Fig 12.

PRRSV nsp12 and N proteins inhibit SHFL transcription. (A) Schematic diagram of truncated SHFL promoter constructs (P1–P8). (B) Mapping the core promoter region of the pig shfl gene. A series of truncated SHFL promoter constructs (P1–P8) was cotransfected with the pRL-TK-Luc in HEK-293T cells. At 24 hpt, cell lysates were collected and analyzed by the dual-luciferase activity. (C–E) Screening for PRRSV proteins that suppress SHFL promoter activity. SHFL promoter construct (P2) was cotransfected with pRL-TK-Luc and individual plasmids expressing each PRRSV viral protein in HEK-293T cells. At 24 hpt, cell lysates were collected and analyzed by the dual-luciferase activity. (F and G) PRRSV nsp12 and N proteins inhibit SHFL transcription. HEK-293T cells were transfected with FLAG-tagged nsp12 or N (1 μg) for 36 h. Cells were harvested for total cellular RNA extraction, and endogenous SHFL mRNA levels were detected by RT-qPCR (F). Cell lysates were collected, and endogenous SHFL protein expression levels were analyzed by Western blot. β-Tubulin was detected as a loading control. Grayscales of SHFL/β-tubulin are shown below the figure panel G. (C–F) Statistical significance between empty vector (EV) and viral protein was analyzed using the two-tailed unpaired Student’s t-test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant. (H and I) The subcellular localization of nsp12 and N. MARC-145 cells in 35 mm dishes were mock-infected or infected with TA-12 (MOI = 1) for 12 h and 24 h, after which cells were fixed and processed for IFA using a mAb against nsp12 or N protein (scale bar, 20 μm). (J) The subcellular localization of PRRSV nsp12 and N in virus-infected MARC-145 cells was analyzed by nuclear and cytoplasmic fractionation.