Nonstructural proteins nsp2TF and nsp2N of porcine reproductive and respiratory syndrome virus (PRRSV) play important roles in suppressing host innate immune responses

Fig. 1. PRRSV nsp2 variants suppress type I IFN production. (A) A schematic diagram of individual PRRSV nsp2 variants (nsp2, nsp2TF and nsp2N) and domains [PLP2, nsp2(599–1233), nsp2TF(599–1402) and nsp2(599–1579)]. The numbers show both N- and C-terminal residues on polyprotein 1a for each individual protein. The catalytic residues of the PLP2 domain are labeled as Cys437 and His506. (B) Expression of nsp2-related proteins were evaluated by western blot analysis using anti-FLAG M2 mAb. GAPDH was detected as loading control. Rabbit pAb against the unique nsp2 C-terminus was used to confirm the expression of nsp2 and nsp2(599–1579), while rabbit pAb against unique nsp2TF C-terminus was used to confirm the expression of nsp2TF and nsp2TF(599–1402). (C) Effect of nsp2-related proteins on the expression of IFN-β promoter-driven luciferase expression. HEK-293T cells were cotransfected with a plasmid expressing individual nsp2-related proteins or domains, p125-Luc reporter plasmid expressing firefly luciferase under the control of the IFN-β promoter (0.5 μg), and pRL-SV40 reporter plasmid (20 ng). An empty vector (EV) was used as a control. At 24 h posttransfection, cells were stimulated with…

Fig. 2. The de-ubiquitination and de-ISGylation activities of PRRSV nsp2 related proteins. (A) Effect of nsp2 related protein expression on ubiquitin conjugation. HEK-293T cells were co-transfected with plasmid DNAs expressing HA-Ub and FLAG-tagged nsp2, nsp2TF, nsp2N, PLP2 or their catalytic site mutants (nsp2-C/H>A, nsp2TF-C/H>A, nsp2N-C/H>A, PLP2-C/H>A). HA-Ub conjugated cellular proteins were visualized by western blot analysis using anti-HA mAb. The expression of FLAG-tagged nsp2 related proteins was detected by anti-FLAG M2 mAb. (B) HEK-293T cells were co-transfected with plasmid DNAs expressing ISG15 and its conjugation enzymes E1/E2/E3, FLAG-tagged nsp2, nsp2TF, nsp2N, PLP2 or their catalytic site mutants (nsp2-C/H>A, nsp2TF-C/H>A, nsp2N-C/H>A, PLP2-C/H>A). ISG15-conjugated cellular proteins were detected by ISG15 specific mAb in western blot analysis. The expression of FLAG-tagged nsp2 related proteins was detected by anti-FLAG M2 mAb. For both assays, the empty p3xFLAG vector plasmid was included as a control, while expression of housekeeping gene GAPDH was detected as a loading control.

Fig. 3. Construction andin vitrocharacterization of nsp2TF/nsp2N-deficient mutants. (A) Nucleotide sequences in the regions of mutations created in nsp2TF/nsp2N-deficient mutants. KO1, knockout mutant 1 (premature termination codons in TF ORF); KO2, knockout mutant 2 (premature termination codon and disrupted frameshift signal). Mutated nucleotides are shown in red. (B) Radioimmunoprecipitation detection of nsp2-related proteins in virus infected cells. MAb 140-68 was used to recognize the common N-terminal PLP2 domain. The truncated nsp2TF in vKO1-infected cells is indicated with a red arrow. (C) Multiple-step virus growth curve. Each data point shown represents the mean value from duplicates, and error bars show SEM.

Fig. 4. nCounter mRNA Profiling of immune gene expression in virus-infected cells. (A) An overview on the number of differentially expressed genes (DEG) in virus-infected cells at 12 hpi. (B) Common and uniquely upregulated DEGs in virus-infected cells at 12 hpi. (C) Enriched functional categories of DEGs in virus-infected cells at 12 hpi determined by KEGG Pathway Enrichment analysis using DAVID (Huang et al., 2009a, Huang et al., 2009b). P-values adjusted by Benjamini-Hochberg correction less than 0.05 were defined as significant enrichment. The x-axis is the negative log10 of P-value.

Fig. 5. Quantitative RT-PCR detection of the expression of selected differentially expressed genes in virus-infected cells. (A) Six selected immune genes with increased expression levels in cells infected by nsp2TF/nsp2N-deficient mutants. Each data point shown represents the mean value from five replicates, and the gene expression levels in mock were treated as 1. (B) The relative expression levels of six immune genes in virus-infected MARC-145 cells at 12 hpi. (C) The relative expression levels of six immune genes in virus-infected PAM at 12 hpi. (B-C) Each data point shown represents the mean value from five replicates, and the gene expression levels in WT infected cells were treated as 1.

Fig. 6. Comparison of viral RNA loads in serum samples from pigs inoculated with the WT virus and nsp2TF/nsp2N-deficient mutants. Pigs were uninfected (mock) or infected with WT PRRSV (WT), vKO1 or vKO2 mutant. Serum samples were collected on the indicated days post-infection. Viral loads in serum samples quantified by quantitative RT-PCR and calculated as viral RNA copies per milliliter. Statistical significance between the WT virus-infected group and mutant virus-infected groups was determined by one-way ANOVA (Tukey's test) and indicated with asterisks (*, P<0.05; **, P<0.01; ***, P<0.001).

Fig. 7. Comparison of IFN-α production levels and NK cell cytotoxicity in pigs inoculated with WT virus and nsp2TF/nsp2N-deficient mutants. Pigs were uninfected (mock) or infected with WT PRRSV (WT), vKO1 or vKO2 mutant. (A) IFN-α levels in serum samples collected at 0, 1, 2 and 6 DPI were analyzed with a ProcartaPlex Porcine IFN alpha Simplex kit. (B) PBMCs (NK effectors) harvested on the day of necropsy (6 DPI) were co-cultured with target cells (K562) at an E:T ratio of 100:1 or 50:1. After overnight incubation, flow cytometry was performed to evaluate the NK cell-specific cytotoxic activity. Each data point represents the mean value and SEM of data from 3 pigs. Statistical significance between the wild-type virus infected group and mutant virus-infected groups was determined by one-way ANOVA (Tukey's test) and indicated with asterisks (*, P<0.05; **, P<0.01; ***, P<0.001).

Fig. 8. T-helper and cytotoxic T cell responses in pigs infected with WT virus and nsp2TF/nsp2N-deficient mutants. PBMCs isolated at 6, 21 and 28 DPI were unstimulated or restimulated with the WT virus. Cells were immunostained for pig specific markers CD3, CD4, CD8α and CD8β, and the frequency of each lymphocyte subset was grouped based on the combination of markers: (A) CD3+CD4+CD8α- (T-helper cells) and (B) CD3+CD4-CD8αβ+ (cytotoxic T cells) after analysis by flow cytometry. Statistical significance between the groups was determined by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. (*P<0.05; **P<0.01).

Fig S1. Expression of nsp2-related proteins in HEK-293T cell. HEK-293T cells seeded in a 24-well plate were transfected with a plasmid (0.5 μg) expressing each of nsp2-related protein. At 24 hpi, immunofluorescence assay was performed to evaluate protein expression. The α-FLAG mAb was used to detect the expression of FLAG-tagged proteins. Rabbit pAb (α-TF) specifically recognizing the unique C-terminal domain of nsp2TF was used to detect the expression of full-length nsp2TF (Li et al., 2014), while rabbit pAb (α-nsp2) specifically recognizing the nsp2C-terminus was used to detect the expression of full-length nsp2 (Guo et al., 2016). Cell nucleus was stained with DAPI.

Fig S2. Effect of nsp2-related proteins on HEK-293T cell viability. Cell viability was determined using a CellTiter 96 AQueous cell proliferation assay (Promega). Viability of transfected cells were compared to that of untreated control cells (100%). The average and standard deviation (SD) of a representative experiment are shown. All experiments were repeated twice, and triplicates were performed at each time.

Fig S3. Protein-protein interaction networks of DEGs in cells infected with WT virus and nsp2TF/nsp2N-deficient mutants. Protein-protein interaction networks were constructed for all DEGs in cells infected with WT virus (A-B), vKO1 (C-H), and vKO2 (I-N) using the search tool for the retrieval of interacting genes/proteins (STRING). Confidence view of protein-protein interaction network from DEGs showing the strength of data support is indicated by the thickness of the grey line connecting genes and nodes. The representative pathways enriched in cells infected with WT virus (A-B), vKO1 (C-H), and vKO2 (I-N) were highlighted with red color.