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. Using the reporter assay, we identified amino acid variations that modulate 3C protease activity. Additionally, we observed that the 3C proteases from distinct EV-A71 strains exhibited differential efficiency in cleaving 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.IMPORTANCEThe family Picornaviridae comprises various biologically distinct viruses that have been linked to life-threatening diseases, including emerging and previously neglected pathogens whose molecular characteristics remain elusive. Although the viral protease encoded by picornaviruses, the 3C protease, is an ideal target for anti-picornaviral pharmacological intervention, the current assay system for evaluating the activity of 3C proteases remains limited to a few specific picornaviruses. To address this, we sought to establish a flexible and straightforward protocol for assessing and comparing various picornaviral 3C proteases. We developed a cell-based assay for 3C proteases using a split-green fluorescent protein (GFP) variant with a specific viral sequence that could be applied to a wide range of human pathogenic and animal-derived picornavirus strains. Furthermore, by swapping the viral sequence inside GFP for a host-derived sequence, this assay could be used to evaluate the 3C protease activity of cleaving host proteins. This assay will facilitate future research comparing functional differences of 3C proteases among picornaviruses, conducting high-throughput screening of anti-picornaviral drugs, or investigating the relationship between protease activity and picornaviral pathogenesis.