Porcine Respirovirus 1 Suppresses Host Type I Interferon Production and the JAK-STAT Signaling Pathway

Figure 1. The expression IFNβ and ISGs in LLC-MK2 cells infected with PRV1 (KS 17-258 strain). (A,B) PRV1 infection was unable to stimulate IFN-β production. LLC-MK2 cells were infected with KS 17-258 at an MOI of 1. LLC-MK2 cells infected with 100 HA units of SeV and with mock infection were used as the positive and negative control. At 24 hpi, cells were harvested with TRIzol reagent or IP lysis buffer. IFNβ expression at the mRNA level was evaluated by real-time RT-PCR with total cellular RNA. The relative mRNA expression level of IFN-β was normalized to the mRNA level of GAPDH, and the relative IFN-β mRNA level of negative control was set as 1 (A). The expression of two ISGs (RIG-I and ISG15) was assessed by Western blot analysis with monoclonal antibodies. F protein was detected to monitor KS 17-258 infection, and housekeeping gene GAPDH was detected as the loading control (B). (C,D) PRV1 infection suppressed IFNβ production. LLC-MK2 cells were infected with KS 17-258 at an MOI of 1 or mock-infected. At 24 hpi, cells were infected with 100 HA units of SeV to induce type I IFN production. Naïve LLC-MK2 cells were included as the negative control. After 12 h, cells were harvested with…

Figure 2. Multiple PRV1 proteins suppress IFN-β production. HEK293T cells cultured in 24-well plates were co-transfected with a plasmid expressing PRV1 proteins, or the empty vector (EV), pRL-SV40, and a luciferase reporter plasmid p125-Luc (A), p55-CIB-luc (B) or pNF-κB-luc (C). At 24 h post-transfection, cells were infected with 100 HA units/mL SeV for 16 h to stimulate the production of interferon. Firefly and Renilla luciferase activities were measured with cell lysate. Relative luciferase activity is defined as the ratio of firefly luciferase reporter activity to Renilla luciferase activity. The relative luciferase activity of EV with stimulation was set as 100%. Each data point shown represents a mean value from two biological replicates, and each experiment was repeated at least three times. Error bars show standard deviations of the normalized data. (D) The expression of PRV1 proteins in HEK-293T cells was evaluated by Western blot analysis with anti-FLAG mAb M2. The SV40 larger T antigen (TAg) was detected as the loading control. ***, p-value < 0.001; ****, p-value < 0.0001.

Figure 3. PRV1 V protein inhibits MDA5-dependent and RIG-I-dependent IFNβ production. (A,B) HEK-293T cells in 24-well plates were co-transfected with the plasmid pEFneo-MDA5 (A) or pEFneo-RIG-I (B), along with pRL-SV40, an increasing amount (0.25, 0.5, and 1.0 μg) of pCAGGS expressing V protein or the empty vector (EV), and p125-luc plasmid for 24 h. (C) HEK-293T cells in 24-well plates were co-transfected with the plasmid pEGFP-C3-MAVS, along with pRL-SV40, 1μg of pCAGGS expressing V protein or the empty vector (EV), and p125-luc plasmid for 24 h. Firefly and Renilla luciferase activities were measured with cell lysate. Relative luciferase activity is the ratio of firefly luciferase reporter activity to Renilla luciferase activity. The relative luciferase activity of EV with stimulation was set as 100%. Each data point shown represents a mean value from two biological replicates, and each experiment was repeated at least three times. Error bars show standard deviations of the normalized data. (D) The interaction between V protein and MDA5. HEK-293T cells were co-transfected with a plasmid expressing V protein or the empty vector (EV), and a plasmid expressing HA-MDA5. At 24 hpt, cell…

Figure 4. PRV1 V protein inhibits RIG-I ubiquitination. (A) The interaction between V protein and TRIM25. HEK-293T cells were co-transfected with a plasmid expressing V protein or the empty vector (EV), and a plasmid expressing c-MYC-TRIM25. At 24 hpt, cell lysates were harvested for immunoprecipitation (IP) with anti-FLAG mAb M2. Western blot analysis with IP eluents and whole-cell lysate (WCL) was performed to detect V protein and RIG-I with anti-FLAG and anti-c-MYC mAbs, respectively. (B) HEK-293T cells were transfected with plasmids expressing HA-Ub, GST, or GST-RIG-IN as indicated. Cells were also co-transfected with the empty vector or a plasmid expressing PRV1 V. A GST pulldown assay was performed. Western blot analysis was performed with anti-HA mAb to show covalent ubiquitin-modified species of GST–RIG-IN as indicated. Western blot analysis with whole-cell lysate (WCL) was performed to verify protein expression. The housekeeping gene GAPDH was detected as the loading control (A,B).

Figure 5. PRV1 P gene products inhibit the type I IFN signaling pathway by interaction with STAT1. (A) PRV1 proteins antagonize the type I IFN signaling pathway. HEK293T cells cultured in 24-well plates were co-transfected with a plasmid expressing PRV1 proteins, or the empty vector (EV), pRL-SV40, and a luciferase reporter plasmid pISRE-Luc. At 24 h post-transfection, cells were treated with 100 units/mL of human IFNα for 16 h to stimulate the production of ISGs. Firefly and Renilla luciferase activities were measured with cell lysate. Relative luciferase activity is the ratio of firefly luciferase reporter activity to Renilla luciferase activity. The relative luciferase activity of EV with stimulation was set as 100%. Each data point shown represents a mean value from two biological replicates, and each experiment was repeated at least three times. Error bars show standard deviations of the normalized data. The expression of PRV1 proteins in HEK-293T cells was evaluated by Western blot analysis with anti-FLAG mAb M2. The SV40 larger T antigen (TAg) was detected as the loading control (B). (C) The nucleus translocation of STAT1. HEK-293T cells were co-transfected with a plasmid expressing…