Figure 5. ORF6 enhances lipolysis by interacting with ATGL.

• A
  HeLa cells were transfected with vector or ORF6‐Flag for 12 h and treated with 200 μM OA for another 12 h, the medium was removed and replaced with fresh complete culture containing 1 μM DGAT1 inhibitor and 2 μM DGAT2 inhibitor for 24 h. Meanwhile, 50 μM ATGL inhibitor or 100 μM Chloroquine (CQ) was added and allowed to incubate for 24 or 4 h to block lipolysis or lipophagy, respectively. Cells were fixed and stained with anti‐Flag (red). LDs were labeled with BODIPY‐493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm. White ROIs indicate cells expressing ORF6 and yellow ROIs indicate the cells without ORF6 expression.
• B
  Mean number of LDs in each cell in (A) was counted from 25 cells of three independent experiments. Two‐tailed Unpaired Student's t‐test, ***P < 0.001, ****P < 0.0001, ns means no significance. Error bars represent the mean ± SD.
• C
  HEK293T cells were transfected with ORF6‐Flag. Protein interactions were detected by immunoprecipitation with anti‐Flag beads and immunoblotting analysis with ATGL, HSL, and CGI58 antibodies.
• D
  Purified GST‐ORF6 or indicated mutants were incubated with purified His tagged ATGL, and analyzed the interactions by GST pull‐down.
• E
  The interactions of Flag‐ATGL with ORF6‐GFP or the ΔNTD mutant were analyzed by immunoprecipitation.
• F
  The effect of ORF6‐Flag expression on the interaction of HA‐CGI58 with ATGL was analyzed by immunoprecipitation.
• G
  Model for the regulation of lipolysis by interacting with ATGL.
• H, I
  The effect of ORF6‐Flag or the ΔNTD mutant on the interactions of Flag‐ATGL with GFP‐Plin2 or GFP‐UBXD8 was analyzed by immunoprecipitation.
• J
  HeLa cells were transfected with ORF6‐Flag or the ΔNTD mutant for 6 h and were treated with 200 μM OA for 24 h. The medium was removed and replaced with serum free DMEM for another 4 h. The concentration of FFA in cells was analyzed. Error bars, mean ± SD of three independent experiments. Two‐tailed Unpaired Student's t‐test, *P < 0.05, **P < 0.01, ns means no significance.
• K, L
  Vero‐E6 cells were transfected with Flag‐ATGL and GFP‐Plin2 or GFP‐UBXD8 for 24 h and infected or non‐infected with SARS‐CoV‐2 for 24 h. Protein interactions were analyzed by immunoprecipitation with anti‐GFP beads and immunoblotting analysis.
• M
  SARS‐CoV‐2‐infected Vero‐E6 cells were treated with 50 μM ATGL inhibitor for 24 h. Cell viability was analyzed. Cell lysates were analyzed via WB. Supernatants were determined by plaque assays. Virus titer values represent mean ± SD for three independent replicates. Two‐tailed Unpaired Student's t‐test, *P < 0.05, ns means no significance. Error bars represent as the mean ± SD.

Source data are available online for this figure.