Figure 1.
Antiviral effect of IFN-α and RBV combination treatment using a sub-genomic replicon cell line (S3-GFP) and HCV infected Huh-7.5 cells.
(A) S3-GFP cells were treated with IFN-α and RBV for 72 hours. GFP expression was examined under a fluorescence microscope. (B) GFP positive cells were quantified by flow cytometric analysis. (C) Infected Huh-7.5 cells were treated with IFN-α alone, RBV alone and combination for 72 hours. Renilla Luciferase activity of infected cells was measured and normalized with 1µg of cellular protein. (D) Expression of HCV core protein was measured by immunostaining and (E) core positive cells in five different high power fields (hpf) at 40X magnification were counted under a light microscope. Quantitative assessment of the number of HCV positive cells with mean and standard deviation of the combination treatment are compared.
Figure 2.
IFN-α and RBV each inhibited the internal ribosome entry site (IRES) mediated translation of green fluorescence protein (GFP).
Huh-7 cells were infected with T7-expressing adenovirus. After 2 hrs, HCV IRES-GFP plasmid was transfected and then treated with indicated concentration of IFN-α and RBV. (A) HCV IRES mediated GFP expression was monitored under fluorescent microscopy. (B) Inhibition of GFP expression was further confirmed by Western blot analysis in both IRES and non-IRES mechanisms. β-actin is used as loading controls.
Figure 3.
Different combinations of IFN-α, RBV, and IFN-λ inhibits HCV IRES Rluc mediated translation.
Huh-7 cells were infected with T7-expressing adenovirus. After 2 hrs, HCV IRES-RLuc plasmid was transfected and then treated with indicated concentration of IFN-α, IFN-λ and RBV. The concentration dependent inhibition of Renilla luciferase activity by (A) IFN-α, RBV, and IFN-λ single treatment;(B) Combination of IFN-α + IFN-λ; (C) Combination of IFN-α + RBV and (D) Combination of IFN-λ + RBV.
Figure 4.
Analysis for synergistic effect of IFN-α + IFN-λ, IFN-α + RBV, and IFN-λ + RBV using Calcusyn and MacSynergyII software.
(A) CalcuSyn software analysis show that IFN-α + RBV combination treatment has a very strong synergy antiviral activity against HCV IRES mediated inhibition with combination index, CI<1. (B) IFN-λ + RBV combination treatment also has a very strong synergy antiviral activity with CI<1. (C) IFN-α + IFN-λ treatment are either additive or slightly antagonistic. Three dimensional inhibition plots of (D) IFN-α + RBV, (E) IFN-λ + RBV and (F) IFN-α + IFN-λ treatment against HCV IRES mediated inhibition of Rluc at 95% confidence interval synergy plot. Three dimensional synergy plot of (G) IFN-α + RBV, (H) IFN-λ + RBV, and (I) IFN-λ + IFN-α.
Figure 5.
The distribution of HCV IRES-GFP mRNA in the monosome and polysomes fractions in the Huh-7 cells with (+) and without (-) IFN-α and RBV treatment.
(A) Illustrates the separation of monosome and polysomes along the sucrose gradient fractions (1 to 14). The values indicate the spectrophotometry of optical density of the polysome fractions at 260 nm wavelengths. The point arrow shows the 60S, 80S and separation between monosomes and polysomes in the gradient fractions. (B) Formaldehyde agarose gel electrophoresis and ethidium bromide staining of RNA samples isolated from the corresponding gradient fractions of untreated Huh-7 cells. The 18S and 28S band appears on the gel throughout the fractions and it become more intense on the 80S fractions of the gradient as expected. (C) Shows the distribution of HCV IRES-GFP mRNA in the monosome and polysome fractions by Northern blot analysis using a riboprobe targeted to the 5’ UTR. In the untreated IFN (-) cells the IRES-GFP mRNA efficiently translated and formed polyribosome complexes (Lane 11-14). But the IFN treatment (+) prevented polysome formation on IRES-GFP mRNA (Lane 11-14). (D) In the RBV untreated Huh-7 cells, the IRES-GFP mRNA efficiently translated and formed polyribosome complexes (Lane 11-14). RBV treatment (+) prevented polysome formation on IRES-GFP mRNA (Lane 10-14). (E) Similar experiment was performed where the effect of IFN-α or RBV treatment on the distribution of EGFP mRNA was examined by Northern blotting using RNA probe specific to GFP. IFN-α treatment did not alter the distribution of EGFP mRNA that translates by non-IRES dependent mechanism. (F) Comparison of the relative amount of HCV IRES and non-IRES mRNAs in monosome and polysome fractions in the sucrose density gradient analysis generated from the transfected cells. Density of the Northern blot was measured using an image analysis computer software (Total Lab, TL120). Values are expressed as percentage of total mRNA recovered from the gradient versus the mRNA present in each fraction. (G) The formation of polyribosome of EGFP mRNA was not altered by IFN-α treatment.
Figure 6.
Western blot analysis of polyribosome fractions of HCV IRES-GFP transfected cells.
(A) Untreated HCV IRES-GFP transfected cells showing IMPDH, PKR, pPKR is bound to the ribosome throughout the gradient. (B) PKR induced peIF2α protein is found in the monosome-containing fractions (lanes 1-5) and absent in the higher density polyribosome fractions (lanes 6-10) due to IFN-α treatment. (C) IMPDH, pPKR and peIF2α proteins were found in the monosome fractions (lanes 1-6) but excluded from the high-density polyribosome fractions (lanes 6-10). The level of actin was detected throughout the gradient.
Figure 7.
IFN-α and RBV synergy antiviral mechanism involves the activation of PKR, eIF2α and inhibition of cellular IMPDH.
(A) IFN-α and RBV each induced phosphorylation of PKR and eIF2α. (B) Flow cytometric analysis showing RBV show a dose dependent inhibition of HCV IRES-GFP translation. (C) Inhibition of IMPDH and PKR levels by siRNA prevented RBV antiviral action against HCV IRES-GFP translation determined by flow cytometric analysis. (D) Dose dependent prevention of RBV action due to increasing concentration of guanosine was determined by flow cytometric analysis. (E) IFN-α inhibits HCV IRES-GFP translation. (F) Inhibition of PKR by siRNA prevented IFN-α mediated inhibition of HCV IRES-GFP translation.
Figure 8.
Diagram summarized the proposed IFN-α and RBV synergy antiviral mechanisms against HCV IRES-GFP translation.
IFN-α binds to the cell surface receptor, which activates the cellular Jak-Stat pathway leading to the activation of PKR. The activated PKR phosphorylates the eIF-2α. Phosphorylation of eIF-2α inhibits the recycling of initiation factors and translation initiation. On the other hand, RBV activates the PKR and eIF2 α phosphorylation and inhibits the translation initiation. Ribavirin inhibits HCV IRES translation by inhibiting IMPDH and GTP pool.