Figure 3.

Induction of autophagy by various coronaviral PLPs that is independent on the protease activity. (A) The plasmids of HCoV-NL63 PLP2-TM, SARS-CoV PLpro-TM, MERS-CoV PLpro-TM and PEDV PLP2-TM were transfected into HEK293T cells. As the positive control for induction of autophagy, HEK293T cells were treated with complete medium supplemented with 400 nmol/L Rapamycin for 6 h. The cells were then lysed for Western blotting analysis using a rabbit anti-LC3 antibody to detect endogenous LC3 expression (top panel in Fig. 3A). The whole cell lysate (WCL) was blotted using anti-V5 antibodies to evaluate expression of PLP2 (PLpro)-TM (middle panel in Fig. 3A), and β-Actin was detected in whole cell lysate (WCL) as a loading control (bottom panel in Fig. 3A). (B) PLP2-TM-V5 and the catalytic mutants (C1678A, H1836A and D1849A) as showed in Fig. 1A were transfected respectively into HEK2923T, and induction of autophagy was detected as described in Fig. 3A