Fig. 2.

PL^pro of SARS-CoV-2 cleaves ULK1 after G499.

(A) Sequence alignment of the PL protease domain of SARS-CoV-2 and PL protease domain 2 of MHV-A59 is shown. The SARS-CoV2-PL^pro was cloned into EGFP-C1 vector. (B) HEK293T cells were transfected with either control or PL^pro-expressing plasmid for 24 h. Lysates were probed by western blotting for endogenous ULK1 using the monoclonal anti-ULK1 antibody and normalized to ACTB as in Fig. 1A. (C) HEK293T cells were co-transfected with 3×Flag-ULK1 and either control vector or PL^pro-expressing plasmid for 24 h. Lysates were probed with anti-Flag antibody to detect exogenous ULK1. Arrow denotes cleavage fragment observed at ∼70 kDa. Densitometry is provided as in Fig. 1A. (D) Purified SARS-CoV-2 3CL^wt (4 μg) or catalytically-inactive C145A 3CL^mut (4 μg) of SARS-CoV-2 and CVB3 3C^wt (0.1 μg) alongside HeLa lysates (30 μg) were used to perform in-vitro cleavage assay. Western blotting was performed to probe for endogenous ULK1 and normalized to ACTB as in Fig. 1A. Arrows denote cleavage fragments. (E) PL^pro consensus cleavage motif in ULK1 from H. sapiens, M. musculus, and R. norvegicus is presented. Highlighted gray boxes denote consensus sites. Dashed arrows denote sites of potential cleavages. (F) HEK293T cells were co-transfected with PL^pro and either 3×Flag-ULK1^WT, 3×Flag-ULK1-G499^mut or 3×Flag-ULK1-G531^mut. Western blotting was conducted with anti-Flag and anti-GFP antibody for detection of ULK1 and PL^pro, respectively. (G) Schematic diagram of ULK1 protein sequence with corresponding functional domains, antibody recognition epitopes and identified PL^pro cleavage site.