Ampliscribe t7 transcription kit

We prepared double-stranded RNA toward both irs and green fluorescent protein (GFP) as described before [11 (link),48 (link)]. Briefly, for dsRNA toward irs we used a fragment of the irs open reading frame cloned using forward and reverse primers 5′-TTTGCAGTCGTTGCTGGTA-3′; 5′-GCTTAAAGCCGGATAACGTG-3′, respectively, into the pCR^® 4-TOPO^® vector as a template for PCR [11 (link)]. PCR primers fused to T7 promoter sequence (underlined)
F: 5′-TAATACGACTCACTATAGGGCGAGCGAACCGGTAGTCGTAAAG-3′ and R: 5′-TAATACGACTCACTATAGGGCGAGCAGTGATCAAACGTGGCTT-3′ were used to produce a 583 bp product. GFP dsRNA was synthesized from AF09833 as a template as described before [41 (link),48 (link),49 (link)]. Underlined segments specify the T7 promotor sequences fused to the Vg-specific primers. We purified the resulting PCR products using the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA.). dsRNA was then prepared using the AmpliScribe T7 transcription kit (Epicentre Biotechnologies, Madison, WI, USA.). We purified the dsRNA with phenol: chloroform extraction and verified product size and purity on a 1% agarose gel. dsRNA was brought to a final working concentration of 10 μg/μL in nuclease-free water [11 (link)].

For in vitro transcription of full-length mRNA for the Luciferase assay, the WT and mutated Phe-IRES plasmids were linearized with XbaI, which cleaves the plasmids after the firefly luciferase coding region. mRNA was transcribed in vitro using the AmpliScribe T7 transcription kit (EPICENTRE) according to the manufacturer. For in vitro transcription of short-length mRNAs, the mutated IRES plasmids were linearized with NarI, which cleaves 33 nt downstream of the ATG start codon of the luciferase coding region.

The plasmid pFL-J6/JFH1 encoding the HCV J6/JFH-1 genome was linearized with
XbaI for in vitro transcription using the Ampliscribe
T₇ transcription kit (Epicentre Technologies, WI). Fifteen
micrograms of J6/JFH-1 RNA/10 cm plate was delivered into Huh-7.5 cells by
electroporation as described previously [4] (link), [35] (link). Cells were passaged every 3–5 days; the
presence of HCV in these cells and the corresponding supernatants were
determined as described previously [4] (link). The cell-free virus was propagated in Huh7.5
cell culture as described previously [4] (link). The expressions of HCV protein in HCV-infected
cells were analyzed using western blot assays. The HCV cell culture supernatant
was collected at appropriate time points and was used to infect naïve
Huh7.5 cells at multiplicity of infection (moi) of 1 for 5–6 h at
37°C and 5% CO₂[4] (link), [35] (link). The viral titer in
cell culture supernatant was expressed as focus forming unit (ffu)
ml^-1, which was determined by the average number of
HCV-NS5A-positive foci detected at the highest dilutions as described previously
[4] (link). The cell culture
supernatant collected from Huh7.5 cells expressing JFH-1/GND (replication
defective virus) were used as a negative control. In most of the experiments,
HCV-infected cells were serum starved for 4 h before harvesting.