Figure 1.

SARS-CoV-2 ORF8 interacts p62 to form condensates

(A) Co-precipitation analysis of endogenous p62 with ORF8-Strep in HEK293T.

(B) Purified MBP-ORF8 was incubated with purified GST or GST-p62, and analysis of the interaction between ORF8 and p62 by in vitro GST pull-down was performed.

(C and D) Cells were co-transfected with ORF8-Strep and mCherry-p62 for 24 h, then stained with anti-Strep antibody and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ^∗∗∗∗p < 0.0001.

(E) p62 WT and KO Vero-E6 cells were transfected with ORF8-Strep for 24 h, then cells were stained with anti-Strep and anti-p62 antibodies, and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ^∗∗∗∗p < 0.0001.

(F) HeLa cells were co-transfected with GFP-ORF8 and mCherry-p62 for 24 h, then cells were mock treated or treated with 1,6-hexanediol for 1 min and imaged using confocal microscopy. Scale bar, 10 μm. The number of ORF8/p62 puncta in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ^∗∗∗∗p < 0.0001.

(G) HeLa cells with exogenous expression of GFP-ORF8 and mCherry-p62 undergo liquid-like behavior; fluorescence recovery after photobleaching (FRAP) images are shown before and at indicated time points after bleaching. Time 0 indicates the time of photobleaching. Scale bar, 10 μm. Quantification of fluorescence intensity recovery of GFP-ORF8 and mCherry-p62 in the bleached droplet.

(H) HeLa cells with exogenous expression of GFP-ORF8 and mCherry-p62 undergo fusion, images are shown at indicated time points after imaging.

(I) mCherry-p62 were purified from Escherichia coli BL21 and analyzed via Coomassie blue.

(J) In vitro phase separation assay of GFP and mCherry-p62/His6-UBx8 or GFP-ORF8 and mCherry-p62/His6-UBx8. Fluorescence images of 10 μM each protein in phase separation assay buffer without PEG8000. Representative fluorescence images of three independent experiments are shown. Scale bar, 10 μm.

(K) GFP-ORF8 truncations were expressed as indicated with p62-FLAG. Cell lysates were subjected to immunoprecipitation with GFP antibody and analyzed using western blotting (WB).

(L) Cells were transfected with GFP-ORF8 or mutant with mCherry-p62 for 24 h, and imaged using confocal microscopy. Scale bar, 10 μm.

(M) p62-FLAG mutations were expressed as indicated with ORF8-Strep. Cell lysates were subjected to immunoprecipitation with FLAG antibody and analyzed using WB.

(N) p62 KO Vero-E6 cells were transfected with p62-FLAG or mutant with ORF8-Strep for 24 h, then stained with anti-FLAG and anti-Strep antibodies and imaged using confocal microscopy. Scale bar, 10 μm.

(O) SARS-CoV-2 infected Vero-E6 cells were stained with anti-ORF8 and anti-p62 antibodies and imaged using confocal microscopy. Scale bar, 10 μm.

(P) Co-precipitation analysis of p62-HA with SARS-CoV-2 ORF8-FLAG or SARS-CoV ORF8-FLAG or MERS-CoV ORF8-FLAG in HEK293T.

(Q) Cells were co-transfected with mCherry-p62 with SARS-CoV ORF8-FLAG or MERS-CoV ORF8-FLAG for 24 h, then stained with anti-FLAG antibody and imaged using confocal microscopy. Scale bar, 10 μm.

See also Figures S1 and S2.