Alpha interferon inhibits translation mediated by the internal ribosome entry site of six different hepatitis C virus genotypes, by S. Hazari, A. Patil, V. Joshi, D. E. Sullivan, C. D. Fermin, R. F. Garry, R. M. Elliott and S. Dash
Journal of General Virology vol. 86, part 11, pp. 3047 - 3053
Supplementary Table S1.
Effect of cytokines on GFP expression in HCVs of different genotypes.[ PDF file ] (34 KB)
Supplementary Fig. S1
Flow analysis showing the effect of IFN-α2b on the expression of GPP. Huh-7 cells transfected with either IRES expression plasmid (HCV-GFP) or non-IRES expression plasmid (pEGFP-N1) using a two-step transfection procedure. Following transfection, cells were treated with different concentrations of IFNs. After 24 hours, cells were harvested, treatment with trypsin-EDTA, washed and resuspended in phosphate-buffered saline, and subjected to flow cytometry. Analysis was performed using CELL QUEST computer software. IFN-α2b inhibited translation of GFP in a concentration-dependent manner in Huh-7 cells of HCV-IRES genotypes 1a, 2a and 3a. IFN-α2b did not inhibit GFP when expressed by a non-IRES mechanism from a pEGFP-N1 clone (26–27 %).
[ PDF file ] (574 KB)
Supplementary Fig. S2
RPA demonstrating intracellular GFP mRNA levels in Huh-7 cells 24 hours after transfection. Huh-7 cells were seeded in 12-well plates. On the following day, cells were incubated with 1 ml D-MEM, 2 % FBS, 2 µl replication defective adenovirus expressing T7 RNA polymerase, and then transfected with 2 µg HCV-GFP chimeras using the FuGENE 6 transfection reagent. Immediately after transfection cells were treated with different concentrations of IFN diluted in serum-free media. RNA extracts were prepared by the guanidine isothiocyanate (GITC) method. Total RNA (20 µg) was subjected to a RPA for positive-strand GFP mRNA using a negative-strand probe targeted to the GFP mRNA. The bands show the presence of a 296 nt protected fragment in HCV-GFP transfected Huh-7 cells treated with different concentrations of IFN.
[ PDF file ] (1122 KB)
