Foot-and-mouth disease virus infection inhibits LGP2 protein expression to exaggerate inflammatory response and promote viral replication

Figure 1. FMDV infection triggers LGP2 mRNA and decreases LGP2 protein expression levels. (a, b) PK-15 cells were mock-infected or infected with FMDV (0.5 MOI) for 0, 2, 4, 8, 12 or 24 h, and LGP2 mRNA and viral RNA were detected by qPCR. (c) PK-15 cells were infected with FMDV (0.5 MOI) for 0, 2, 4, 8, 12 or 24 h, and endogenous LGP2 and viral VP1 proteins were detected by western blotting. (d) PK-15 cells were transfected with Myc-vector (2 μg) or Myc-LGP2 plasmids (2 μg), and Myc-LGP2-transfected cells were mock-infected or infected with 0.05 or 0.5 MOI of FMDV at 12 hpt. Expression of Myc-LGP2 and viral VP1 proteins was detected by western blotting at 12 hpi. The change in abundance of Myc-LGP2 in the transfectants was analysed by densitometric analysis using ImageJ Software and normalized to β-actin (right panel).

Figure 2. Overexpression of LGP2 suppresses FMDV replication. (a) PK-15 cells were transfected with Myc-vector (2 μg) or Myc-LGP2 plasmids (2 μg), and the transfected cells were infected with FMDV (0.5 MOI) at 24 hpt. Expression of Myc-LGP2 and viral VP1 proteins was detected by western blotting, and viral RNA was detected by qPCR, at 12 hpi. (b) PK-15 cells were transfected with increasing amounts of Myc-LGP2 plasmids (0, 1, 2 or 3 μg) and the Myc-vector was used in the transfection process to ensure that the cells received the same amounts of total DNA plasmids. The transfected cells were infected with FMDV (0.5 MOI) at 24 hpt. Expression of Myc-LGP2 and viral VP1 proteins was detected by western blotting, and viral RNA was detected by qPCR, at 12 hpi; (c) and the viral titres were detected by TCID50 assay. All the experiments were repeated three times with similar results.

Figure 3. LGP2 suppresses several cytokines expression during FMDV infection. PK-15 cells were transfected with Myc-vector or Myc-LGP2 plasmids, and transfected cells were infected with FMDV at 24 hpt. Cytokine expression levels in the cell lysates was detected at 18 hpi using porcine cytokine array kits (RayBiotech). (a) Downregulation of cytokines was greater in LGP2 overexpressing cells than vector-transfected cells after FMDV infection. (b) Upregulation of cytokines was greater in LGP2-overexpressing than vector-transfected cells after FMDV infection. (c) qPCR analysis of mRNA abundance of six determined cytokines.

Figure 4. Loss of LGP2 promotes inflammatory response and FMDV replication. (a) PK-15 cells were transfected with NC siRNA or LGP2 siRNA, and expression of endogenous LGP2 protein was detected at 0, 36 and 48 hpt. (b) PK-15 cells were transfected with NC siRNA or LGP2 siRNA for 36 h, followed by infection with equal amounts of FMDV (0.5 MOI) for 0, 10 and 18 h. Expression of endogenous LGP2 and viral proteins was detected by western blotting. Expression of LGP2 mRNA and viral RNA was detected by qPCR. (c) Expression of CCL3L1 and TNF-α mRNA in the NC siRNA- and LGP2 siRNA-transfected cells infected by FMDV for 18 h was detected by qPCR. (d) Alignment of the LGP2 genomic reference sequence, the LGP2-WT, LGP2-KO-1 and LGP2-KO-2 DNA sequences using LaserGene software. The red box indicates the regions that were mutated. (e) Confirmation of successful knockout of LGP2 in the LGP2-KO cell line by western blotting. (f) Equal amounts of LGP2-WT and LGP2-KO cells were infected by FMDV (0.5 MOI) for 10 h, and viral protein (left panel) and RNA (right panel) were detected. (g) Expression of CCL3L1 and TNF-α mRNA were determined.

Figure 5. FMDV 2B protein induces reduction of LGP2. (a) PK-15 cells were transfected with Myc-LGP2 plasmids for 24 h followed by mock infection or infection with FMDV (0.5 MOI) for 12 h, and Myc-LGP2 mRNA levels were detected by qPCR. (b) HEK293T cells were transfected with Myc-LGP2 plasmids (2 μg) along with various plasmids expressing Flag-tagged viral proteins (Lpro, VP0, VP1, VP2, VP3, 2B, 2C, 3A, 3B, 3Cpro or 3Dpol) or empty Flag vector plasmid (2 μg). Expression of Myc-LGP2 and Flag-tagged viral proteins was detected by western blotting at 36 hpt. (c) HEK293T cells were co-transfected with Myc-LGP2 plasmids (2 μg) and increasing amounts of Flag-L, Flag-3C or Flag-2B plasmids (0, 0.5, 1 or 2 μg) for 36 h. Empty vector was used in the transfection process to ensure that the cells received the same amounts of total plasmids. Expression of Myc-LGP2 and Flag-tagged viral proteins was detected by western blotting. (d) HEK293T cells were co-transfected with Myc-LGP2 plasmids (2 μg) and increasing amounts of Flag-2B plasmids (0, 0.5, 1 or 2 μg) for 36 h. Empty vector was used in the transfection process to ensure that the cells received the same amounts of total plasmids. Expression of…

Figure 6. Functional domain of LGP2 to suppress FMDV replication. (a) Schematic representation of a series of Myc-tagged LGP2 mutant constructs. (b) PK-15 cells were transfected with the empty vector (2 μg), Myc-LGP2 (2 μg) or various LGP2 mutant plasmids (2 μg) for 24 h, and followed by infection with FMDV (0.5 MOI) for 12 h. Viral RNA levels were detected by qPCR. (c) HEK293T cells were co-transfected with Myc-LGP2 plasmids (2 μg) or various LGP2 mutant plasmids (2 μg) and Flag-2B plasmid for 36 h. Expression of Myc-LGP2, Myc-tagged LGP2 mutants and Flag-2B proteins was detected by western blotting.

Figure 7. Functional region of 2B responsible for inducing reduction of LGP2. (a) Schematics of a series of Flag-tagged 2B mutant constructs. (b) HEK293T cells were co-transfected with Myc-LGP2 plasmid (2 μg) and Flag-2B or Flag-tagged 2B mutant plasmids (2 μg) for 36 h. Expression of Myc-LGP2, Flag-2B and Flag-tagged 2B mutant proteins were detected by western blotting. (c) PK-15 cells were transfected with the plasmids expressing Flag-2B or truncated mutants, and the cells were infected with equal amounts of FMDV at 12 hpt. Expression of CCL3L1 and TNF-α was detected by qPCR at 18 hpi.

Figure 8. Exploration of the mechanism of 2B-induced reduction of LGP2. (a) HEK-293T cells were co-transfected with Myc-LGP2 and empty vector, Flag-2B or Flag-VP2 plasmids for 36 h. Cell lysates were immunoprecipitated with mouse anti-Myc or mouse normal IgG antibody and subjected to western blotting (upper panel). Whole-cell lysates were also directly detected by western blotting to confirm expression of target proteins (lower panel). (b) HEK-293T cells were co-transfected with Myc-LGP2 and empty vector or Flag-2B plasmids for 36 h. The cell lysates were immunoprecipitated with rabbit anti-Flag or rabbit normal IgG antibody and subjected to western blotting (upper panel). Whole-cell lysates were also directly detected by western blotting to confirm expression of target proteins (lower panel). (c) HEK-293T cells were co-transfected with Myc-LGP2 and Flag-2B mutant plasmids for 36 h. Cell lysates were immunoprecipitated with mouse anti-Myc or mouse normal IgG antibody and subjected to western blotting (left panel). Whole-cell lysates were also directly detected by western blotting to confirm the expression of the target proteins (right panel). (d–f) HEK293T cells were co-transfected with…