Regulation of Hepatitis C Virion Production via Phosphorylation of the NS5A Protein
Figure 5
Levels of CKII α and α′ subunits affects infectious virus production.
A). Western blots of cells electroporated with CKI α, CKIα′, or both CKIα and CKIα′ siRNAs are indicated and probed with antibodies recognizing CKI α, CKIα′, or both CKIα and CKIα′. A time course of 72 hours post siRNA delivery is shown. β-actin loading control panels are shown in the lower row of images. B). Cell viability, expressed as percent viability of cells electroporated with no siRNA, for cells electroporated with irrelevant siRNA (IRR), CKI α (CKIA), CKIα′ (CKIA′), or both CKIα and CKIα′ (MIX) as determined by ATP activity assay. C). CKI α and CKIα′ messenger RNA levels, relative to GAPDH in arbitrary units. Dark lines and text cues indicate which real time PCR probe set was used for each set of bars. Designations under bars indicate the siRNA delivered to the cells. Designations of siRNAs are as described in B. D). HCV RNA replication 72 hours post siRNA treatment for cells treated with various siRNAs (labels below columns) and infected with HCV viruses indicated below the dark bars (J6/JFH-1 or J6/JFH-1 with the DEED mutation). Column colors further highlight the different viruses used for these experiments, with light gray for J6/JFH-1 infected samples, and dark gray for J6/JFH-1 DEED infected samples. E). Infectivity release 72 hours post treatment with the indicated siRNAs (column labels) for cells infected with viruses indicated by dark bars (J6/JFH-1 or J6/JFH-1 with the DEED mutation). Column bar colors are as designated in D. F). CKII α and CKIIα′ catalytic subunit over-expression (OE) relative to cells transfected with an empty vector (M). Western blots using CKII α and CKIIα′ antisera as indicated. β-actin Western blot of samples shown in over-expression samples as a loading control.