ORF52 possesses LLPS properties and is required for cVACs formation. (A) ORF52 forms puncta in COS-7 cells after transfection. Plasmids expressing RFP-ORF52 or mCherry-ORF52 were individually transfected into COS-7 cells. Cells were observed with a fluorescence microscope. Bar: 5 μm. (B) mCherry-labeled ORF52 forms spherical bodies in cells. The XY, XZ, and YZ planes of a representative ORF52 body are shown. Bar: 2 μm. (C) A plot showing the sphericity of ORF52 bodies (n = 94). Error bars represent SD. (D) Fusion of ORF52 puncta. Plasmids expressing mCherry-ORF52 were transfected into COS-7 cells. Cells were observed with a time-lapse microscope at 24 h post transfection. Bar: 2 μm. (E) FRAP of mCherry-ORF52 in COS-7 cells at 37°C. Bar: 2 μm. (F) Formation of cVACs relies on ORF52. 293T cells were transfected with ORF52-null BAC and detected by indirect immunofluorescence at 48 h post transfection. ORF33 was detected using a mouse anti-ORF33 monoclonal antibody, followed by an Alexa Fluor 488-conjugated secondary antibody (green channel). ORF45 was detected using a rabbit anti-ORF45 polyclonal antibody, followed by an Alexa Fluor 647-conjugated secondary antibody (red channel). Nuclei were stained with DAPI (blue channel). Bar: 5 μm. (G) TEM of a 293T cell transfected with ORF52-null BAC. The right panel shows an enlargement of the boxed area from the left panel. Scale bars: 3 µm (main image); 0.5 µm (zoomed-in image). White arrowheads: capsids.