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

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.