NS5A but not PI4P can transcomplement replication-deficient replicons with NS5A mutations affecting PI4KIIIα interaction.
A: Experimental setup of transcomplementation experiments: Huh7-Lunet cells bearing either a persistent HCV replicon (I) or constitutively expressing HCV NS3 to NS5A (II) or NS5A (III), respectively, are transfected with luciferase reporter replicons harboring the HIT triple alanine mutation to analyze for conditions rescuing RNA replication. Wt replicons and a replication deficient NS5B mutant (ΔGDD) are used for positive and negative control, respectively. B. Huh7-Lunet cells with persistent replicons (neoJFH) or constitutive expression protein of genotype 2a NS3-5A or NS5A, respectively, were subjected to immunofluorescence analysis. NS5A (red) or PI4P (green), respectively, were detected with specific antibodies and DAPI was used to stain nuclei (blue). C. Quantitation of intracellular PI4P levels by measuring PI4P fluorescence intensity using ImageJ analysis (IntDen read-out) on cells as shown in panel B and on equivalent cell lines harboring genotype 1b (Con1) replicons and proteins. PI4P levels were normalized to the mean value of naïve Huh7-Lunet cells. Data represent mean +/− SD of 35 NS5A positive cells analyzed per condition. D. Detection of NS5A and β-actin in Huh7-Lunet cells with persistent replicons or constitutive expression of NS3-5A or NS5A of genotype (Gt) 1b and 2a, respectively, by western-blot. Equal amounts of NS5A were loaded to judge variations in NS5A phosphorylation. Differences in β-actin, therefore, reflect varying NS5A expression levels. E, F: Wt (dark color), repHIT (medium color) and ΔGDD reporter replicons (light color) of either genotype 2a (panel E, JFH-1, red) or genotype 1b (panel F, Con1ET, blue) were transfected into Huh7-Lunet cell lines harboring a persistent replicon (Repl.) or constitutively expressing NS3-5A (3-5A) or NS5A (5A) of genotype 1b or 2a, as indicated. RNA replication of replicons was determined by measuring luciferase activity in cell lysates 72 h post transfection relative to 4 h to normalize for transfection efficiency. Diagrams show mean values +/−SD of at least two experiments performed in duplicates.