Structure-Guided Mutagenesis Alters Deubiquitinating Activity and Attenuates Pathogenesis of a Murine Coronavirus

Xufang Deng; Yafang Chen; Anna M. Mielech; Matthew Hackbart; Kristina R. Kesely; Robert C. Mettelman; Amornrat O’Brien; Mackenzie E. Chapman; Andrew D. Mesecar; Susan C. Baker

146 culture based assays. The in vitro biochemical studies presented here support the notion that we 147 are able to use a structure-guided mutagenesis to uncouple the DUB enzymatic activity from MHV 148 PLP2 while preserving the peptide hydrolysis and deISGylating activities of PLP2. Next, we 149 focused on comparing the activity of the mutant enzyme to its wild-type counterpart for the ability 150 to remove Flag-tagged-ubiquitin conjugated to host proteins in cultured cells (Fig. 3A) . We found 8 that in cells, wild-type PLP2 exhibits robust DUB activity and removes ubiquitin modifications 152 from multiple cellular proteins. On the other hand, the PLP2-D1772A mutant exhibits reduced 153 DUB activity, similar to that of the previously documented catalytic cysteine to alanine mutant, 154 PLP2-CA (19). To determine if this impaired DUB activity altered the ability of PLP2 to act as an 155 interferon antagonist, we transfected cells with a RIG-I expression plasmid, an interferon-156 luciferase reporter construct, and either wild-type or mutant PLP2 plasmid and measured luciferase 157 activity at 18 hours post-transfection. In agreement with previous reports (13, 25, 31), we find that 158 wild-type PLP2 acts as an interferon antagonist, reducing reporter activity by 50-80%. In contrast, 159 PLP2-D1772A is unable to significantly reduce interferon activation in this assay despite similar 160 expression levels of the wild-type and mutant versions of the protein (Fig. 3B ). We also evaluated 161 the protease activity of the enzymes in cells using two independent trans-cleavage assays and 162 found that the wild-type and DUB-mutant enzymes produce similar levels of cleaved products. 163 These results indicate that the D1772A substitution did not alter protease activity (Fig. 3C and D), 164 in agreement with the in vitro kinetic results described above (Fig. 2) . Together, these studies 165 reveal that aspartic acid residue 1772 of MHV-PLP2 is important for DUB activity and interferon 166 antagonism, but not for protease activity.