Coronavirus membrane protein with a fluorescent protein tag enables particle tracking for the study of virus assembly and egress in live cells.

Coronavirus particles assemble at endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membranes and exit from host cells via secretory organelles that are not well defined. The interplay between viral components and intracellular transport pathways that facilitate assembly and egress is not fully understood, and recent studies suggest that multiple pathways may be involved. Reverse genetics was used to develop a model system to further understand the assembly and egress processes. Mouse hepatitis coronavirus (MHV-A59) was genetically engineered to express the membrane (M) protein fused to green fluorescent protein (M-GFP), with the chimeric gene cloned in place of the open reading frame (ORF) 4 coding region in the RNA genome. The recovered M-GFP virus also expresses wild-type (WT) M protein (M WT) from its native ORF. The M-GFP virus exhibited morphology and growth properties like the WT virus. M-GFP and WT M proteins colocalized early in infection, but less M-GFP trafficked toward the cell surface at later times, suggesting that the fusion protein is incorporated less efficiently into virus particles. M-GFP was stably expressed through at least four virus passages. Early passage M-GFP virus particles were visualized by confocal and total internal reflection fluorescence (TIRF) microscopy in live cells. The fluorescently labeled virus particles represent a new tool for coronavirus intracellular trafficking and egress studies. Such studies can also help provide a more detailed understanding of infection and disease processes to provide insight for the development of new therapeutic strategies. IMPORTANCE: Coronavirus assembly, intracellular transport, egress, and the virus-host interactions involved in these processes are not fully understood. The membrane (M) protein is the most abundant virion structural component, which forms the scaffold for the assembly of the viral envelope. It facilitates the incorporation of the viral nucleocapsid and the other viral membrane proteins, spike (S) and envelope (E). In this study, we engineered the M protein tagged with green fluorescent protein (GFP), which is expressed along with wild-type (WT) M from the viral genome. M-GFP interacted with WT M protein and co-assembled into virus particles. The resulting fluorescently labeled coronavirus particles open opportunities to use state-of-the-art microscopy to monitor virus assembly, trafficking, and egress in live cells to facilitate a deeper understanding of the molecular and cell biology of these processes. The ability to visualize particles also opens opportunities to help further understanding of steps in viral replication and pathogenesis for potential therapeutic targets.