Article Figures & Data
Figures
- FIG 1
Three antibodies specific to NS5A phosphorylation at S222, S235, and S238 were characterized. (A) Partial peptide sequences of the LCS I region of NS5A from six major HCV genotypes. The UniProt accession numbers are the following: 1a, P27958 ; 2a, Q99IB8 ; 3a, Q81258 ; 4a, O39929 ; 5a, O91936 ; and 6a, O39927 . Phosphorylation events at S222, S235, and S238 were identified with high confidence scores in a previous study (22). (B) Dot blot tests for the specificity of the three antibodies. Synthetic peptides with or without phosphorylation at the designated serine residues (numbered) were dotted on nitrocellulose membranes before detection with the antibodies.
- FIG 2
NS5A S235 phosphorylation and S238 phosphorylation showed parallel time-dependent increases in the infected cells. (A to F) Immunoblots for NS5A phosphorylation at S222, S235, and S238 in the HCV-infected Huh7.5.1 cells at an MOI of 0.001 (A to C) or 0.01 (D to F). ß-Actin served as a loading control. (G and H) Line plots summarizing total NS5A and NS5A phosphorylation at S235 and S238 from three independent experiments. Relative protein abundance was quantified with the Li-Core Odyssey scanner and software. Values are means ± standard errors (SE). (I) Pearson's correlation analysis for S235 and S238 phosphorylation. (J) Dot blot analysis of the S222 phosphorylation-specific antibody for detection interference by phosphorylation at S225 and S229. Phosphorylated serine residues are numbered.
- FIG 3
NS5A S235 phosphorylation was a prerequisite to S238 phosphorylation. Immunoblots for NS5A phosphorylation at S222 (A), S235 (B), and S238 (C) in the HEK293T cells. The HEK293T cells were transfected with various NS3-NS5A expression constructs: the wild types (WT) and single-, double-, or triple-alanine mutations at S222, S235, or S238 for 2 days before the cell lysates were collected for immunoblotting. ß-Actin staining served as a loading control. (D) A bar diagram summarizing the immunoblotting results from three experiments. Values are means ± SE (n = 3). The asterisk indicates significance: P value of <0.05 by Student's t test. (E and F) Representative immunoblot images (E) and summary (F) for NS5A phosphorylation at S235 and S238 in the HEK293T cells transfected with an S235D mutant NS3-NS5A expression construct.
- FIG 4
NS5A S235 phosphorylation could occur alone, while S238 phosphorylation occurred only with S235 phosphorylation on the same NS5A molecule. (A) Immunoblots for NS5A and NS5A phosphorylation at S235 (left) or S238 (right) in the immunoprecipitate (IP) of the S235 phosphorylation-specific antibody. (B) Immunoblots for NS5A and NS5A phosphorylation at S235 (left) or S238 (right) in the immunoprecipitate of the S238 phosphorylation-specific antibody. In, input, i.e., HCV (J6/JFH1)-infected Huh7.5.1 cell lysate; Unb, unbound; W, wash; E, eluate.
- FIG 5
S235- and S238-phosphorylated NS5A colocalized with double-stranded RNA. Confocal immunofluorescence micrographs of NS5A phosphorylated at S222, S235, or S238 costained for dsRNA (A) or lipid droplet (B) in the Huh7.5.1 cells infected with HCV for 6 days. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (in blue). Boxes indicate the areas enlarged. Mander's colocalization coefficient for S235 and S238 phosphorylated NS5A with dsRNA (C) and lipid droplet (D). Numbers of cells analyzed are indicated.
- FIG 6
NS5A S235 phosphorylation is essential for HCV replication. Confocal immunofluorescence micrographs of NS5A phosphorylated at S235 or S238 costained for dsRNA (A) or lipid droplet (B) in the wild-type (WT) JFH1 replicon-transfected Huh7.5.1 cells. (C and D) Confocal immunofluorescence micrographs of NS5A phosphorylated at S235 or S238 costained for dsRNA in the Huh7.5.1 cells transfected with S235A (C) or S238A (D) mutant replicon. Nuclei were stained with DAPI (in blue). Boxes indicates the areas enlarged.
- FIG 7
CKIα was responsible for NS5A S235 and S238 phosphorylation. Immunoblots for PI4KIIIα (A), CKIα (D), NS5A S235 phosphorylation (B and E), and NS5A S238 phosphorylation (C and F) in the T7-Huh7 cells. Control (shCon), PI4KIIIα knockdown (shPI4KIIIα), or CKIα (shCKIα) knockdown T7-Huh7 cells were transfected with the NS3-NS5B expression construct for 1 day before the immunoblot analysis. The bar diagrams summarize results from three experiments. Values are means ± SE normalized against values under the control conditions.
- FIG 8
CKIα inhibition reduced NS5A phosphorylation at S235 and S238. Immunoblots for NS5A phosphorylated at S235 (A) and S238 (B) in T7-Huh7 cells. The cells were transfected with an NS3-NS5B expression construct and treated with the CKIα inhibitor D4476 for 1 day prior to immunoblotting. β-Actin served as a loading control. (C) Summary for three independent experiments. Values are means ± SE, normalized against values of the vehicle control (0 μM). Asterisks indicate significance: P value of <0.05 by Student's t test against values of the vehicle control.
