Fig 1: Blocking HCV secretion partially rescues intracellular RNA accumulation during PCBP1 knockdown. Three days post-JFH-1T infection (MOI 0.05), the cell culture medium was replaced with media containing Brefeldin A (BFA) or DMSO (vehicle control). Total intracellular RNA, intracellular virus, and extracellular virus were collected at 0 h and 6 h post-treatment. (A) Extracellular (secreted) viral titers and (B) intracellular viral titers from untreated cells (0 h) and after 6h of DMSO or BFA treatment, quantified by FFU assay. (C) Intracellular RNA accumulation relative to the siCTRL condition, assessed by RT-qPCR analysis. All data are representative of three independent biological replicates, and error bars represent the standard deviation of the mean. Statistical significance was calculated by paired t-test (ns, not significant; ** p < 0.01; **** p < 0.0001).
Fig 2: Model for PCBP1’s effects on HCV assembly and secretion. Under wild-type conditions, endogenous PCBP1 limits HCV assembly and secretion, and the majority of intracellular viral RNAs are engaged in translation and viral genome replication. In contrast, PCBP1 knockdown promotes virion assembly and secretion. This results in a shift in viral RNA utilization such that an increased proportion of viral RNAs are engaged in viral particle production, leaving a smaller proportion of viral RNAs to engage in the translating/replicating pool.
Fig 3: PCBP1 knockdown enhances infectious virion secretion even in the absence of RNA replication. (A) Schematic representation of the experimental approach for 2'CMA experiments: two days post-siRNA transfection, Huh-7.5 cells were infected with JFH-1T (MOI = 0.05). Three days post-infection, the cell culture medium was replaced with media containing 2'CMA or DMSO (vehicle control). Total RNA, intracellular and extracellular virus were collected at t = 0, 6 and 12 h post-treatment. (B) Quantitative RT-PCR analysis after 2'CMA treatment. (C) Intracellular viral titers and (D) intracellular virus accumulation rate calculated by linear regression (see also Figure S4). (E) Extracellular viral titers and (F) virus secretion rates calculated by linear regression. All data are representative of three independent replicates and error bars in (B,C,E) represent the standard deviation of the mean. Error bars in (D,F) represent the slopes of the linear regressions ± standard error. Statistical significance was calculated by two-way ANOVA. (ns, not significant; ** p < 0.01; **** p < 0.0001).
Fig 4: PCBP1 knockdown decreases HCV protein expression and intracellular viral RNA accumulation, but increases secreted virus titers. (A) Schematic representation of the HCVcc infectious particles and genomic RNA used in infections. Huh-7.5 cells were transfected with siPCBP1 or siCTRL two days prior to infection with JFH-1T or J6/JFH-1 (MOI = 0.05). Total protein, RNA, and intracellular and extracellular infectious virus were harvested at day 3 post-infection. (B) Viral protein expression analysis by Western blot (ns, non-specific band; arrow, core and NS5A bands). (C) Viral RNA accumulation analysis by Northern blot; the relative HCV/actin signal, expressed as a percentage of the siCTRL HCV/actin signal, is indicated for the siPCBP1 lanes. (D) Viral RNA quantification by RT-qPCR. (E) Intracellular and (F) extracellular (secreted) virus titers, quantified by FFU assay. All data are representative of at least three independent biological replicates, and error bars represent the standard deviation of the mean. Statistical significance was calculated by paired t-test (ns, not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001).
Fig 5: PCBP1 knockdown has no direct effect on HCV entry, translation, genome stability or RNA replication. (A) siRNA-transfected Huh-7.5 cells were spinoculated with luciferase reporter pseudoparticles expressing the HCV E1/E2 glycoproteins (HCVpp) or the VSV-G glycoprotein (VSVpp). In parallel, cells were transfected with a firefly luciferase expression plasmid. Samples were harvested 3 days post-infection/transfection and analyzed by luciferase assay. (B) siRNA-transfected Huh-7.5 cells were electroporated with replication-defective J6/JFH-1-RLuc-GNN RNAs, and luciferase activity was monitored at several timepoints in the two days post-electroporation. (C) Huh-7.5 cells were electroporated with the replication-competent ?core-p7 J6/JFH RLuc RNA, and luciferase activity was monitored at several timepoints post-electroporation. RLuc values were normalized to the early timepoint (3 h), to control for disparities in electroporation efficiency between experiments. All data are representative of at least three independent biological replicates and error bars represent the standard deviation of the mean.
Supplier Page from Abcam for Anti-PCBP1 antibody [EPR11055]