Viable polioviruses that encode 2A proteins with fluorescent protein tags

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Picornaviridae enterovirus 2A proteins are cysteine proteinases crucial for viral polyprotein cleavage.
• 2A proteins are also implicated in viral RNA replication, independent of their protease activity.

Purpose of the Study:

• To investigate the function of 2A proteins in poliovirus replication using fluorescently tagged variants.
• To assess the impact of fluorescent protein insertions on 2A protease activity and viral replication.

Main Methods:

• Generation of viable polioviruses encoding 2A proteins with Discosoma sp. red fluorescent protein (DsRed) insertions.
• Analysis of viral plaque formation, polyprotein cleavage efficiency, and viral RNA replication in vitro and in infected cells.
• Confocal microscopy to visualize the localization of the 2A-DsRed fusion protein within infected cells.

Main Results:

• Polioviruses with a DsRed insertion near the 2A C terminus produced viable viruses with slightly smaller plaques than wild-type.
• The 2A-DsRed fusion protein retained protease activity, efficiently cleaved polyproteins, and supported viral RNA translation and replication.
• Intracellular membrane reorganization was similar to wild-type, and 2A-DsRed localized to perinuclear foci and a cytoplasmic matrix.

Conclusions:

• Fluorescent tagging of 2A proteins is a viable method to study their function during poliovirus infection.
• The 2A proteinase retains essential enzymatic and replication-supporting functions when fused to a fluorescent protein.
• The study provides insights into the localization and function of 2A protein in viral replication complexes.