Establishment of a green fluorescent protein (GFP)-based reporter for picornaviral 3C proteases

Picornaviruses represent a diverse group of plus-stranded RNA viruses, many of which have been linked to severe diseases in both humans and animals. The viral 3C protease is essential for the maturation of viral proteins and the propagation of picornaviruses and, owing to its cleavage activity against multiple host proteins, is associated with the pathogenesis of picornaviruses. The picornaviral 3C protease is an ideal drug target for inhibiting viral propagation and mitigating pathogenesis; however, methodology to evaluate and compare the activity of phylogenetically diverse proteases remains lacking. To address this, herein, we propose a novel green fluorescent protein (GFP)-based reporter optimized to visualize the enzymatic activity of picornaviral 3C proteases in cells by using the conformational change of a GFP variant induced by the 3C protease, generating fluorescence emission linked to the enzymatic activity. Upon treatment of picornaviruses with a known 3C protease inhibitor, the fluorescence decreased in a dose-dependent manner, demonstrating that the signal depended on the activity of the 3C protease. The reporter system for the 3C protease can be applied to major pathogenic human picornaviruses, such as those in the genera Enterovirus, Rhinovirus, Cosavirus, Salivirus, and Kobuvirus. Furthermore, the fluorescent signal from the reporter was confirmed in various animal- derived picornaviruses, such as those from bats, rodents, and primates. Therefore, the reporter could be widely used to analyze the activity of several 3C proteases from currently prevalent picornaviruses and those that may emerge in the future. To demonstrate the flexibility of the reporter in comparing phylogenetically different proteases, the enzymatic activity of the 3C protease derived from clinical strains of enterovirus A71 (EV-A71) was tested and compared. The results showed that the amino acid residues of the 3C protease affect its activity by utilizing the reporter system. Additionally, clinical EV- A71 strains had different effects on the activity of 3C protease against host proteins. Our findings will aid in elucidating the molecular characteristics of 3C proteases among picornaviruses and developing therapeutics to mitigate the pathogenesis of these viruses.