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Identification of a critical horseshoe-shaped region in the nsp5 (Mpro, 3CLpro) protease interdomain loop (IDL) of coronavirus mouse hepatitis virus (MHV)

By Benjamin C. Nick, Mansi C. Pandya, Xiaotao Lu, Megan E. Franke, Sean M. Callahan, Emily F. Hasik, Sean T. Berthrong, Mark R. Denison, Christopher C. Stobart

Posted 19 Jun 2020
bioRxiv DOI: 10.1101/2020.06.18.160671

Human coronaviruses are enveloped, positive-strand RNA viruses which cause respiratory diseases ranging in severity from the seasonal common cold to SARS and COVID-19. Of the 7 human coronaviruses discovered to date, 3 emergent and severe human coronavirus strains (SARS-CoV, MERS-CoV, and SARS-CoV-2) have recently jumped to humans in the last 20 years. The COVID-19 pandemic spawned by the emergence of SARS-CoV-2 in late 2019 has highlighted the importance for development of effective therapeutics to target emerging coronaviruses. Upon entry, the replicase genes of coronaviruses are translated and subsequently proteolytically processed by virus-encoded proteases. Of these proteases, nonstructural protein 5 (nsp5, Mpro, or 3CLpro), mediates the majority of these cleavages and remains a key drug target for therapeutic inhibitors. Efforts to develop nsp5 active-site inhibitors for human coronaviruses have thus far been unsuccessful, establishing the need for identification of other critical and conserved non-active-site regions of the protease. In this study, we describe the identification of an essential, conserved horseshoe-shaped region in the nsp5 interdomain loop (IDL) of mouse hepatitis virus (MHV), a common coronavirus replication model. Using site-directed mutagenesis and replication studies, we show that several residues comprising this horseshoe-shaped region either fail to tolerate mutagenesis or were associated with viral temperature-sensitivity. Structural modeling and sequence analysis of these sites in other coronaviruses, including all 7 human coronaviruses, suggests that the identified structure and sequence of this horseshoe regions is highly conserved and may represent a new, non-active-site regulatory region of the nsp5 (3CLpro) protease to target with coronavirus inhibitors.

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