Nucleic acids drive phase separation of ORF52. (A) In vitro phase separation assay of ORF52 with DNA. Phase separation of ORF52 with 45-bp ds-DNA was performed in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl. 10 μM ORF52 protein was mixed with 5 μM 45-bp ds-DNA (2% Cy3-labeled) in 96-well plates coated with 20 mg/ml BSA. Mixtures were incubated and images were captured by confocal microscopy. Bar: 20 μm. (B) ORF52 phase separation in vitro in the presence of total RNA isolated from COS-7 cells. Phase separation of ORF52 with total RNA was performed in physiological buffer. 5 μM ORF52 protein (3% Alexa 488-labeled) was mixed with 100 ng/μl total RNA in 96-well plates coated with 20 mg/ml BSA. Mixtures were incubated and images were captured by confocal microscopy. Bar: 10 μm. (C) Fusion of ORF52-DNA droplets formed during the in vitro phase separation process. Bar: 5 μm. (D) FRAP of ORF52-DNA liquid droplets. Bleaching was performed during the in vitro phase separation process. (E) Phase separation diagram of ORF52 with ds-DNA. Bar: 5 μm. (F) Fusion of ORF52-RNA droplets formed during the in vitro phase separation process. Bar: 5 μm. (G) FRAP of ORF52-RNA liquid droplets. Bleaching was performed during the in vitro phase separation process. Bar: 5 μm. (H) Phase separation diagram of ORF52 with total cellular RNAs. (I) cVACs contain little DNA. After infection with mEosEM-ORF52 virus (MOI = 3), COS-7 cells were fixed and stained with DAPI. Bar: 5 μm. (J) RNA is concentrated in cVACs. EU was added to cultured COS-7 cells to label nascent RNA 24 h before the cells were infected with mCherry-ORF52 virus. EU-labeled RNA was detected by click chemistry reaction. Bar: 5 μm.