Figure S3.
Analyses of ORF52 mutants and the LLPS properties of ORF52 from other γ-herpesviruses. (A) Empty vector or plasmids expressing mCherry-ORF52 or plasmids expressing mCherry-tagged mutants were individually transfected into 293T cells. Cells were collected for Western blot at 48 h post transfection. (B) 293T cells were transfected with ORF52 mutant 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. (C) KSHV-ORF52 and EBV-BLRF2 form puncta in COS-7 cells after transfection. Plasmids expressing mCherry-ORF52 (KSHV) or mCherry-BLRF2 (EBV) were transfected into COS-7 cells. Cells were observed with a fluorescence microscope. Bar: 5 μm. (D) FRAP of mCherry-ORF52 homologues in COS-7 cells at 37°C. Bar: 2 μm. (E) A conserved cluster of basic amino acids in the IDR is also required for phase separation of KSHV-ORF52 and EBV-BLRF2. Plasmids expressing KSHV mCherry-ORF52(4A) or EBV mCherry-BLRF2(4A) were transfected into COS-7 cells. Cells were observed with a fluorescence microscope. Bar: 10 μm. (F) Formation of cVACs relies on the phase separation properties of KSHV ORF52. 293T cells were transfected with MHV-68-ORF52-null BAC plus a plasmid expressing KSHV-ORF52 or its 4A mutant. At 48 h post transfection, MHV-68-ORF33 and KSHV-ORF52 were visualized by indirect immunofluorescence. ORF33 was detected using a mouse anti-ORF33 monoclonal antibody, followed by an Alexa Fluor 488-conjugated secondary antibody (green channel). ORF52 was detected using a rabbit anti-flag monoclonal antibody, followed by an Alexa Fluor 555-conjugated secondary antibody (red channel). Nuclei were labeled with DAPI (blue). Bar: 5 μm. (G) Plot of the normalized fluorescence intensity of KSHV-ORF52 (red) and MHV-68-ORF33 (green) along the line in F. Source data are available for this figure: SourceData FS3.

Analyses of ORF52 mutants and the LLPS properties of ORF52 from other γ-herpesviruses. (A) Empty vector or plasmids expressing mCherry-ORF52 or plasmids expressing mCherry-tagged mutants were individually transfected into 293T cells. Cells were collected for Western blot at 48 h post transfection. (B) 293T cells were transfected with ORF52 mutant 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. (C) KSHV-ORF52 and EBV-BLRF2 form puncta in COS-7 cells after transfection. Plasmids expressing mCherry-ORF52 (KSHV) or mCherry-BLRF2 (EBV) were transfected into COS-7 cells. Cells were observed with a fluorescence microscope. Bar: 5 μm. (D) FRAP of mCherry-ORF52 homologues in COS-7 cells at 37°C. Bar: 2 μm. (E) A conserved cluster of basic amino acids in the IDR is also required for phase separation of KSHV-ORF52 and EBV-BLRF2. Plasmids expressing KSHV mCherry-ORF52(4A) or EBV mCherry-BLRF2(4A) were transfected into COS-7 cells. Cells were observed with a fluorescence microscope. Bar: 10 μm. (F) Formation of cVACs relies on the phase separation properties of KSHV ORF52. 293T cells were transfected with MHV-68-ORF52-null BAC plus a plasmid expressing KSHV-ORF52 or its 4A mutant. At 48 h post transfection, MHV-68-ORF33 and KSHV-ORF52 were visualized by indirect immunofluorescence. ORF33 was detected using a mouse anti-ORF33 monoclonal antibody, followed by an Alexa Fluor 488-conjugated secondary antibody (green channel). ORF52 was detected using a rabbit anti-flag monoclonal antibody, followed by an Alexa Fluor 555-conjugated secondary antibody (red channel). Nuclei were labeled with DAPI (blue). Bar: 5 μm. (G) Plot of the normalized fluorescence intensity of KSHV-ORF52 (red) and MHV-68-ORF33 (green) along the line in F. Source data are available for this figure: SourceData FS3.

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