The LLPS properties of ORF52 are critical for cVACs formation and virion production. (A) Schematic presentation of full-length and mutant ORF52 proteins. (B and C) ORF52 mutants have different phase separation properties. Plasmids expressing mCherry-ORF52 or mCherry-tagged mutants were individually transfected into COS-7 cells. Cells were observed with a fluorescent microscope at 24 h post transfection (B). Bar: 5 μm. In each experiment, 30 cells expressing mCherry-ORF52 or mutants were randomly chosen. Cells with ORF52 puncta were counted, and the percentage of cells with ORF52 puncta among all cells expressing fluorescent protein was calculated (C). Values shown are means ± SD (n = 3). (D and E) Phase separation of ORF52 is critical for complete viral replication and mature virion formation. WT BAC or BAC containing the indicated ORF52 mutation was transfected individually into 293T cells. After 96 h, viral genome copies in the supernatant were determined by real-time PCR (D) and viral titers were examined by plaque formation assay (E). Values shown are means ± SD (n = 3). (F) ORF52 phase separation is required for cVACs formation. 293T cells were individually transfected with WT BAC or BAC harboring the indicated ORF52 mutation. cVAC structure was analyzed by TEM. Bar: 0.2 μm. Red boxes: cVACs; white arrowheads: capsids or immature viral particles; red arrowheads: mature viral particles.