Fig. 4: PA accumulation on HCV DMVs and autophagy-related structures and role of alternative PA synthesis pathway for HCV replication.

a PA accumulation at NS5A containing structures. Huh7-Lunet/T7 cells were transfected with a construct analogous to the one in Fig. 3a, but containing a mCherry insertion in lieu of EGFP, along with an EGFP-tagged wildtype (WT) or mutant (4E) PA sensor (construct pTM-EGFP-PABD-Raf1-WT or −4E). Twenty-four hours later, EGFP-PABD and NS5A-mCherry were visualized by fluorescence microscopy. White boxes indicate regions magnified in the lower right of each panel. Two biologically independent experiments showed similar results. b Top panel: Alternate PA biosynthesis pathways via lysophosphatidic acid (LPA) or phosphatidylcholine (PC) catalyzed by AGPATs or PLDs, respectively. Bottom panel: Huh7-Lunet/T7 cells were electroporated with in vitro transcripts of a subgenomic HCV reporter replicon encoding the firefly luciferase. Four hours after transfection, different concentrations of PA synthesis inhibitors were added to the cells and luciferase activities were analyzed 48 h after electroporation. Graph shows average and SD from three independent experiments. Cell viability determined by CellTiter-Glo luminescent assay is indicated with the red line. Data are represented as mean ± SD from three biologically independent experiments. c PA recruitment to autophagy-related structures in selective and non-selective autophagy. Top panel: Huh7-derived cells expressing EGFP-PABD-Raf-1 were incubated in growth medium (top left panels) or in serum-free medium with 200 nM BafA1 (top right panels) for 3 h. Cells were fixed and stained with a LC3 specific antibody. Bottom panel: For selective autophagy, mCherry-tagged Parkin was co-expressed with EGFP-PABD-Raf1, followed by incubation with 10 µM Valinomycin to induce mitophagy. Cells were fixed after 3 h, and EGFP-PABD and mCherry-Parkin were visualized by fluorescence microscopy. Images in panels e and g are maximum intensity projections. Two biologically independent experiments showed similar results.