker™ Red probe were obtained from Invitrogen (USA).

Primers NS2R (5'-CCCATAGTGTCACATACCAG-3'), F1 (5'-GAAGTCGACGGAAAGATAGATGGCGGTTGGCAGC-3') (the underlined sequences are the Sal I restriction enzyme recognition sites), R1 (5'-GAAGGATCCTCTAAGCACCCAGCCAAGGTGTTCCA-3') (the underlined sequences are the Bam H I restriction enzyme recognition sites), F2 (5'-GAAAAGCTTGGAAAGATAGATGGCGGTTGGCAGC-3') (the underlined sequences are the Hind III restriction enzyme recognition sites), R2 (5'-GAACCGCGGTCTAAGCACCCAGCCAAGGTGTTCCA-3') (the underlined sequences are the Sac II restriction enzyme recognition sites), were designed to amplify the CSFV NS2 gene according to the archived CSFV Shimen strain nucleotide sequence (GenBank: AF092448). Primer NS2R was used for the first cDNA strand synthesis and the primers F1 and R1 were used for the PCR amplification and were designed with 5' terminal restriction enzyme recognition sites for aid cloning into pEGFP-C1, F2 and R2 were also used for the PCR amplification and were designed with 5' terminal restriction enzyme recognition sites for aid cloning into pEGFP-N1. Primers were synthesized by Shanghai Invitrogen (China). In order to synthesize CSFV NS2 cDNA, total RNA was extracted from PK-15 cells infected with CSFV Shimen strain using Trizol Reagent (Invitrogen, USA) and reverse transcribed using a first-strand cDNA synthesis kit (Takara Bio., Dalian China). The PCR was performed in a total volume of 25 μL using a thermocycling protocol of 94°C for 4 min, 94°C for 30 s, 60°C for 30 s and 72°C for 1.5 min for 35 cycles, followed by 72°C for 10 min. The RT-PCR product was detected by 1.0% agarose gel electrophoresis, purified from the gel and digested with restriction enzymes to be cloned into the pEGFP-C1 and pEGFP-N1 expression vector. The resulting recombinant plasmid, named pEGFP-NS2 and pNS2-EGFP, was recovered from transformed E. coli using a plasmid mini-kit (Axygen, China) and identified by restriction enzyme digestion and sequence analysis.

SUVEC cells were seeded into 12-well dishes 24 h before being transfected (up to 60-70% confluence). Cells were transfected with pEGFP-NS2, pNS2-EGFP and pEGFP-C1 control vector by Lipofectamine 2000 (Invitrogen, USA) and passaged (up to 80% confluence) in selection media containing 1500 μg/mL G418 for two weeks. When all control cells had evidence of death in the presence of the selection agent, cultures transfected with pEGFP-NS2, pNS2-EGFP and pEGFP-C1 were propagated for two further weeks in medium containing 400 μg/mL G418. The resulting stably transfected cell lines expressing either GFP, GFP-NS2 or NS2-GFP fusion proteins were used for subsequent analyses.

To examine the expression and subcellular localization of CSFV NS2 protein, the stable cell lines expressing GFP-NS2, NS2-GFP protein or control cells (GFP and untransfected cells) were grown on glass coverslips in 6-well tissue culture plates. After pretreatment with 2.5 μM MG132 proteasome inhibitor (Calbiochem, USA) for 16 h, cells were washed with Hank's balanced salt solution (HBSS) and incubated with Hoechst33342 at 37°C for 15 min, and then washed twice with HBSS, after that, all the cells were incubated with ER-Tracker™ Red probe (Invitrogen, USA) at 37°C for 30 min. Cells were subsequently washed with DMEM without serum and images were viewed by laser confocal scanning microscopy (Model LSM510 META, Zeiss, Germany) and the images were processed by Adobe Photoshop software.

Whole cell extracts were prepared by washing cells with PBS, harvested by scraping and then suspended in 1 mL PBS. Following centrifugation, the cells were resuspended in cell lysis buffer (50 mM Tris-HCl, 5 mM EDTA, 150 mM NaCl, 0.1% NP-40, 0.5% deoxycholic acid, 1 mM sodium orthovanadate, 100 μg/mL PMSF and protease inhibitors) and centrifuged at 15 000 × g for 30 min at 4°C. Cell extracts were resolved by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore, USA). The membrane was blocked overnight with 5% skim milk in TNT buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.05% Tween 20) and then incubated with mouse anti-GFP-tag mAb, or the anti-swine cyclin A rabbit polyclonal antiserum for 2 h. Porcine GAPDH proteins were detected using mouse anti-porcine GAPDH antibody. Detection of primary antibodies was performed with either horseradish peroxidase-conjugated goat anti-rabbit antibody or horseradish peroxidase-conjugated goat anti-mouse antibody, as appropriate. The protein bands were visualized by enhanced chemiluminescence methods as per the manufacturer's instructions (Millipore, USA).

The MTS cell proliferation assay was performed to determine the growth properties of CSFV NS2-expressing and control cells according to the manufacturer's instructions. Briefly, cells were seeded in 96-well culture plates at a concentration of 4 × 10³ cells/well in 100 μL culture medium. After incubation at 37°C for 24, 48, 72 and 96 h, the culture medium was carefully replaced with 100 μL of a fresh medium without disturbing the cells. Twenty microlitres of MTS (Promega, USA) reagent was added to each well and incubated in a CO₂ incubator at 37°C for 4 h. The absorbance at a wavelength of 492 nm was read on a microplate reader (Model 680, Bio-Rad, USA) at appropriate time intervals.

Since DNA fragmentation suggests apoptotic DNA damage and indicated by a distinct sub-G0/G1 peak in flow cytometry assay [14], the cell cycle and apoptosis was measured as previously described [15]. In brief, approximately 2 × 10⁶ cells of the stable cell lines and control cells were trypsinised and collected. Following two washes with PBS, cells were resuspended in 70% ethanol and fixed at 4°C for 18 h. Cells were washed and resuspended in PBS containing 20 μg/mL of RNase A and 50 μg/mL of propidium iodide (PI) and incubated on ice for 30 min. Finally, the nuclear DNA content was determined using a Coulter Epics XL flow cytometer (Beckman Coulter, USA).

Since the sequence of porcine cyclin A mRNA has not been previously published, it was necessary to determine this sequence for the development of the quantitative real-time RT-PCR assay for porcine cyclin A mRNA. Two primers were designed to amplify the human cyclin A nucleotide sequences (GenBank: X51688) and used to amplify the porcine cyclin A gene by RT-PCR given that significant homology is likely to exist between the two species in this gene. The sequences of these primers were as follows: R: 5'-GATTTACATCTTAGAAAACAAAGG-3'; PC1: 5'-GGTGATCCCGCCGTCCACT-3' and PC2: 5'-GATTTACATCTTAGAAAACAAAGGCAGTC-3'. Total RNA was extracted from PK-15 cells and reverse transcribed using a first-strand cDNA synthesis kit (Takara, Japan). The PCR reaction was performed in a total volume of 25 μL using a thermocycling protocol of 94°C for 4 min, 94°C for 30 s, 62°C for 30 s and 72°C for 1.5 min for 35 cycles, followed by 72°C for 10 min. The PCR product was purified and cloned into the pMD-19T vector (Takara, Dalian China) and positive recombinant plasmids were identified by restriction enzyme digestion and sequenced. The sequences of porcine cyclin A were submitted to GenBank, and are available under the accession number, GQ265874. To quantify cyclin A mRNA, total RNA was extracted from cells using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions. Synthesis of cDNA was performed with 2 μg of total RNA in a 25 μL reaction containing 20 U/μL M-MuLV reverse transcriptase (Takara Bio., China), 0.5 μM dNTP and 0.25 μM Oligo(dT)18 primer at 42°C for 3 h. Real-time PCR of cDNA was carried out using the SYBR real-time PCR kit (Takara Bio., Dalian China) on a real-time PCR system (model 7500; ABI, USA) with the following cycling profile: 5 min at 95°C followed by 40 cycles of 10 s at 95°C and 20 s at 60°C. The individual samples were normalized for genome equivalents using the respective CT value for the porcine β-actin housekeeping gene. One pair of primers was designed for the quantification of porcine cyclin A mRNA levels by real-time PCR according to the obtained porcine cyclin A sequences. The sequences for the two primers were PC1: 5'-AAGTTTGATAGATGCTGACCCGTAC-3' and PC2: 5'-GCTGTGGTGCTCTGAGGTAGGT-3'. Additionally, primers for detecting the porcine β-actin housekeeping gene (GenBank: DQ845171 and SSU07786), ActinF (5'-CAAGGACCTCTACGCCAACAC-3') and ActinR (5'-TGGAGGCGCGATGATCTT-3') were synthesized.

To analyze the expression of glucose regulated protein, 78 kDa (GRP78), a well characterized ER chaperone protein that is a marker of ER stress, we developed a real-time PCR assay for its detection [16‐21]. A pair of primers, GRP78F (5'-AATGGCCGTGTGGAGATCA-3') and GRP78R (5'-GAGCTGGTTCTTGGCTGCAT-3'), were designed according to the available porcine GRP78 sequences (GenBank: X92446) to detect the porcine GRP78 mRNA level in cells.

To determine the alteration of NF-κB activity by GFP, GFP-NS2 and NS2-GFP proteins in the established cell lines, the level of p50 activity was measured using the NF-κB TransAM kit (Active Motif) according to the manufacturer's instructions. Briefly, cells nuclear extraction was prepared by using the Nuclear Extract Kit (Active Motif) and protein concentrations were measured using the Bradford assay (Bio-Rad). Lysates (20 mg total proteins) were incubated in ELISA wells coated with the oligo-nucleotide motif recognized by active p50, p50 was then detected using a specific antibody, followed by a secondary antibody conjugated to peroxidase. The colorimetric reaction was measured at A450, with 620 nm acting as the reference wavelength. This experiment was repeated three times.

SPSS^®15.0J (SPSS Inc, USA) was used to perform the statistical analyses including one-way analysis of variance (ANOVA) followed by Dunnett's test.

The expression and subcellular localization of CSFV NS2 protein in SUVEC stable cell lines were analyzed. Western blot analysis showed that the GFP-NS2 and NS2-GFP fusion protein had a molecular weight of approximately 80 kDa and was present in all cells transfected with the pEGFP-NS2 plasmid and pNS2-EGFP plasmid, and no signal can be detected from the negative SUVEC control cells (Fig. 1A). Since the molecular weight of GFP is known to be approximately 27 kDa, the molecular weight of the putative CSFV NS2 protein is approximately 53 kDa. The subcellular localization of NS2 was investigated by confocal fluorescence microscopy and showed that GFPNS2 protein and NS2GFP protein were both distributed in the endoplasmic reticulum, by contrast, the GFP protein was distributed in the whole cells (Figs. 1B). These results revealed that CSFV NS2 protein localized to the ER, the report prtein GFP which either fused to the -NH2 terminal or the -COOH terminal of CSFV NS2 protein did not affect the trans-localization of CSFV NS2 protein.

Compared with control cells (pEGFP-C1 transfected and untransfected), the CSFV NS2 expressing cells divided much more slowly leading to a significantly decreased cell number after a period of time. Cell proliferation of NS2 expressing cells measured by the MTS assay was decreased by approximately 20 - 30% over a time course of 72 to 96 h (Fig. 2).

To determine whether the growth inhibition of CSFV NS2-expressing cells was due to the arrest of the cell cycle at a certain phase(s) of cell division, flow cytometric analysis was performed based on DNA content in nuclei stained with PI. The proportions of G0/G1 phase, S-phase and G2/M phases for the control cells were 57.96%, 35.98% and 6.06%, respectively. For SUVEC expressing GFP, the proportions of the phases were G0/G1: 60.84%, S-phase: 34.19%, and G2/M: 4.96%, whereas for GFP-NS2-expressing SUVEC stable cells, the proportions were G0/G1: 52.96%, S-phase: 42.29% and G2/M: 4.76%, consistently, for the NS2-GFP-expressing SUVEC stable cells, the proportions were G0/G1: 54.40%, S-phase: 41.40% and G2/M: 4.19% (Fig. 3A). Apoptosis was also analyzed by flow cytometry but no differences were observed between untransfected cells or pEGFP-C1 transfected cells and cells expressing GFP-NS2 or NS2-GFP fusion protein. A sub-G0/G1 peak was not detected by flow cytometry for both the GFP-NS2-expressing, NS2-GFP-expressing and control cells and suggests that the CSFV NS2 protein induced cell cycle arrest in the S-phase, rather than inducing apoptosis. The results showed that relative to control cells, NS2 expression causes a significant increase in the proportion of cells in the S-phase accompanied by a decrease in the cell proportion in the G0/G1 phase (Fig. 3B). Taken together, these results strongly suggest that NS2 protein causes the inhibition of cell growth by the induction of cell cycle arrest in the S-phase.

Since cyclin A is required for the entry into G2/M phase, the cyclin A protein levels in NS2 expressing cell lines and control cells were analyzed by western blot assay. Cyclin A expression level was significantly decreased in both cell lines that expressed NS2 protein compared with control cells. Furthermore, when the GFP-NS2-expressing and NS2-GFP-expressing cell lines were pre-treated with MG132, blocking proteasomal degradation of proteins, the relative intensity of cyclin A western blot signal was significantly increased (Fig. 4), suggesting that cyclin A is rapidly degraded in the presence of NS2. To further support these findings, quantitative real-time RT-PCR was employed and showed that cyclin A mRNA levels in the GFP-NS2-expressing and NS2-GFP-expressing cell lines were significant higher than in control cells (Fig. 5A). This suggests that the induction of cell cycle arrest in the S-phase by CSFV NS2 protein is associated with the increase of cyclin A proteasomal degradation rather than a decrease of cyclin A transcription. Moreover, transcription of GRP78, the ER molecular chaperone was also increased in GFP-NS2-expressing cell lines compared with controls (Fig. 5B). Analysis of the activity of NF-κB in GFP-NS2-expressing and NS2-GFP-expressing cell lines using a TransAMTM NF-κB p50 Transcription Factor Assay Kit demonstrated that the NF-κB was significantly activated compared with control cells (Fig. 6). Together, these analyses have shown that expression of CSFV NS2 results in the up-regulation of GRP78, cyclin A and NF-κB suggesting a role of NS2 in ER stress activation. Additionally, NS2 induces cell cycle arrest at the S-phase and is associated with the increased proteasomal degradation of cyclin A.