Fig. 1.

The PRRSV replicase nsp1β is a critically viral antagonist of PKR. (A and B) Production of dsRNAs in MARC-145 and PAMs infected with PRRSV strain JXwn06 at an MOI of 0.1. The cells were stained with antibodies to dsRNA and to PRRSV N protein at 18 hpi and examined by a Nikon A1 confocal microscope, and the images are representative of at three independent experiments. Oil objective: 100×; zoom in 1× (A) or 2× (B). (C and D) Western blot analysis of PKR phosphorylation in PRRSV-infected MARC-145 cells and PAMs with antibodies to phosphorylated-PKR (p-PKR), PKR, β-actin, and N protein. Poly(I:C) (1.5 µg/mL) as a positive control was used to treat MARC-145 cells for 12 h or PAMs for 6 h. (E and F) Effect of PRRSV infection on poly(I:C)-induced PKR phosphorylation. MARC-145 and PAMs were infected or mock-infected with PRRSV at an MOI of 0.1, and at 24 (MARC-145) or 12 hpi (PAMs), the cells were treated with poly(I:C) at a concentration of 1.5 µg/mL for 12 or 6 h before being collected for Western blot analyses. (G) Dose-dependent effect of nsp1β on PKR phosphorylation. HEK-293FT cells seeded in six-well plates were transfected to express increasing amount of HA-nsp1β. At 24 h posttransfection, the cells were treated with poly(I:C) (1.5 µg/mL) for 12 h before being collected for Western blot analysis. The relative band density of p-PKR was normalized to the total PKR and then the loading control β-actin, and then compared to the corresponding mock control, and expressed as p-PKR(+/−).