Figure 2.

ORF8/p62 condensates subvert ER-phagy

(A) Co-precipitation analysis of GFP-FAM134B with ORF8-Strep in HEK293T.

(B) Purified MBP or MBP-ORF8 was incubated with purified GST or GST-FAM134B, and analyzed the interaction between ORF8 and FAM134B by GST pull-down.

(C) Immunofluorescence of cells expressing FAM134B-HA or FAM134B-HA and GFP-ORF8 or FAM134B-HA, p62-FLAG and GFP-ORF8 with anti-HA and anti-FLAG antibodies. Scale bar, 10 μm. The number of FAM134B puncta (>1 μm) in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ^∗∗∗∗p < 0.0001.

(D) In vitro phase separation assay of GFP-FAM134B alone or GFP-FAM134B and MBP-ORF8 or GFP-FAM134B and mCherry-p62/His-UBx8 or GFP-FAM134B, MBP-ORF8 and mCherry-p62/His-UBx8. Fluorescence images of 10 μM each protein in phase separation assay buffer without PEG8000. Representative images of three independent experiments are shown. Scale bar, 10 μm.

(E) GFP-ORF8 truncations were expressed as indicated with FAM134B-HA. Cell lysates were subjected to immunoprecipitation with GFP antibody and analyzed using WB.

(F) HeLa cells were transfected with GFP-ORF8 truncations with FAM134B-HA for 24 h, then stained with anti-HA antibody and imaged using confocal microscopy. Scale bar, 10 μm.

(G and H) U2OS cells expressing mCherry-GFP-RAMP4 were transfected with p62-FLAG with or without ORF8-Strep or mutants for 24 h, then cells were starved in EBSS for 12 h and imaged using confocal microscopy (G). Scale bar, 10 μm. The number of red puncta in each cell (H) was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ^∗∗∗∗p < 0.0001.

(I and J) U2OS cells expressing mCherry-Sec61B were transfected with indicated plasmids for 24 h and then starved in EBSS for 12 h. Cell lysates were analyzed using WB.

(K) U2OS cells expressing mCherry-Sec61B were transfected with indicated plasmids for 24 h. Cell lysates were analyzed using WB.

(L) Vero-E6 cells were transfected with control or ORF8-KD plasmid for 36 h and analyzed the knockdown efficiency using WB.

(M and N) Vero-E6 WT cells were transfected with control or ORF8-KD plasmid, p62 KO Vero-E6 cells were transfected with p62^EHGG-AAAA-FLAG for 12 h and then infected with SARS-CoV-2 for 24 h, then cells were stained with anti-FAM134B and anti-p62 or anti-FLAG antibodies and imaged using confocal microscopy. Scale bar, 10 μm. The number of colocalization between FAM134B and p62 droplets in each cell was counted from 20 cells of two independent experiments. Two-tailed unpaired Student’s t test, ^∗∗∗∗p < 0.0001.

(O) Vero-E6 cells were transfected with ORF8-KD plasmid with or without si-ORF3a or si-Atg7 for 24 h and then infected with SARS-CoV-2 for another 24 h. Cell lysates were analyzed using WB.

See also Figure S3.