Supplementary Materials and Methods
Quantification of HCV RNA level by Real-Time PCR (RT-qPCR): Assay was
performed by using a modified published protocol.16 Briefly, 2 µg of cellular
RNA or total RNA from 1 ml culture supernatant was used to amplify the 5´-UTR
region
of
HCV
genome
using
sense
primer
5΄-
TCTTCACGCAGAAAGCGTCTA-3΄(60-80; HCV/S) and anti-sense primer 5΄CGGTTCCG CAGACCACTATG-3´ (157-138; HCV/AS). The probe (5΄-/56FAM/TGAGTG TCG/ZEN/TGCAGCCTCCAGGA/3IBκFQ/-3΄) labeled at the 5´
end with a FAM (6-carboxyfluorescein) fluorophore reporter molecule and ZENIowa Black FQ (IBFQ) double quenchers was used to reduce the background and
increase signal than traditional dye-quencher combinations (Integrated DNA
Technologies Inc., San Jose, CA). RT-qPCR assay was performed in 20 µl
containing 10 µl of iQ supermix (Bio-Rad Laboratories Inc., Hercules, CA), 0.25
µM of each primers and probe and 4 µl of cDNA product obtained from the RT
reaction. The amplification was carried out using the standard program that
includes: the first cycle at 48oC for 30 minutes, 95oC for 10 minutes, then
additional 45 cycles each cycle consists of a denaturation step at 95°C for 15
seconds, annealing and extension step at 60oC for 1 minute. HCV cDNA
standards were used starting at 109 copies of virus and decreasing in 10-fold serial
dilutions. Amplification, data acquisition, and analysis were performed on a
CFX96 Real-Time instrument using CFX manager software (Bio-Rad
Laboratories Inc., Hercules, CA).
siRNA-nanosome treatment of HCV infected cells: Huh-7.5 cells (1.2х105
cells) were infected with 1 ml of culture supernatant containing either JFH1-GFP
or JFH1-∆V3-Rluc virus (MOI 0.1) for 24 hours. On the following day, the
infected culture was washed with PBS and then incubated with freshly prepared
media. To screen the antiviral efficacy of 13 designed siRNAs, 100 pmole of
individual siRNA was transfected and after 72 hours measured the luciferase
activity. The complete clearance or the development of an escape mutant virus
was assessed by infectivity assay. Cells received two consecutive treatments
within 5 days were then cultured up to 30 days. The effect of the siRNA treatment
on the replication of the full-length virus in the culture was measured by NS5ARluc activity. Total protein concentration was measured by the Bradford method
and the luciferase activity was expressed in per micro-gram of total protein.
Western blots and immunostaining for HCV-core were performed using
monoclonal antibody (Thermo Fisher Scientific, Logan, UT). The HCV-RNA
level in the infected cells was measured by RT-qPCR.
Escape Mutant Analysis: Total cellular RNA was isolated from resistant as well
as untreated replicon cell lines by the GITC method. The HCV RNA sequences in
the sub-genomic RNA clone (Accession No AB114136), starting from nucleotide
10 to nucleotide 496 that include the siRNA targets, were amplified by reverse
transcription hemi-nested PCR reaction using oligonucleotides 5΄-UTR/10S: 5΄AATAGGGGCGACAC
TCCGCCA-3΄,
ATCAGAGCAGCCGATTGTCTG-3΄
and
G-418/99AS:
primer
5΄-UTR/43S:
5΄5΄-
CTGTGAGGAACTACTGTC-3΄. The amplified DNA fragment was purified
from an agarose gel and cloned into the plasmid pCR2.1 using the TA cloning kit
according to the manufacturer’s recommendation (Invitrogen, Carlsbad, CA).
Three plasmid clones from each cell line were sequenced using the M13 forward
and reverse primers then sequences were edited, aligned and analyzed using
Bioedit version 7.0.4.1.50 Presence of the escape mutant clone was determined by
comparing the DNA sequence of the mutant clone with the wild type replicon.
Appearance of escape mutant virus in the infected cell culture was determined by
muticycle infectivity assay using the Renilla luciferase.
Immunostaining for HCV core: Infected Huh-7.5 cells with or without siRNA
treatment were mounted onto a glass slide via the cytospin method. The cells were
washed twice with 10 mM PBS pH 7.4 (Sigma-Aldrich, St Louis, MO) for 5
minutes. The cells were fixed in chilled acetone for 15 minutes and then
permeabilized by treatment with Reveal Decloaker RTU (Biocare Medical, RV
100) for 25 minutes at boiling point. Slide were then cool down to room
temperature for 25 minutes. Blocking was performed utilizing Background Sniper
(Biocare Medical, BS966) for 10 minute at room temperature. The cells were
incubated with monoclonal anti-core antibody (Thermo Scientific, Pierce hepatitis
C virus core antigen specific mouse monoclonal antibody, Ma1-080) at 1:200
diluted with Da Vinci Green Diluent (Biocare Medical, PD900) for 1 hour at
room temperature. Following the primary antibody incubation, the cells were
washed 3 times in Tris Buffered Saline ( pH 8.0), and incubated with MACH 4
mouse probe (Biocare Medical, UP534) for 10 minutes. After mouse probe
treated, the cells were incubated with MACH4 HRP Polymer (Biocare Medical,
MRH534) for 30 minutes, and washed the cells with TBS 3 times. Next, the cells
were treated with diaminobenzidine (DAB) chromogen (Dako Cytomation,
Carpinteria, CA) for 5 minutes. The slides were counterstained with hematoxylin
for 30 seconds and Tacha’s bluing Solution (Biocare Medical, HTBLU) for 30
seconds, dehydrated, mounted and observed by light microscopy.