Materials

The Cell Counting Kit-8 (CCK-8) was ordered from Yeasen BioTechnologies co, Ltd. (Shanghai, China). Anti-GAPDH was purchased from Proteintech Group, Inc. (Chicago, USA). Anti RhoA was purchased from Novus Biologicals (Colorado, USA). Antiserum against PRV glycoprotein gB was generated by immunization of mice with purified recombinant gB. Goat anti-Mouse IgG, goat anti-Rabbit IgG, Alexa-Fluor-488-conjugated goat anti-mouse were purchased from Thermo Fisher Scientific (Shanghai, China).

Cells and viruses

Porcine kidney epithelial PK-15 (CCL-33, ATCC) were cultured in monolayers at 37℃ under 5% CO2 in DMEM medium (10566-016, GIBCO) supplemented with 10% FBS (10099141C, GIBCO), 100 U/mL penicillin and 100 µg/mL streptomycin sulfate (15070063, GIBCO). The virulent PRV isolate QXX (PRV-QXX) and the recombinant PRV strain of PRV-GFP, derived from PRV Hubei strain with TK gene replaced by GFP expression cassette from the pEGFP-N1 plasmid, was kindly donated by Professor Bei-bei Chu from the College of Veterinary Medicine, Henan Agricultural University.

Cell viability assay

PK-15 cells were seeded into 96-well plates with 1 × 104 cells/well. On the next day, the medium was changed to DMEM/10% FBS supplemented with various concentrations of certain chemical drugs for the indicated time. Cell viability was determined with CCK-8 according to the manufacturer's instructions. The absorbance at 450 nm was detected with a microplate reader (Awareness Technology Inc, FL, USA).

Western blotting

Whole-cell lysates were extracted with lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM MgCl2) supplemented with protease and phosphatase inhibitors (Roche, Mannheim, Germany). The protein concentrations in the lysates were quantified with a BCA Protein Assay Kit (DingGuo, Beijing, China), detected with a microplate reader (Awareness Technology Inc, FL, USA). Protein samples were separated by SDS-PAGE, transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA), and incubated in 5% non-fat milk (Sangon, Shanghai, China) for 1 h at room temperature. The membranes were incubated with primary antibody overnight at 4℃and then incubated with horseradish-peroxidase- conjugated secondary antibody (Thermo Fisher Scientific, Shanghai, China) for 1 h at room temperature. Immunoblotting results were visualized using Luminata Crescendo Western HRP Substrate (Millipore) on GE AI600 imaging system (Boston, MA, USA).

Quantitative real-time PCR (RT-qPCR)

Total RNA was isolated by using Trizol Reagent (Takara, Shiga, Japan) and subjected to cDNA synthesis with the PrimeScript™ RT Reagent Kit (Takara). RT-qPCR was performed in triplicate by using SYBR Premix Ex Taq (Takara, Shiga, Japan), and data were normalized by the level of GAPDH expression in each individual sample. Melting curve analysis indicated formation of a single product in all cases. The 2−ΔΔCt method was used to calculate relative expression changes. For quantification of PRV genome copy number, PCR product of 187 bp from the gene of PRV glycoprotein H (gH) was cloned into pGEM-T vector. Serial tenfold dilutions of this plasmid were used to construct a standard curve. The total number of PRV genomic equivalents was determined by comparison with the standard curve. Primers used for RT-qPCR are presented in Table 1.

Table 1 Primers used for gene cloning and RT-qPCR analysisRNA interference (RNAi)

Negative control and RhoA-specific siRNAs were designed with BLOCK-iT™ RNAi Designer (Life Technologies, Carlsbad, CA) and commercially synthesized (Genepharma, Shanghai) (Table 2). 20 pmol/well siRNAs were transfected into PK-15 cells using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Grand Island, NY), according to the manufacturer’s instructions. For a transfection in six-well plate, PK-15 cells were grown to 70–80% confluence before transfection. siRNAs and Lipofectamine RNAiMAX were diluted with DMEM (Gibco, Grand Island, NY) and incubated at room temperature for 5 min. Lipid-siRNA complexes were mixed and incubated for an additional 20 min and added drop-wise to cells. The knockdown efficiency of RhoA was determined by RT-qPCR and Western blot at 48 h post-transfection. Each assay was performed in triplicate.

Table 2 Sequence of siRNA used for gene knockdownPlasmid and transfection

The coding sequence of porcine RhoA gene was amplified from the cDNA of PK-15 cells with the specific primers. The sequences of the primers were: FLAG-Fw, 5′-CAAGCTTGCGGCCGCGAATTCATGGCTGCCATCAGGAAGAA-3′ and FLAG-Rv, 5′-CCTCTAGAGTCGACTGGTACCTCACAAGACAAGGCACCCAGA-3′. The PCR product was cloned into p3 × FLAG-CMV-10 (Sigma-Aldrich) to generate FLAG-RhoA. Transfection of plasmid was performed with Lipofectamine®3000 Transfection Reagent (Invitrogen, Grand Island, NY), according to the manufacturer’s instructions. Each assay was performed in triplicate.

Flow cytometry

For viral proliferation assays, cells were infected with recombinant virus expression the GFP reporter gene for 24–48 h and digested with trypsin–EDTA (25200072, GIBCO). Then, cells were collected by centrifugation at 1000 g for 5 min and suspended in phosphate-buffered saline (PBS). The percentage of GFP positive cells was measured by flow cytometry on CytoFLEX (Beckman, Atlanta, GA, USA). All data were analyzed by CytExpert software.

Immunofluorescence assay (IFA)

Cells grown on glass coverslips (Thermo Fisher Scientific) were fixed with 4% paraformaldehyde for 10 min. After washing with PBS, the cells were permeabilized with PBS containing 0.1% Triton X-100 for 10 min and then incubated in PBS/10% FBS for 60 min. And then incubated with PBS containing 10% FBS with the primary antibody for 1 h at room temperature. After washing with PBS, the cells were further incubated with fluorescent secondary antibodies (1:500, Invitrogen) in PBS/10% FBS for 1 h. After the cells were washed three times with PBS and then were labeled with phalloidin (1:200, Invitrogen) in PBS/1% BSA for 20 min. The cells were finally washed in PBS and mounted in ProLong Diamond with DAPI (#P36971, Invitrogen). Images were captured on a Zeiss LSM 800 microscope.

Plaque forming unit assay (PFU)

The virus suspension was serially diluted by tenfold serial dilutions for 4 times, and then inoculated on the monolayer PK-15 cells in 24-well plates (3 × 105 cells per well). The viruses were adsorbed at 37 ℃ for 1 h, and changed with 1% FBS DMEM for 4–7 days. The supernatant was discarded, and 4% PFA was added to fix the cells at room temperature for 30 min. Then, the cells were stained with 1% crystal violet for 30 min, and the plaques and non-shedding cells were observed after 30 min of water immersion. After drying, the number of plaques under the microscope was counted.

Statistical analysis

All data were obtained from at least three independent experiments for quantitative analyses and are expressed as means ± standard errors of the means. Western blot signal intensity was analyzed using Image J software. All data were analyzed using the Prism 8.0.2 software (GraphPad, CA, USA). All statistical analyses were performed with one-way analysis of variance (ANOVA). Significant differences relative to the corresponding controls were accepted at *P < 0.05, **P < 0.01 and ***P < 0.001.