PK15 cells obtained from the China Center for Type Culture Collection were cultured in Modified Eagle's Medium (MEM; Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS; HyClone) and 1% non-essential amino acids (Gibco-BRL Life Technologies, Grand Island, NY, USA). The cells were cultured in an incubator at 37°C and 5% CO₂.

Reverse transcription of RNA to make cDNA for Rapid Amplification of cDNA Ends (RACE) assays was performed using a SMARTer® RACE 3′/5′ Kit (Takara, Dalian, China) according to the manufacturer's instructions. The reactions consisted of 1 μL RNA (1 μg/μL), 1 μL 3′-CDS Primer A (12 μM/L), and 10 μL sterile H₂O (3′ RACE) or 1 μL RNA (1 μg/μL), 1 μL 5′-CDS Primer A (12 μM/L), and 9 μL sterile H₂O (5′ RACE). The solution was mixed, centrifuged, and incubated at 72°C for 3 min. The tubes were cooled to 42°C for 2 min. For the 5′ RACE cDNA synthesis reaction, 1 μL SMARTer II A Oligonucleotide (24 μM/L) was added. Then, 4 μL 5 × First-Strand Buffer, 0.5 μL 100 μmol/L DTT, 20 U of RNase Inhibitor, and 200 U of SMARTScribe Reverse Transcriptase were added. The reaction was performed in a thermal cycler (T100; Bio-Rad, Hercules, CA, USA) for 90 min at 42°C, followed by heat inactivation for 10 min at 70°C. Samples were stored at −20°C for subsequent use.

The 3′ and 5′ RACE gene-specific primers (GSPs) of the first-step PCR amplification were designed based on the conserved regions of porcine PML sequences in NCBI (GenBank Accession No: XM_001925572.5) (Table 1). The reaction consisted of 10 μL 3′ or 5′ RACE-Ready cDNA, 50 μL 2 × Vazyme LAmp Master Mix (Vazyme, Nanjing, China), 2.5 μL 10 × Universal Primer (UPM) long (10 μM/L), 2.5 μL 3′ Race-GSP (10 μM/L) or 5′ Race-GSP (10 μM/L), and 35 μL ddH₂O. The PCR consisted of 5 min at 94°C followed by 35 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 2 min, with a final step of 72°C for 5 min.

If the primary PCR failed to yield the distinct bands of interest, nested PCR was used. The reaction consisted of 30 μL the 3′ RACE first-step PCR product, 50 μL 2 × Vazyme LAmp Master Mix, 2.5 μL 10 × UPM short (10 μM/μL), 2.5 μL 3′ Race-NGSP (10 μM/μL), and 35 μL ddH₂O.

The products of 5′ RACE first-step PCR or 3′ RACE nested PCR were ligated with pMD19-T vector, and the recombinant vector was transformed into DH5α competent cells. We selected positive clones for sequence analysis. Sequencing results were assembled using DNAStar 8.0 software (DNAStar Inc., Madison, WI, USA). The sequences were compared to the published PML gene sequence, and the splicing and retention of exons and introns to obtain full-length cDNA sequences of alternative splicing variants were analyzed.

DNAMAN 5.0 software (Lynnon Biosoft, Vandreuil, QC, Canada) was used for amino acid sequence alignment. The amino acid sequences of Homo sapiens (GenBank Accession No: NP_150241.2), Mus musculus (GenBank Accession No: NP_835188.2), Bos taurus (GenBank Accession No: XP_005221963.1), and Capra hircus (GenBank Accession No: XP_005695206.1) PML were downloaded from NCBI (https://www.ncbi.nlm.nih.gov). The open reading frames (ORFs) of the PML isoforms were analyzed using ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder). Analyses of the conserved domains were performed using CD-Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and SMART (http://smart.embl-heidelberg.de). The NLS was predicted using NucPred (https://nucpred.bioinfo.se/nucpred). The small ubiquitin-like modifier modification (sumoylation) sites were predicted using SUMOsp 2.0 software (http://sumosp.biocuckoo.org).

PML isoforms were amplified from cDNA of PK15 cells with the same forward primer and corresponding reverse primers (Table 2). The amplified fragments (PML-1/2, PML-3, PML-4/5, PML-6, and PML-7) were inserted into the pEGFP-C1 vector. PK15 cells were cultured in 12-well plates. The five PML recombinant eukaryotic vectors were transfected into the cells using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. At 36 h post-transfection, cells were harvested for RT-qPCR and fluorescence analysis.

PK15 cells were washed three times and fixed with 4% paraformaldehyde for 20 min at room temperature. Then the cells were permeabilized with 0.25% Triton X-100 for 15 min. After blocking with PBS containing 5% bovine serum albumin (BSA), the cells were stained with rabbit anti-PML polyclonal antibody (1:100; GeneCreate, Wuhan, China) at room temperature for 1 h. After washing three times with PBS, the cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibody (A-11008, 1:1,000; Invitrogen, Carlsbad, CA, USA). The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).

PK15 cells were cultured in 12-well cell culture plates, and infected with JEV strain SA14–14–2 (MOI = 1) diluted in MEM (21). The infected cells were washed twice with PBS after 1 h of adsorption, and then maintained in MEM with 2% FBS. The infected cells were collected for RNA isolation at the indicated time points after infection.

Total RNA was extracted using TRIzol reagent (Invitrogen). Total RNA (1 μg) was used to synthesize cDNA with an RT-PCR reagent kit (CWBiotech, Beijing, China). Quantitative analysis was performed using SYBR Premix Ex Taq II (Takara) on a CFX96 Touch instrument (Bio-Rad). The sequences of the qPCR primers used in the present study are listed in Table 3. The following PCR cycling conditions were used: denaturation at 95°C for 2 min, followed by 40 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 20 s, and then melting curve analysis. Relative expression levels were calculated using the 2^−ΔΔCt method and GAPDH, ACTB, 18srRNA, and RPL32 as reference genes (22).

Total proteins of JEV-infected and uninfected PK15 cells were extracted using RIPA lysis buffer with phosphatase and protease inhibitor (CWBiotech) respectively. Then the cell lysates were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk for 1 h, the membranes were incubated with rabbit anti-PML polyclonal antibody (1:1,000; GeneCreate) and rabbit anti-JEV (NS3) polyclonal antibody (GTX125868, 1:5,000; GeneTex, Irvine, CA, USA) overnight at 4°C. After washing three times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (A21020; 1:10,000; Abbkine, Wuhan, China) for 1 h at room temperature. Then the membranes were imaged using a Tanon 5200 system (Biotanon, Shanghai, China) after treatment with HRP Substrate (Merck Millipore, Darmstadt, Germany). β-Actin was used as an internal control.

All data are presented as the mean ± SEM of three independent experiments. Statistical significance was assessed using Student's t test. In all analyses, p < 0.05 was taken to indicate statistical significance.

cDNA for RACE assay was amplified from PK15 cells by RT-PCR. The products of the first cycle (using 3′ Race-GSP and 5′ Race-GSP) and the second cycle of nested PCR (using 3′ Race-NGSP) were analyzed by agarose gel electrophoresis (Figure 1). As shown in Figure 1, specific bands were obtained from 3′ RACE nested PCR and 5′ RACE first-step PCR, and the PCR products were cloned into the pMD19-T vector. After transformation into DH5α cells, positive clones were selected and sequenced. From the sequencing and alignment results, we identified seven types of PML cDNA clone, which we designated, from longest to shortest, as PML-1, PML-2, PML-3, PML-4, PML-5, PML-6, and PML-7 (Figure 2A).

Sequencing results were assembled using DNAStar 8.0 software (DNAStar Inc.), and the gene structures of the seven cloned different isoforms of the porcine PML gene are shown in Figure 2A. The full-length mRNA sequence of PML (PML-1) was 3,659 bp in length, and consisted of nine exons. The coding DNA sequence (CDS) of PML-1 was 2,625 bp and encoded 874 amino acids. The 3′ untranslated region (UTR) was 1,009 bp, and the 5′ UTR was much shorter at only 25 bp. PML-2 also encoded a product of 874 amino acids, but the 3′ UTR of PML-2 only retained 400 bp of the PML-1 3′ UTR. PML-3 contained exons 1–7 and retained part of the intron 7 sequence (7b: 641–1,788 bp), and encoded a product of 810 amino acids. PML-4 contained exons 1–7 and retained the partial sequence of intron 7 (7a: 1–1,785 bp), and encoded a product of 602 amino acids. PML-5 also contained exons 1–7 and retained part of the intron 7 sequence (7c: 1–434 bp, 7d: 1,245–1,788 bp). PML-6 contained exons 1–5 and retained a partial sequence of exon 6 (at 1–109 bp) and intron 7 (7e: 1,685–1,781 bp). PML-6 encoded a product of 522 amino acids. PML-7 contained exons 1–4 and retained a partial sequence of intron 4 (4a: 540–865 bp), and it encoded a product of only 420 amino acids. The PML-1 and PML-3 alternative splicing variants cloned here were the same as sPML-I and sPML-II cloned in a previous study (20).

All of the PML isoforms shared exons 1–4 and contained the same N-terminal domain but different C-terminal domains. The PML gene is a member of the TRIM gene family, and is also called TRIM19. Therefore, the first three exons of the PML isoforms contained the RING zinc finger, two B-box domains, and a coiled-coil motif (Figure 2B). Like other mammals, porcine PML-1 to PML-6 proteins included three sumoylation sites (at residues 65, 160, and 482), and PML-7 contained two sumoylation sites (at residues 65 and 160). Exon 6 encoded the NLS (Figure 2B). Therefore, six nuclear PML isoforms PML-1 to PML-6 were localized in the nucleus, while PML-7 was localized in the cytoplasm.

To understand the evolutionary characteristics of the PML gene, we selected the porcine PML-1 protein and the longest PML isoform of human (H. sapiens), cattle (B. taurus), goat (C. hircus), and mouse (M. musculus) for amino acid sequence alignment using DNAMAN 5.0 software (Figure 2C). The amino acid sequence of porcine PML-1 showed 76.21, 77.17, 77.05, and 61.78% identity those of human, cattle, goat, and mouse, respectively. The identity of the PML gene varied highly at the N- and C-terminals. The RBCC motif and three sumoylation sites were very highly conserved. These results indicate that the PML protein was moderately conserved in these species and the functional domains were highly conserved.

PML is a critical component of PML-NB formation, and the PML-NBs exhibit a dot-like structure in immunofluorescence images (23). To determine the roles of the seven PML isoforms in the formation and distribution of PML-NBs, PML isoforms were overexpressed in PK15 cells by transfection with the recombinant eukaryotic vectors pEGFP-C1-PML1/2, PML3, PML4/5, PML6, and PML7, respectively. The transfection efficiency was determined by the expression of enhanced green fluorescent protein (EGFP). The transfection efficiency reached approximately 30%, as demonstrated by EGFP expression (Figure 3A). The expression of PML isoforms was examined by RT-qPCR. Compared to that of cells transfected with pEGFP-C1 as an empty vector control, PML was significantly overexpressed in pEGFP-C1-PML isoform-transfected cells (Figure 3B). Further, the typical PML-NBs could be observed after overexpression of the five PML isoforms in PK15 cells (Figure 3C). PML-1/2, PML-3, PML-4/5, and PML-6 formed typical PML-NBs in the nucleus, and the EGFP-PML fusion proteins were also expressed in the cytoplasm. However, PML-7 formed PML-NBs that were only observed in the cytoplasm.

PML and PML-NBs play important roles in the innate immune response and antiviral defense (9). To investigate the porcine PML gene expression patterns after JEV infection, PK15 cells were infected with JEV, and the cells were harvested at the indicated time points. Viral replication was monitored by RT-qPCR, and the viral mRNA in PK15 cells peaked at 36 h post-infection (hpi) (Figure 4A). The expression of PML isoforms and PML-NB-associated genes (Daxx and SP100) were examined (Figure 4A). JEV infection significantly upregulated the mRNA expression of PML isoforms, Daxx and SP100 at 36 and 48 hpi. Among them, the PML-1/2 isoform expression was upregulated by 48-fold at 36 hpi. The PML protein levels were examined by Western blotting analysis using a polyclonal antibody against the N-terminal shared amino acids of the PML isoforms (Figure 4B). PML isoforms were upregulated at 36 and 48 hpi. To further determine the characteristics of the PML-NBs in JEV-infected PK15 cells, the cells were infected with JEV for 36 h and then we performed immunofluorescence analysis of the infected cells using anti-PML polyclonal antibody (Figure 4C). Compared to non-infected cells, the number of PML-NBs was significantly increased in JEV-infected cells, with approximately 20–30 PML-NBs in each cell. These results suggest that PML and PML-NBs may play important roles in JEV infection.