Fig. 6: Role of AGPATs in SARS-CoV-2 DMV formation and viral replication.

a AGPATs are localized at SARS-CoV-2 HA-nsp3-4-V5 induced DMVs. Huh7-derived cells were transiently transfected with AGPAT2-EGFP HA-nsp3-4-V5 and subjected to CLEM. Light and EM images were correlated by using lipid droplets as fiducial markers. White and yellow boxes indicate areas magnified in the corresponding panels on the right. Several regions from two cells of the same experiment showed similar relocalisation. b AGPAT1/2 contribute to SARS-CoV-2 replication. Huh7-Lunet control, AGPAT2 single (SKO) and AGPAT1/2 double (D)KO cells were infected with SARS-CoV-2 (MOI = 0.1). Twenty-four hours later, cells were fixed and immunostained for nucleocapsid, and the percentage of N-positive cells was determined using CellProfiler. Normalized data from three biologically independent experiments are plotted. Significance was calculated using ordinary one-way ANOVA. (top right panel). Total RNA was isolated from infected cells, and SARS-CoV-2 RNA levels were determined using RT-qPCR. Data were normalized to cellular GAPDH mRNA (bottom right panel). Significance was calculated using ordinary one-way ANOVA. Data are represented as mean ± SD from three biologically independent experiments. c Aberrant SARS-CoV-2 DMVs in AGPAT1/2 DKO cells. Huh7-Lunet cells with single (SKO) or double knock-out (DKO) and stably expressing T7 polymerase were transfected with a plasmid encoding SARS-CoV-2 HA-nsp3-4-V5 and fluorescent neon-green. Twenty-four hours later, cells were fixed and NeonGreen positive cells were recorded and examined by EM. Scalebar 500 nm. d HA-nsp3-4-V5 induced DMVs and multi-membrane vesicles (MMVs) were quantified. Shown are the number and diameter of DMVs and MMVs in these cells as observed from n = 8 cell profiles per condition. Statistical significance was calculated using ordinary one-way ANOVA. ****p = 1.67453E-49. Data are represented as mean ± SD. Light microscopy image in panel a is a maximum intensity projection.