Download A hepatitis C virus (HCV)

Characterization of the inhibition of hepatitis C virus entry by in vitro generated and
patient derived oxidized low density lipoprotein
Supplemental online material
Materials and Methods
Cell culture and cell lines
Huh-7.5/SR-BI knock down cells (7.5/SR-BIkd) harboring a doxycycline inducible shRNA
against SR-BI were a kind gift from A. Nicosia (Okairos, Basel, Switzerland (1)). Huh-7.5, Huh7.5/SR-BIkd and 293T cells were cultured in DMEM with 10% FCS, non-essential amino acids,
L-glutamine and Penicillin/ Streptomycin. CHO-745 cells were cultured in RPMI with the
same additives.
DNA constructs
For lentiviral transduction the pTRIP (2) and pWPI expression (3) systems were used.
Mutants were generated by standard PCR-based site-directed mutagenesis and confirmed
by sequencing. Detailed cloning strategies and oligonucleotide sequences are available upon
request. Full length HCV reporter constructs pFK_Luc-Jc1, pFK_Luc-Jc1/ΔHVR1 and FlucJc1/G451R encoding firefly luciferase were described recently (4). The intergenotypic JFH1
chimeras were a kind gift of Jens Bukh (Copenhagen University Hospital, Copenhagen,
Denmark; (5)). sE2 of genotype 1a was generated by pcDNA3.1 tpa.E2-661.6xHis construct
(6).
Virus separation over a density gradient
Density gradient centrifugation was performed as described previously (7). Briefly, virus was
separated by stepwise iodixanol gradient (0-40%) cetrifugation at 154.000xg in TH-641
swingout rotor at 4°C using Sorvall Ultra Wx80 centrifuge. 10 Fractions of 1ml volume were
collected and their in vtro infectivity without or with oxLDL treatment were determined.
Density of each fraction was quantified by refractometry.
Soluble E2 and HCVcc binding assays
CHO-745 cells were transduced with SR-BI receptor variants (wt, S101A, E210A, mSR-BI and
CD36). sE2 was produced by transfection of 293T cells with pcDNA3.1 tpa.E2-661.6xHis and
harvesting of supernatant after 48 hours. Transduced CHO-745 cells were incubated with sE2
for 2h. Cells were stained with anti-6xHis-tag (ab9108, Abcam) followed by Alexa-Fluor 488
goat anti-rabbit secondary antibody (Life Technology). We used a BD FACSCalibur flow
cytometer for data acquisition. For HCV binding assay CHO-745 cells transduced with SR-BI
receptor variants were incubated with Jc1-wt for two hours at 37°C. Total RNA war isolated
by Qiagen RNeasy Mini Kit (Hilden, Germany) as indicated in the manufacturers’ manuals.
HCV specific RNA was quantified by quantitative reverse transcription polymerase chain
reaction (qRT-PCR) using a Light Cycler 480 device (Roche, Mannheim, Germany). qRT-PCR
was performed as previously described(4) .
Time course experiments
To evaluate time kinetics of cell entry cells were inoculated with HCVcc on ice for one hour.
Then supernatant was aspirated and replaced with pre-warmed media to start synchronized
cell entry of cell-bound virions (8). Inhibitors oxLDL, anti-SR-BI (C167 (1)) or anti-CD81 (JS-81;
BD Pharmingen, Heidelberg, Germany) were added at defined time points.
SDS-PAGE and immunoblotting
Immunoblotting followed standard protocol except that for detection of NPC1L1 lysis buffer
without 2-mercaptoethanol was used. Detection was performed with primary antibodies
against SR-BI (Novus Biologicals), CLDN1 (clone 2H10D10, Zymed/Life Technology), OCLN
(clone OC-3F10; Zymed), NPC1L1 (New England Biolabs) and β-actin (Sigma Aldrich).
Flow cytometry
Trypsinized cells were stained with anti-CD81 (JS-81; BD Pharmingen) followed by AlexaFluor 488 goat anti-mouse secondary antibody (Life Technology). We used a BD FACSCalibur
flow cytometer for data acquisition.
Serum samples
Sera were from the HepNet serum bank or were collected at either MHH outpatient clinic or
at the University of Frankfurt Hospital. Approval from the institutional ethics boards was
obtained and all patients and healthy controls gave informed consent.
Statistics
As this study shall be deemed to be exploratory, p-values are assessed descriptively. Values
p≤0.05 are referred to as statistically significant and are indicated by an asterisk.
In most experiments results are presented as means ± standard deviation. Student’s t-test
for unpaired data, Fisher’s exact test or one-way ANOVA was used to test for significance as
appropriate and indicated in the text. The 50% tissue culture infectious dose (TCID50) was
calculated as described (8).
Concentration-response-curves were fitted by a nonlinear four-parameter regression model.
50% inhibitory concentrations (IC50) between groups were compared by a t-test for
independent samples.
Time course experiments, where infectivity is measured with inhibitors added at defined
timepoints, were evaluated by a mixed model regression analysis with repeated measures.
Replications at each day of experiment were handled as random effects. Post-hoc
comparisons of time points for each inhibitor were adjusted by Bonferroni.
Statistical analyses were conducted using Microsoft Excel (Micorsoft, Redmond, WA, USA)
and SAS 9.3 (SAS Institute Inc., Cary, NC, USA) and R 2.13.2 including the Bioassay Analysis
package.
Legends for supplemental online figures and tables
Supplementary Figure 1: Inhibition of different gradient fractions of Jc1 by oxLDL.
A preparation of Jc1 was separated according to density over a gradient. Then infectivity in
the different fractions without oxLDL (black line) and after the addition of oxLDL (50µg/ml)
(grey dots) was measured. A representative of three independent experiments is shown.
Supplementary Figure 2: Modulation of cellular SR-BI expression levels does not affect
oxLDL inhibition of HCV entry.
Naïve Huh-7.5, Huh-7.5 overexpressing SR-BI, uninduced Huh-7.5 SR-BI knock-down (7.5/SRBIkd) cells, induced 7.5/SR-BIkd cells and induced 7.5/SR-BIkd cells transduced with SR-BI
were inoculated with virus with or without oxLDL (50 µg/ml) for four hours. Means +/- SD
from a representative experiment done in duplicate is shown.
Supplementary Figure 3: oxLDL inhibiton in the presence of SR-BI receptor variants .
SR-BI mutants where 5 amino acid residues in the putative oxLDL binding site were replaced
with alanine were expressed in Huh-7.5 cells and Fluc-Jc1 infectivity with or without oxLDL
(50 µg/ml) was measured.
Supplementary Figure 4: HCV and sE2 binding to SR-BI variants.
(A) CHO-745 cells were transduced to express the indicated SR-BI variants and then
incubated with Jc1. Bound virus was quantified after washing repeatedly by quantitative
PCR. (B) sE2 bearing a His-tag were incubated with CHO-745 cells expressing the indicated
SR-BI variants for 2 hours. sE2 binding was quantified by FACS as described in the methods
section.
Supplementary Fig. 5: Lack of correlation between Mercodia and Immundiagnostik oxLDL
ELISA systems.
oxLDL content in sera from the HCV cohort as determined with the two commercially
available ELISA systems offered by Mercodia and Immundiagnostik.
Supplementary Table 1: Characteristics of HCV positive patients.
Supplemental online references
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