Fig. 5
Identification of amino acid residues within the conserved WVPRA motif within the SARS-CoV-2 NSP2 that are required for efficient processing of the NSP1/NSP2 junction by PLpro (NSP3). Cell lysates from BHK cells infected with vTF7-315 and transfected with plasmids that express the SARS-CoV-2 NSP1-NSP2-Flag (wt or with single amino acid substitutions within the conserved WVPRA motif) either alone (odd numbered lanes) or with the plasmid encoding the SARS-CoV-2 PLpro (even numbered lanes) were analyzed by immunoblotting as in Fig. 4. Molecular mass markers (kDa) are indicated on the left. A negative control was included (lane 9). The NSP1-NSP2-Flag and the NSP2-Flag products are indicated on the right side of the western blot picture and were detected using anti-Flag-antibodies. Panel (a) shows the wt and various substitutions of the NSP2 W243 and a negative control. Panel (b) shows the wt, the NSP2 R246A, the NSP2 P247A, NSP2 P247G and a negative control. The uncut membranes are shown in Supplementary Figs. S4 and S7. This experiment has been repeated three times in total with separate transfections and western blots and resulted in very similar results. The additional experiments are shown in Supplementary Figs. S5, S6, S8 and S9. Band intensities for each experiment were quantified using pixel count in ImageJ. Each lane was set to 100%, and the relative percentage of each of the two bands was calculated. The mean values of the triplicates were plotted, and the bar diagram with error bars representing the standard deviation (SD) are shown on the right side of panels a and b.
