Supporting Material
ELISA titration of plasma anti-HCV E2 Abs. Anti-HCV E2 Abs in these plasma
samples were titrated by an ELISA assay modified from a previous report19. Briefly, 96well microplates were coated with 100 µl of purified recombinant HCV E2
(MyBioSource, San Diego, CA) at 1 µg/ml in the CB2 Antigen Plate Coating Buffer
(ImmunoChemistry Technologies, Bloomington, MN) overnight at 4oC. Plates were
washed six times with wash buffer (phosphate buffered saline or PBS at pH7.2 plus
0.05% Tween-20), and blocked with 2% bovine serum albumin (BSA) in wash buffer at
4oC for 2 h. Plates were then washed three times with wash buffer. Each plasma sample
was serially diluted fourfold starting from 1:1000 to give dilutions of 1:4000, 1:16000,
1:64000, 1:256000, and 1:1024000 in wash buffer containing 2 % BSA. Each dilution
(100 µl) was added to the well and incubated at 37 °C for 1 h. Four HCV-negative
plasma samples were diluted in the same pattern and added as negative controls. After
washing, 100 µl of 1:50000-diluted horseradish peroxidase (HRP)-conjugated antihuman whole IgG monoclonal Ab (Millipore, Temecula, CA) was added per well and
incubated at 37 °C for 1 h. Plates were washed and substrate was added, followed by
incubation at room temperature (RT) in darkness for 20 min. The reaction was stopped
by 2 M sulfuric acid and the absorbance of each well was read at 450 nm. The cut-off
was determined as the mean absorbance value of four plasma samples from HCVnegative donors. The Ab titer was determined as the highest dilution with an absorbance
value above the cut-off.
Real-time qPCR. Real-time qPCR was performed in an ABI Prism 7000 Sequence
Detection SystemApplied Biosystems7000 (Applied Biosystems, Foster City, CA). The
plasmid pFL-J6/JFH, which contains the full-length cDNA of JFH-1 isolate cloned into
the plasmid vector pCR2, was a gift from Apath (Brooklyn, NY) and Dr. Charles M. Rice
at Rockefeller University (New York, NY) and was used to establish standard curves in
each real-time qPCR performance for quantitative analysis of HCV RNA copy number.
The number of construct copies in the plasmid solution was calculated, based on
plasmid vector size plus insert size of JFH-1 cDNA. A plasmid-based calibration curve
was generated with 10-fold serial dilutions of plasmid pFL-J6/JFH; to control pipetting
steps, three 10-fold serial dilutions were prepared, and concentrations were checked by
real-time qPCR. The quantification was linear over a range of 0 to 10 5 starting plasmid
copy numbers, and the detection limit was five copies per reaction. Each sample,
control, and standard were set up in triplicate containing 10 l of PCR-grade water,
12.5 l of 2 × Sybr Green Master Mix, 0.15 pmol each primer, and 2 l of sample cDNA
or diluted plasmid pFL-J6/JFH. PCR cycling conditions were 95 °C for 10 min, followed
by 40 cycles of 95 °C for 15 s, 60 °C for 1 min, and then a final extension step at 72 °C
for 10 min. The fluorescence intensity of SYBR green was measured automatically
during the annealing steps. At the end of each run, a melting curve analysis was
performed. The PCR primers used: 5’-CACGCAGAAAGCGCCTAGC-3’ (forward) and
5’-CAAGCGCCCTATCAGGCAGT-3’ (reverse).
As a control, a plasma vector pcDNA3.1 (Invitrogen, Carlsbad, CA) inserted with
full-length HIV-1 gag cDNA was generated in our lab and was used to establish
standard curves in real-time qPCR for quantitative analysis of HIV-1 RNA copy number.
The HIV-1 qPCR primers used were: 5’-GGCAAGAGTTTTGGCTGAAG-3’ (forward)
and 5’-CACATTTCCAACAGCCCTTT-3’ (reverse).
Western blot. PHHs and Huh7.5.1 cells treated or untreated with PI-PLC were washed
3 times with PBS, followed by lysis with 1X cell lysis buffer (Cell Signaling Technology,
Danvers, MA). Protein extraction was subjected to NuPAGE Novex high-performance,
precast gel (Invitrogen, Carlsbad, CA) and blotted onto polyvinylidene difluoride
membranes (Millipore, Billerica, MA). After blocking, the membranes were incubated
overnight at 4oC with BRIC229 or mouse anti-human -actin Ab (Biolegend, San Diego,
CA) at 1 g/ml. After washing, blots were visualized with the appropriate HRPconjugated secondary Abs and an ECL detection system (Pierce, Rockford, IL).
HCV particles purified from cell-free supernatant of JFH-1-infected Huh7.5.1 cells
or from plasma samples of HCV-infected patients (Pt1 to Pt5, Table 1) were lysed with
15 l of 1% Triton X-100. The lysed virus solution was mixed with 5 l of 4 X NuPAGE
SDS sample buffer (Invitrogen, Carlsbad, CA) and boiled for 5 min, and then subjected
to Western blot as described above.
Hemolytic assay of plasma complement activity. Complement activity of the plasma
samples from HCV-infected patients (Pt28, Pt42, Pt49, Pt84, Pt99 and Pt369)(Table 1)
was determined by a hemolytic assay as described in our previous report 6. Briefly, 5 x
106 human erythrocytes from a healthy blood donor were resuspended in 160 l of PBS,
and then were incubated with 20 g of BRIC229 at RT for 30 min to neutralize CD59
function. PBS and IgG at 20 g were included as background and isotype controls,
respectively. After washing, cells were incubated with rabbit anti-human erythrocyte
polyclonal Abs (Fitzgerald, Acton, MA) at 1 g/ml, followed by an addition of 40 l of
either diluted plasma or heat-inactivated plasma from HCV-infected patients. Samples
were then incubated at 37°C for 40 min. The amount of hemoglobin released from lysed
erythrocytes was determined by measuring the absorbance of the supernatant at 414
nm, and the percent lysis was calculated as follows: [(experimental OD414 – PBS
OD414)/(total lysis OD414 - PBS OD414)] X 100%. Total lysis was obtained by adding
pure water to the erythrocytes. Heat-inactivated plasma was included as a negative
control of complement activity. All assays were performed in triplicate.
Infectivity assay. The number of infectious HCV virions remaining in the virolysis
solution samples of the ADCML experiments was determined by a HCV focus-forming
unit (FFU) assay as described previously with slight modifications (Yi. 2008, J Virol 81,
629-638). Briefly, 1 ml of 10-fold serially diluted samples (the dilution factors generally
ranged from 1:10 to 1:1000) were inoculated onto fresh Huh7.5.1 cells seeded in 24well plates. After 2 h of incubation at 37°C, cells were extensively washed with complete
RPMI 1640 medium to remove unbound HCV particles and were cultured in 1 ml of
fresh complete RPMI1640 medium per well at 37°C for 3 days in a 5% CO 2 incubator.
Cells were then fixed in 2% paraformaldehyde at RT for 30 min, blocked for 60 min in a
blocking buffer (3% BSA, 0.3% Triton X-100, 10% FBS in PBS), followed by incubation
with mouse anti-HCV core monoclonal Ab (Clone: C7-50, Thermo Fisher Scientific,
Rockford, IL) at 1:500 dilution. After 1 h incubation at RT, cells were extensively washed
with PBS and then incubated with an Alexa Fluor 488 goat anti-mouse IgG (Invitrogen,
Carlsbad, CA) at 1:500 dilution for 30 min. Following PBS washes, the numbers of
fluorescent foci per well at appropriate dilutions were counted. Viral infectivity titers are
expressed as numbers of FFU per milliliter of input supernatant (FFU/ml), determined
by the average number of HCV core-positive foci in triplicate samples at the highest
HCV-positive dilution.
Figure legend
Fig. S1. Infection of Huh7.5.1 cells with GFP-Ad5. Huh7.5.1 cells were infected with
1, 2, 10 MOI of GFP-Ad5 overnight (16 h) at 37oC in a 5% CO2 incubator. Cells were
harvested by scratching and centrifugation. Cells were then fixed in 2%
paraformaldehyde and subjected to FACS analysis. Uninfected Huh7.5.1 cells were
used as a negative control of infection. Data were analyzed using FlowJo software.
Solid gray histogram represents uninfected cells. Histograms with dot, thin, and thick
lines represent 1, 2, 10 MOI of Ad5 infections, respectively.
Fig. S2. Determination of the number of infectious HCV virions. HCV focus-forming
unit (FFU) assay was performed to determine the number of infectious HCV virions
remaining in the virolysis samples of the ADCML experiments. One ml of 10-fold serially
diluted samples was inoculated onto fresh Huh7.5.1 cells seeded in 24-well plates. After
2 h of incubation at 37°C, cells were extensively washed and were then cultured in 1 ml
per well of fresh complete RPMI1640 medium for 4 days. Cells were then fixed and
stained with mouse anti-HCV core monoclonal Ab (Clone: C7-50, Thermo Scientific,
Rockford, IL), followed by incubating with an Alexa Fluor 488 goat anti-mouse IgG
(Invitrogen, Carlsbad, CA). The numbers of fluorescent HCV foci per well at appropriate
dilutions were counted. Viral infectivity titers are expressed as numbers of FFU per
milliliter of supernatant (FFU/ml), determined by the average number of HCV corepositive foci in triplicate samples at the highest HCV-positive dilution. Pictures were
taken using a fluorescence microscope by double exposure of the same fields with or
without filters for Alexa 488.
Fig. 3S. Hemolytic assay of plasma complement activity. Complement activity of the
plasma samples from HCV-infected patients (Pt28, Pt42, Pt49, Pt84, Pt99 and Pt369)
was determined by a hemolytic assay. Undiluted plasma samples or plasma samples
diluted at 1:2, 1:4, 1:8, and 1:16 were tested. The amount of hemoglobin released from
lysed erythrocytes was determined by measuring the absorbance of the supernatant at
414 nm, and the percent lysis was calculated as follows: [(experimental OD414 – PBS
OD414)/(total lysis OD414 - PBS OD414)] X 100%. Total lysis was obtained by adding
pure water to the erythrocytes. Heat-inactivated plasma was included as a negative
control of complement activity. All assays were performed in triplicate. Each value
represents the mean  SD of triplicate determinations. The experiments were repeated
twice with similar results. P, plasma; HIP, heat-inactivated plasma.