Transmissible Gastroenteritis Virus Papain-Like Protease 1 Antagonizes Production of Interferon-β through Its Deubiquitinase Activity

Figure 4

TGEV PL1 displays DUB activity that is dependent on its catalytic activity and reduces the ubiquitinated forms of RIG-I and STING. (a) HEK293T cells were transfected with HA-tagged ubiquitin and TGEV PL1 or the catalytic mutants C32A, H183A, and D196A. Proteins were assayed using Western blot with anti-HA and anti-Myc antibodies. ((b) and (c)) HEK293T cells were transfected with Myc-TGEVPL1 together with Flag-RIG-1 (b) and Flag-STING (c) for 24 h, and lysates were subjected to coimmunoprecipitation and Western blot to determine the ubiquitination status of immunoprecipitated proteins. ((d) and (e)) HEK293T cells were transfected with Flag-RIG-1 (d) or Flag-STING (e), together with HA-Ub in the presence or absence of Myc-TGEVPL1. Cells were incubated for 24 h after transfection and then lysates were harvested. Lysates were immunoprecipitated with Flag, HA, and Myc antibodies and the products subjected to immunoblotting with anti-HA to evaluate ubiquitinated proteins.

(a)

(b)

(c)

(d)

(e)