Figure 5.

Recognition of DiUb^Lys48 and ISG15 by SARS PLpro Appears Distinct

(A) Michaelis-Menten kinetics of WT (black) and selected SARS PLpro mutants (M209S, S1 mutant, light blue; R167S/E168R, S1 mutant, dark blue; F70S/E71K/H74G, S2 mutant, green) using ISG15-AMC. Extracted kinetic parameters (k_cat and K_M) in Table 2.

(B) Cleavage of HMW-Ub^Lys48 (top, WB anti-K48) and ISG15-conjugates (bottom, WB ISG15) in lysates prepared from IFNβ/MG132-treated cells by SARS PLpro WT and S2 and S1 mutants.

(C) Quantification of loss of HMW-Ub^Lys48 (left) and appearance of free ISG15 (right) from duplicate experiments shown in Figure 5B. Error bars represent ±SEM.

(D) Schematic representation of SARS PLpro substrate specificity. Dashed lines can indicate the -AMC substrate, a non-Lys48-linked Ub unit, or a protein substrate.

See also Figure S6.