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Supporting Materials
Supporting Figure 1: HCV J6/JFH-1 infection of Huh7.5 cells modulates Ezrin,
Moesin and Radixin mRNA expression.
(A) HCV RNA and (B) HCV NS5A protein analysis of Huh7.5 cells and HCV J6/JFH-1
infected Huh7.5 cells 96h after infection. (C)Huh7.5 cell were infected with HCV J6/JFH1 virus (MOI of 1) over the indicated time points and analyzed for percentage HCV
infected cells measuring HCV NS3 protein by flow cytometry. Data expressed as mean
+ SEM, and p<0.05 was considered statistically significant by the Mann-Whitney test
representative of 3-4 independent repeat experiments.
Supporting Figure 2: Chronic HCV infection modulates EMR mRNA expression in
Huh7.5 cell and induces significant liver injury in HCV infected patients.
(A, B&C) Total RNA was extracted from Huh7.5 cells and chronic HCV J6/JFH-1
infected Huh7.5 cells. Total RNA extracted was subjected to SYBR green-based realtime quantitative PCR for (A) moesin mRNA, (B) radixin mRNA and (C) ezrin mRNA
expression. 18S rRNA used as a normalization control. (D)Total RNA was extracted
from patient liver biopsy samples and subjected to SYBR green quantitative real time
PCR analysis for HCV RNA expression using 18S rRNA as normalization control. (E)
Serum samples were extracted from patient whole blood and analyzed for aspartate
aminotransferase (AST) as a marker for liver injury using a kinetic assay. Results
presented as mean + SEM is representative 4 independently repeated experiments and
from 12 non-HCV patients and 12 HCV-infected patients. p<0.05 was considered
statistically significant by the Mann-Whitney test.
Supporting Figure 3: Chronic HCV infection or transient EMR knockdown in
Huh7.5 cell induces significant stable microtubule formation.
(A) Western blot analysis of Glu-Tubulin expression in chronic HCV J6/JFH-1 infected
Huh7.5 cells (96h). (B) Huh7.5 cells were subjected to siRNA transfection (48h) as
indicated and extracted proteins subjected to western blot for ezrin, moesin and radixin.
(C) Huh7.5 cells were subjected to siRNA transfection (48h) as indicated and western
blot analysis for Glu-Tubulin determined by western blot. β-actin western blot analysis
was used as protein loading control for all blots. Results are expressed as mean + SEM,
and p<0.05 was considered statistically significant by the Mann-Whitney test for 3
independent repeat experiments
Supporting Figure 4: p-SYK interacts with p-Ezrin
HCV J6/JFH-1 virus (MOI of 10) was co-cultured with Huh7.5 cells over the indicated
time course. Cells were then lysed, total protein extracted and analyzed by coimmunoprecipitation (co-IP)-western blotting for phosphorylated SYK (pY323) and Ezrin
(Y353) following p-SYK IP and western blot for p-Ezrin and vice versa. β-actin analysis
by western blot was used as input control for the IP-western blot analysis. A
representative blot of 4 independent repeat experiments is shown.
Supporting Figure 5: Cell toxicity assay for Bay61-3606 and Cytochalasin B.
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(A&B) Cellular toxicity of Bay61-3606 and cytochalasin B in Huh7.5 cells was analyzed
24h after treatment at concentrations indicated using the LDH-cytotoxicity assay
(Abcam cat.#ab65393) according to the manufacturers’ specifications. Staurosporine
(20nM) treatment or complete lysis of Huh7.5 cells was used as internal positive
controls as indicated. Toxicity assay data are expressed as mean + SEM, and p<0.05
was considered statistically significant by the Mann-Whitney test for 3 independent
repeat experiments.
Supporting Figure 6: Western Blot analysis of HCV RNA in Huh7.5 cells after
transient knockdown of Ezrin, Moesin and Radixin.
Real time quantitative PCR analysis of HCV RNA in Huh7.5 cells 48hrs after transient
knockdown of ezrin, moesin, radixin and 24h after HCV J6/JFH-1 infection. Results are
expressed as mean + SEM, and p<0.05 was considered statistically significant by the
Mann-Whitney test for 3 independent repeat experiments.
Supporting Figure 7: Cellular signaling and molecular aspects of ezrin-moesinradixin in HCV infection
HCV E1/E2 protein engagement of CD81 activates down-stream signaling adaptors
initially leading to spleen tyrosine kinase (SYK) activation. Activated SYK in turn induces
ezrin phosphorylation which leads to its cellular redistribution with F-actin. Post-entry,
the HCV RNA replication complex gets associated with and utilities F-actin which is
redistributed with ezrin for initial short distance virus transport towards the endoplasmic
reticulum(ER). Additionally, HCV proteins within the RNA replication complex induces
the down-regulation of moesin and radixin resulting in increased stable microtubules.
The HCV RNA replication complex then hitchhikes on stable microtubules to facilitate its
macromolecular diffusion to the ER for effective infection. Disrupting any of these
signaling events using chemical agents or gene regulation methods impedes effective
HCV infection.
Supporting Material and Methods
Cell lines and HCV J6/JFH-1 Virus
RIG-I-deficient Huh7.5 cells and Huh7.5 cells harboring Con1 HCV full length replicon
(Genotype 1b) (from Dr. Charles Rice, Rockefeller University, New York, NY) were
cultured as previously described (30). Briefly, all cells were maintained in low glucose
DMEM medium supplemented with 10% FBS, 10µg/mL of ciprofloxacin, and nonessential amino acids. Additionally, Con1 full length replicon cells were maintained in
the presence of 500 µg/mL of G418. All cells were kept in a humidified incubator at
37°C in air supplemented with 5% CO2. Infectious and replication competent HCV
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J6/JFH-1 virions were generated by pFL-J6/JFH-1 plasmid transfection into Huh7.5
cells as previously described (31). The pFL-J6/JFH-1 plasmid was a gift of Dr. Charles
Rice and Dr. Takaji Wakita (National Institute of Infectious Diseases, Tokyo, Japan).
Multiplicity of infection of virus supernatants were determined as previously described
(45).
Patient Liver biopsy samples
This study used 12 chronic HCV-infected and 12 HCV-negative patients between the
ages of 31 to 78 years with a mean age of 53.76 years. Liver specimens were obtained
from the NIH liver tissue cell distribution system (LTCDS), [Minneapolis, Minnesota]
[Pittsburgh, Pennsylvania] [Richmond, Virginia], which was funded by NIH contract #
N01-DK-7-004/HHSN26700700004C. Patient genotypes included types 1a and 3.
Liver function biochemical assay
Patient serum aspartate aminotransferase (AST) was determined using a kinetic
method (D-TEK LLC, Bensalem, PA, United States) according to the manufacturer’s
specification.
Recombinant DNA constructs, siRNA constructs and cell transfections
The following expression plasmids were used: human GFP-ezrin (46) (Addgene plasmid
20680), human moesin-GFP (Origene cat. # RG205674) and human radixin-GFP
(Origene cat. # RG207953); pCMV6-XL5-ezrin (Origene Cat. # SC117991), pCMV6XL5-moesin
(Origene
Cat.
#
SC118629),
pCMV6-XL5-radixin
(Origene
Cat.
#SC110995); ezrin siRNA (Santa Cruz Biotechnology cat. # sc-35349); moesin siRNA
(Santa Cruz Biotechnology cat. # sc-35955); radixin siRNA (Santa Cruz Biotechnology
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cat. # sc-36366); Syk siRNA (Santa Cruz cat. # Sc-29501); Control siRNA-H (Santa
Cruz Biotechnology cat # sc-44236). Plasmids and siRNA were transfected into cells as
indicated using FugeneHD (Roche cat # 04709713001) according to the manufacturer′s
specification. Transfected cells after transient knockdown or over-expression of a
specific gene using the indicated plasmids were used 24h post transfections for HCV
J6/JFH-1 infection as indicated.
RNA analysis
Total RNA from cells or liver tissue was purified using the RNeasy® Micro Kit (QIAGEN,
cat. #74004) with on-column DNA digestion according to the manufacturers’
specification.
Isolated
RNA
was
quantified
using
the
NanoDrop
1000
Spectrophotometer (Thermo Scientific). cDNA was transcribed with the iScript™ cDNA
Synthesis Kit (BioRad cat. # 170-8891).
Quantitative Real-Time polymerase chain reaction analysis
Real-time quantitative polymerase chain reactions (RT-qPCR) for transcribed cDNA
were performed using the
CFX96 Real-Time System (Bio-Rad Laboratories, Inc,
Hercules, CA) and iTaq SYBR Green Supermix with ROX (Bio-Rad, cat # 172-5851 )
using 18S rRNA for normalization of relative gene expression of indicated genes. Gene
expression was determined using the comparative delta-Ct method as previously
described(45). The following primers were used:
HCV-Forward Primer: 5′-TCTGCGGAACCGGTGAGTAC-3′(45)
HCV-Reverse primer: 5′-TCAGGCAGTACCACAAGGCC-3′(45)
Ezrin-Forward Primer: 5′-CTGAGACTGCCGTGCTCTTG-3′
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Ezrin-Reverse primer: 5′-GTAAGTTTGTGCTGGTCCATCACT-3′
Moesin Forward primer: 5′-GAGGATGTGTCCGAGGAATTG-3′
Moesin Reverse primer: 5′-GTCTCAGGCGGGCAGTAAA-3′
Radixin Forward Primer: 5′-AATTGTGGCTAGGTGTTGATGC-3′
Radixin Reverse Primer: 5′-GGTGCCTTTTTGTCGATTGGC-3
Antibodies and reagents
The antibodies and reagents used included: anti-NS5A antibody (9E10, a generous gift
from Dr. Charles Rice, Rockefeller University, New York, NY); anti-HCV core antibody
(Abcam Cat # ab2740), anti-NS3 antibody (Abcam cat # ab13830); anti-moesin
antibody (Cell signaling Cat #3146); anti-moesin antibody (Abcam cat # ab52490); antiezrin antibody(cell signaling cat #3145); anti-radixin antibody (cell signaling cat #2636);
anti-Phospho ezrin Tyr 353 (Cells signaling cat #3144); phospho-specific antiezrin(Thr567)/radixin(Thr564)/moesin(Thr558) antibody( Millipore cat #AB3832); antiSYK antibody(cell signaling cat # 2712); anti-phospho-SYK (pY323) (Cell signaling cat #
2715); anti-Syk phospho (pY323) (Epitomics cat # 2173-1); anti-GFP antibody (Abcam
cat # 290, Santa Cruz Biotechnology cat # sc-8334); Anti-tubulin detyrosinated
(Millipore cat. # AB3201); normal rabbit IgG-AC antibody ( Santa Cruz Biotechnology
Cat # sc-2345); normal mouse IgG-AC antibody (Santa Cruz Biotechnology cat # sc2343); Anti-beta Actin antibody [AC-15] (Abcam, Cat # ab6276); protein A/G PLUSAgarose beads (Santa Cruz Biotechnology cat. # sc-2003); goat anti-mouse IgG-HRP
(Santa Cruz Cat. # sc-2005); goat anti-rabbit IgG-HRP (Santa Cruz cat # sc-2004); BAY
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61-3606 hydrochloride hydrate (Sigma cat. # B9685); Cytochalasin B (Sigma cat. #
6762).
Immunoprecipitation and Western Blot analysis
For western blot and immunoprecipitation, treated cells were washed twice in ice cold
phosphate buffer saline then lysed in RIPA (Boston Bio-products cat # BP-115) or
immunoprecipitation(IP) lysis buffer (Thermo Scientific cat. #87788) respectively,
supplemented with protease inhibitor cocktail (Roche Cat. # 11836153001). Cell lysates
were then centrifuged for 5minutes at 2000rpm to remove cell debris. Cell lysate
supernatants containing total cell protein was quantified using the Bio-Rad protein
assay to determine total soluble protein concentration following the manufacturer's
protocol. After quantification, proteins used for immuno-precipitation experiments were
pre-cleared for 1h with protein A/G Plus beads (Santa Cruz cat # Sc-2003) and
immunoprecipitated with specific antibodies as indicated. After at least 4 washes with a
1X wash buffer (BD Bioscience cat # 51-9003739), proteins bound to protein A/G beads
were resuspended in 100µL of IP lysis buffer. Immunoprecipitated proteins were eluted
by boiling samples with added Laemmli’s buffer 10minutes. Proteins were resolved in
10% SDS-PAGE gel under reducing conditions and resolved proteins transferred onto a
nitrocellulose membrane. Membranes were later probed with indicated primary
antibodies followed by an appropriate secondary HRP-conjugated IgG antibody as
previously described (45). Protein bands were developed and visualized using the
Fujifilm LAS-4000 luminescent image analyzer.
Immunofluorescence Microscopy
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Cells were fixed for 20min with 2% paraformaldehyde in phosphate buffered saline and
then permeabilized with a 1X Cytofix/Cytoperm solution (BD Bioscience cat. #555028)
for 30 minutes at 4oC. After washing with a 1X Cytoperm solution (BD Bioscience cat.
#555028), cells were blocked for 1 h with a 1X perm/wash solution (BD Bioscience cat.
# 555028) supplemented 5% w/v bovine albumin (Sigma Aldrich cat # A7906). Specific
proteins as indicated were checked by incubation with HCV NS3 antibody (Abcam cat. #
ab13830) and human Glu-Tubulin antibody (Millipore cat # AB3201) as primary antibody
overnight at 4oC. Cells were then washed three times with a 1X perm/wash solution
followed by incubation with a 1:1000 dilution of corresponding secondary fluorescent
antibody [Alexa Fluor® 594 Goat Anti-mouse IgG (H+L) Invitrogen cat. # A-11005;
Alexa Fluor® 488 Goat Anti-Rabbit IgG (H+L) Invitrogen cat # A-11008] for 1 h at room
temperature. Images were then acquired using an Olympus BX51 fluorescence
microscope and the Nixon NIS-Element BR3.10 software.
Intracellular HCV NS3-protein staining:
Time dependent analysis of the percentage of HCV J6/JFH 1 infected of Huh7.5 cells
using an MOI of 1 was determined by Fluorescence-activated cell sorting (FACS)
analysis of HCV NS3-protein expression. Briefly, control uninfected Huh7.5cells or HCV
J6/JFH-1 infected Huh7.5 cells were harvested after days, 1,2,3,4 and 5 post infection
then washed twice with phosphate buffer saline (PBS). Harvested cells were then fixed
and permeabilized with a 1X Fixation and permeabilization solution (BD Bioscience, cat
# 554714) for 10 minutes at room temperature. After fixation and permeabilization, cells
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were stained with a primary monoclonal mouse-anti NS3 antibody (ViroStat, cat # 1878)
for 1 hour at RT. Alexa fluor 488 conjugated goat-anti mouse antibody (Invitrogen cat #)
was used as secondary antibodies. The percentage of HCV NS3 proteins was
determined by FACS analysis using the BD-LSR II (BD Biosciences) for acquisition and
WinMDI 2.9 for data analysis.
HCVpp infection assays
HCV E1/E2 pseudo viruses were generated using an HIV backbone construct (pNL43.Luc.R-E-) with induced mutations that prevented the expression of HIV envelope
glycoprotein coupled to a luciferase gene to direct luciferase expression in infected
cells. The expression of HCV E1/E2 glycoproteins were provided by co-transfection of
HEK-293T/17 cells with pcDNA-E1/E2-H77-1a with pNL4-3.Luc.R-E- as previously
reported(47, 48). Supernatant containing virus particles was harvested 48 to 72 hrs
post-transfection. HCVpp were used to infect cells as indicated. After 72h of infection
cells were lysed in RIPA buffer supplemented with protease inhibitor cocktail and
luciferase activity determined using the Dual Luciferase reporter assay kit (Promega, cat
#E1910) according to the manufacturers specification and luciferase activity read using
the GloMax®-Multi, Promega.