ZNF283, a Krüppel-associated box zinc finger protein, inhibits RNA synthesis of porcine reproductive and respiratory syndrome virus by interacting with Nsp9 and Nsp10

Figure 1. The expression of ZNF283 is upregulated by PRRSV infection. Marc-145 cells and PAMs were mock-infected or infected with PRRSV at an MOI of 0.2 for various durations. The cells were then collected and analysed for ZNF283 mRNA and protein expression levels using RT‒qPCR (A and C) and Western blotting (B and D), respectively. E Marc-145 cells were transfected with HA-tagged ZNF283 for 24 h and then infected with PRRSV at an MOI of 0.2. Mock-infected cells served as controls. At 24 h post-infection, the cells were fixed and immunostained with mouse anti-N protein and rabbit anti-HA antibodies. Nuclei were counterstained with DAPI. F Marc-145 cells were transfected with HA-ZNF283 for 24 h and then infected with PRRSV at an MOI of 0.2. At 36 h post-infection, cellular fractionation followed by Western blotting was performed. HDAC1 and GAPDH were used as control proteins for the nuclear and cytosolic fractions, respectively.

Figure 2. Overexpression of ZNF283 inhibits PRRSV replication. Marc-145 cells were transfected with HA-tagged ZNF283 or HA-tagged empty vectors for 24 h and then infected with PRRSV at an MOI of 0.2. At 12, 24, 36, and 48 h post-infection, viral titres in the cell supernatant (A) and the expression levels of the N protein (B and C) were assessed using TCID50 and Western blotting, respectively.

Figure 3. ZNF283 knockdown facilitates the proliferation of PRRSV. Marc-145 cells were transfected with three independent shRNAs targeting ZNF283 for 24 h, and RT‒qPCR (A) and Western blotting (B) were used to evaluate the knockdown efficiency of ZNF283. Marc-145 cells were transfected with shmZNF283 or scrambled shRNA for 24 h and then infected with PRRSV at an MOI of 0.5 for various time periods. The culture supernatant was used for the TCID50 assay (C), and N protein expression levels were analysed using Western blotting (D and E).

Figure 4. ZNF283 suppresses PRRSV RNA synthesis. Marc-145 cells were transfected with HA-tagged ZNF283 or shmZNF283 and then infected with PRRSV at an MOI of 0.2. An empty vector or scrambled shRNA was used as a control. A and B Cells were harvested at 36 h post-infection for RT‒qPCR analysis of viral RNA expression. C and D At 36 h post-infection, the cells were fixed for immunofluorescence staining of dsRNA (green). The nuclei were counterstained with DAPI (blue).

Figure 5. ZNF283 interacts with PRRSV Nsp9 and Nsp10. A Marc-145 cells were cotransfected with HA-tagged ZNF283 and plasmid-encoded Flag-tagged PRRSV viral proteins (Nsp9, Nsp10, Nsp11, and Nsp12) for 24 h. The cells were then fixed, and immunofluorescence analysis was performed using rabbit anti-HA and mouse anti-Flag antibodies. Nuclei were stained with DAPI. B and C HEK-293T cells were cotransfected with HA-tagged ZNF283 and Flag-tagged Nsp9 or Nsp10 for 36 h, followed by immunoprecipitation with mouse anti-Flag or mouse anti-HA antibodies. Cell lysates were analysed by Western blotting using rabbit anti-HA or rabbit anti-Flag antibodies.

Figure 6. PRRSV Nsp9 interacts with the ZNF283 KRAB domain from aa 56–97. A Diagrammatic illustration of diverse fragments of ZNF283. B Marc-145 cells were cotransfected with Flag-tagged Nsp9 and plasmid-encoded HA-tagged ZNF283 truncations (aa 1–191, 1–296, 1–408, 1–520, and 192–663) or EGFP-tagged ZNF283 aa 56–97 for 24 h. The cells were fixed and immunostained using either a mouse anti-Flag or rabbit anti-HA antibody. Nuclei were counterstained with DAPI. C HEK-293T cells were cotransfected with the plasmids Flag-tagged Nsp9 and full-length HA-tagged ZNF283, EGFP-tagged ZNF283 truncations (aa 56–97), or HA-tagged ZNF283 truncations (aa 192–663) for 36 h. Following cell lysis, immunoprecipitation analysis was conducted using a mouse anti-Flag antibody.

Figure 7. ZNF283 interacts with the aa 178–449 region of Nsp9. A Schematic representation of Nsp9 truncation mutants. B Marc-145 cells were cotransfected with HA-tagged ZNF283 and plasmid-encoded Flag-tagged Nsp9 truncations (aa 1–177, 178–449, 450–643, 1–499, and 178–643) for 24 h. The cells were fixed and immunostained with mouse anti-Flag and rabbit anti-HA antibodies. Nuclei were detected by counterstaining with DAPI. C HEK-293T cells were cotransfected with the HA-tagged ZNF283 plasmid and Flag-tagged truncated Nsp9 (aa 178–449) for 36 h. Immunoprecipitation of cell lysates was performed using either mouse anti-Flag or mouse anti-HA antibodies, followed by Western blotting analysis using either rabbit anti-HA or rabbit anti-Flag antibodies.

Figure 8. The KRAB domain of ZNF283 interacts with the aa 178–449 region of Nsp9. A Marc-145 cells were cotransfected with Flag-tagged truncated Nsp9 spanning aa 178–449 and EGFP-tagged ZNF283 KRAB domain spanning aa 56–97 or an EGFP-tagged vector. After 24 h, the cells were stained with DAPI and incubated with antibodies specific for the Flag-tagged protein. B HEK-293T cells were cotransfected with Flag-tagged truncated Nsp9 (aa 178–449) and EGFP-tagged ZNF283 aa 56–97. Thirty-six hours after transfection, the cell lysate was immunoprecipitated with mouse anti-Flag or mouse anti-GFP antibodies and then immunoblotted using the indicated antibodies from different species. C Schematic representation of the interaction between ZNF283 and PRRSV Nsp9.

Figure 9. PRRSV Nsp10 interacts with the ZNF283 KRAB domain from aa 56–97. A Marc-145 cells were cotransfected with Flag-tagged Nsp10 and EGFP-tagged ZNF283 truncations (aa 56–97) or HA-tagged ZNF283 truncations (aa 192–663) for 24 h. The cells were fixed and immunostained using either mouse anti-Flag or rabbit anti-HA antibodies. Nuclei were counterstained with DAPI. B HEK-293T cells were cotransfected with Flag-tagged Nsp10 and HA-tagged ZNF283, EGFP-tagged ZNF283 truncations (aa 56–97), or HA-tagged ZNF283 truncations (aa 192–663) for 36 h. After cell lysis, immunoprecipitation analysis was performed using a mouse anti-Flag antibody.

Figure 10. ZNF283 has a specific affinity for the PRRSV 3'UTR. A Marc-145 cells were transfected with HA-tagged ZNF283 for 24 h and then mock-infected or infected with PRRSV at an MOI of 0.2 for 36 h. The cells were fixed for immunofluorescence staining with HA-tagged (green) and dsRNA (red). The nuclei were labelled with DAPI. B The 5'UTR or 3'UTR of PRRSV was cloned and inserted into the control reporter pGL3-Luc, as indicated. The resulting constructs, HA-tagged ZNF283 and pRL-TK, which are plasmids expressing Renilla luciferase and were used to normalize transfection efficiency, were cotransfected into Marc-145 cells. After 24 h of transfection, the cells were lysed, and luciferase activities were quantified.