Fig 4.

The PRRSV PLP2-DUB domain inhibits ISG15 expression and conjugation. (A) HeLa cells were transfected with an expression plasmid for nsp2(386–578) or an empty plasmid vector and stimulated with 300 HA/ml of SeV at 24 h posttransfection. Cells were harvested at 24 h poststimulation, and lysates were immunoblotted for detection of ISG15 or nsp2(386–578). An anti-ISG15 MAb was used to detect ISG15 expression, and an anti-nsp2 MAb was used to detect the expression of nsp2(386–578). (B) HeLa cells were cotransfected with plasmid DNAs expressing conjugation enzymes E1/E2/E3, ISG15, and nsp2(386-578). The empty pCAGGS vector plasmid was included as a control. At 6 h posttransfection, cells were stimulated with 1,000 U/ml of IFN-β. Cells were harvested at 24 h poststimulation and analyzed by immunoblotting. The membrane was probed with anti-PRRSV nsp2 MAb to detect the expression of nsp2(386-578). Free and conjugated forms of ISG15 were detected by anti-ISG15 polyclonal antiserum. The anti-β-tubulin antibody was used to detect the expression of β-tubulin (loading control).