Thelonevirologist (@thelonevirologist.bsky.social)

Establishment of a green fluorescent protein (GFP)-based reporter for picornaviral 3C proteases | Journal of Virology The 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. https://journals.asm.org/doi/10.1128/jvi.01936-25