Adoption of the 2A Ribosomal Skip Principle to Track Assembled Virions of Pepper Mild Mottle Virus in Nicotiana benthamiana .

The coat protein (CP) is an important structural protein that plays many functional roles during the viral cycle. In this study, the CP of pepper mild mottle virus (PMMoV) was genetically fused to GFP using the foot-and-mouth disease virus peptide 2A linker peptide and the construct (PMMoV-GFP 2A ) was shown to be infectious. The systemic spread of the virus was monitored by its fluorescence in infected plants. Electron microscopy and immunocolloidal gold labelling confirmed that PMMoV-GFP 2A forms rod-shaped particles on which GFP is displayed. Studies of tissue ultrastructure and virion self-assembly confirmed that PMMoV-GFP 2A could be used to monitor the real-time dynamic changes of CP location during virus infection. Aggregations of GFP-tagged virions appeared as fluorescent plaques in confocal laser microscopy. Altogether, PMMoV-GFP 2A is a useful tool for studying the spatial and temporal changes of PMMoV CP during viral infection.