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

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/782409 doi: bioRxiv preprint under normal experimental conditions (Fig. 2B ). The net result is a significant reduction in the 129 catalytic efficiency (kcat/Km) compared to the wild-type enzyme, which was the goal of these trials. 130 The kinetic response of MHV PLP2 toward another substrate, ISG15-AMC, was also 131 determined. ISG15 is an important ubiquitin-like modifier that is upregulated and used to ISGylate 132 host proteins during viral infection. A number of viruses, including coronaviruses, engender 133 ISGylation during infection but the function(s) and importance of this activity are not clear 30). For MHV, neither the wild-type nor the D1772A mutant PLP2 enzyme can be saturated with 135 ISG15-AMC, suggesting weak binding with this ubiquitin-like modifier (Fig. 2B) . Moreover, the 136 R1772A mutation does not disrupt the interaction with ISG15 but in fact enhances it to some 137 degree. A potential explanation for the observed selective disruption of ubiquitin binding stems 138 from our analysis of a primary sequence alignment of ubiquitin and ISG15 and the residues that 139 interact with D1772 (Fig. 2C ). The interaction between MHV PLP2 D1772 and the R42 residue in 140 human and mouse ubiquitin is absent in human and mouse ISG15 since this residue is a tryptophan 141 in human and mouse ISG15. Therefore, in line with our observations, D1772A mutation would 142 not be expected to alter ISG15 binding. In contrast to R42, residue R72 is conserved in both 143 ubiquitin and ISG15 and its interaction with MHV PLP2 for ubiquitin is likely weaker than that 144 with ISG15.