Sureau, C., Salisse, J. Supporting Information
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EXPERIMENTAL PROCEDURES
Reagents: Tris-(2-carboxyethyl) phosphine (TCEP) was from Toronto Research Chemicals, Inc.
IGEPAL
CA-630
(Nonidet-P40)
was
from
Sigma.
ETI-MAK-4
HBsAg,
enzyme-linked
immunosorbent assay (ELISA) kit was from Dia-Sorin. One ml HiTrap Heparin HP columns were
from GE Healthcare. Heparin (H3149) and Dextran sulfate (D8906) were from Sigma. Myristoylated
preS1-specific peptide (pre-S1/2-48myr) was obtained from S. Urban (University Hospital
Heidelberg, Germany).
Purification of HBV particles. HBV particles (Genotype D, ayw2 subtype) were purified from a
human serum using sucrose density gradient centrifugation (1). Fifteen ml of clarified serum
sample were layered onto a 2-step sucrose gradient consisting of 2 ml 60% sucrose (w/v) and 20
ml 30% sucrose in TNE. Centrifugation was conducted in a SW 28 rotor at 27 000 rpm for 24 hr at
10°C. After centrifugation, 3-ml fractions were collected from the bottom, and each fraction was
analyzed by western blotting for detection of HBV envelope proteins, and by SDS-PAGE and
Coomassie blue staining. Fractions 1 and 2, which contain more than 90% of the total HBV
particles while excluding most of the plasma proteins, were pooled. Sucrose was removed by
ultrafiltration (Amicon Ultra Filter devices, 100 000 Da cutoff) with TNE buffer. Volume was
adjusted to 1.5 ml with TNE corresponding to a 10 x concentration of the initial serum volume.
Heparin-affinity chromatography analysis of SVPs. Heparin-binding assays were also
conducted on a panel of SVPs mutants, or TCEP- or NP-40-treated HDV particles. In these cases,
we made use of a 96-well filter plate (Whatman Schleicher&Schuell) to create 400 µl bed columns
of Heparin-Sepharose 6 Fast Flow. After three washes of each column with binding buffer, sample
was passed through the column by gravity flow, it was reloaded once, and each well was washed 5
times with binding buffer. Elution was performed with 400 µl of 20 mM Tris-HCl pH: 7.4, 500 mM
NaCl and 400 µl-volume eluants were collected in a 96-well plate and analyzed using a preS2ELISA as described (2).
HDV binding/uptake assay. HepaRG cells were exposed to inoculum for 16 hr as described (3).
After exposure, cells were washed 3 times with PBS, and lysed in RNA extraction buffer
(PolyATtract® System 1000 Promega) according to the manufacturer's procedure. After lysis, 50
pmol of a biotinylated oligonucleotide specific for genomic HDV RNA (nt 54-84 HDV genotype-1)
and 5 pmol of biotinylated oligo(dT) were added before incubation at 70°C for 5 min and capture
with 200 µl of Streptavidin MagneSphere® Paramagnetic Particles (Promega). Captured RNAs
were analyzed for genomic HDV RNA and GAPDH mRNA (3). For the experiment presented in Fig.
2B, total RNA was extracted after inoculum removal and washes, and analyzed directly by
Northern blotting.
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Treatment of HDV particles with TCEP and NP-40. Ten µl of a 100 X preparation of HDV
particles were mock treated or treated with 1:2 dilutions of 0.5 mM TCEP, or 0.1% NP-40 for 1 h at
37°C. After treatment, preparations were diluted 100-fold in 20 mM Tris-HCl pH: 7.4, 150 mM NaCl
before being subjected to a preS2-ELISA as described (2) or to an a-determinant-specific ELISA
using monoclonal anti-HBsAg antibody A1.2 (4). HDV RNA was detected by Northern blot analysis
and used as a marker of HDV particles integrity. Note that antibodies (R257) used in the "pre-S2
ELISA" are specific for a linear epitope in the pre-S2 domain of M- and L-HBsAg. The ELISA tests
consisted in coating each well of a 96-well Maxisorp plate (Nunc) with 2 µl of each preparation in
100 µl of 50 mM NaHCO3 buffer, pH: 9.6 as described (2), before detection with anti-pre-S2 or
anti"a" mAb A1.2.
Native agarose gel electrophoresis. Precipitation of viral particles from cell culture fluids was
achieved by adding PEG 8000 to a final concentration of 12% followed by incubation at 4° C for 1
h. The precipitates were collected by centrifugation at 5000 rpm for 1 hr and dissolved in TNE (10
mM Tris-HCl pH: 7.4, 1 mM EDTA, 140 mM NaCl). Free viral DNA was removed from the
suspension by the addition of 200 IU/ml of DNase I (Roche) and incubation for 1 hr at 37° C. The
samples were subjected to electrophoresis through a 0.8% agarose gel, in 20 mM Tris-acetate pH:
8.3, 1 mM EDTA (TAE) with recirculation. After electrophoresis, virus particles were transferred to a
PVDF membrane by blotting in TNE buffer. After transfer, the membrane was incubated in 25 mM
Tris-HCl pH: 8.3, 200 mM glycine, 0.1% SDS. The membrane was washed in 25 mM Tris-HCl pH:
8.3, 200 mM glycine before immunological detection of viral core or envelope proteins as described
(3).
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RESULTS:
Basic residues R122 and K141 contribute to the surface charge of HBV virions. To
substantiate the importance of R122 and K141, we tried to determine their exposure on the surface
of HBV virions and their contribution to the global charge of the particle. This could eventually be
documented by measurement of the isoelectric point (IEP) but, to our knowledge, IEPs of HBV or
HDV particles have not been established, and their precise measurement would require
preparations of highly purified virus. As an alternative, we chose to conduct native agarose gel
electrophoresis of HBV particles under the assumption that their electrophoretic mobility in agarose
gels be directly related to their overall charge, or IEP (5). As shown in Fig. S2, both wt virions and
non-enveloped nucleocapsids (NCs) are negatively charged at pH: 8.3 (IEP<8.3) and migrate
toward the anode. Note that the presence of non-enveloped NCs in addition to virions and SPVs is
often observed in the culture medium of transfected Huh-7 cells. In native agarose gel
electrophoresis, the faster migration of NCs, relative to virions, probably reflects a higher density of
negative charges at the NC surface because, as reported previously (6), there is no contribution of
the NC inner nucleic acid to its IEP; nucleic acid-free core particles having the same electrophoretic
mobility as nucleic acid-containing particles. For enveloped viruses, such as HBV, the IEP of the
whole virion is to an extent greater than it is for non-enveloped viruses, contributed by amino acids
exposed on the virion surface, assuming that a double leaflet lipid membrane has a high electrical
capacitance. Thus a substitution of surface-exposed basic or acidic amino acids in the viral
envelope proteins is expected to impact the virion IEP.
Six types of HBV particles were produced: wt, G145R, R122A, K141A, D144A and
R122A/K141A particles and first analyzed by immunoblotting for the detection of envelope proteins
(mainly
contributed
by
SVPs),
and
core
protein
(contributed
by
HBV
virions)
after
immunoprecipitation with anti-pre-S1 antibodies (Fig. S2A). All mutant proteins were expressed to
equivalent levels and were equally competent for virions assembly. Particles were then subjected
to electrophoresis in agarose gels to determine their relative electrophoretic mobility. The
equivalent of 2 ml supernatant of Huh-7 cells was applied in each well in the order indicated in Fig.
S2B, and this set of samples was duplicated in the same gel. After blotting on PVDF membranes,
one blot was stained for envelope proteins (upper panel) and the duplicate for core proteins (lower
panel) after its soaking in SDS-containing buffer for exposure of virion-associated core proteins.
Core proteins were detected at the position of both non-enveloped NC and that of enveloped
virions (V), whereas surface proteins – the signal of which is mainly contributed by SVPs - were
detected only at the position of virions suggesting no difference in IEPs of SVPs and virions. Most
interestingly, the removal of positive charges (R122A, K141A and R222A/K141A) increased
mobility relative to that of the wt, whereas removal of a negative charge (D144A) or introduction of
an additional positive residue (G145R) reduced mobility (most of the material remained at the top
of the gel), indicating an IEP for D144A and G145R ≥ 8.3. Altogether, these results clearly
demonstrate that R122 and K141 are solvent-exposed and greatly contribute to the overall charge
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of SVPs and virions. Furthermore, SVPs and virions have apparently identical IEPs as evidence by
the exact same mobility of virion-associated core and envelope proteins in the native agarose gel.
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Fig. S1. Inhibition of HDV infection by heparin and dextran sulfate. Infection assays were
conducted as described (3) in the absence, or presence, of 1:2 dilutions of heparin or dextran
sulfate at the indicated concentrations. At day 8 postinoculation, cellular RNA was extracted for
measurement of intracellular HDV RNA by Northern blot analysis. The position of genomic HDV
RNA is indicated. rRNA, Ribosomal RNA. Included in the experiment were pre-S1-specific
lipopeptide (pre-S1/2-48myr) and membrane-impermeable alkylator (AMS). Inhibitors were added
to the cell supernatant with the inoculum for 16 hr (coinoculation). The strongest effect was
observed with dextran sulfate (IC50 <31,25 µg). Control experiments were conducted with cells
exposed to the drugs for 24 hr at day one postinoculation (lower panel). Postinoculation treatment
had no inhibitory effect, demonstrating that heparin or dextran sulfate were not interfering with cell
metabolism or HDV RNA replication. As expected pre-S1/2-48myr and AMS demonstrated an
effiency entry inhibition, with IC50s at <1.25 nM and 0.5 mM and respectively.
Fig. S2 Characterization of HDV particles coated with HBV envelope proteins bearing
substitutions in the AGL amino acid sequence. Culture fluids from Huh-7 cells were harvested
after transfection with a mixture of pSVLD3 coding for HDV RNPs and pT7HB2.7, or derivatives,
coding for wt, or HBV envelope protein mutants, respectively. After normalization using an ELISA
specific for pre-S2, particles from 1 ml of each preparation were concentrated and assayed for the
presence of HBV envelope proteins by immunoblotting. Note that detection of HBV envelope
proteins was achieved using a rabbit anti-S antibody (R247) that recognizes a linear epitope in the
cytosolic domain-I of the three envelope proteins, and a rabbit anti-pre-S2 antibody for specific
detection of L- and M-HBsAg proteins. The relative levels of the immunoblotting signals for HBV
envelope proteins thus do not reflect the real ratio of L-/M-/S-HBsAg. Particles from 140 µl of each
preparation were assayed for the presence of genomic HDV RNA by the Northern blot
hybridization. Wt SML-HDV particles coated with wt S-, M- and L-HBsAg. The position of genomic
HDV RNA is indicated. The glycosylated (gp) and nonglycosylated (p) forms of S-HBsAg (p24 and
gp27) M-HBsAg (gp36) and L-HBsAg (p39 and gp42) proteins are indicated.
Fig. S3. Separation of HBV particles bearing substitutions is the envelope proteins by native
agarose gel electrophoresis. A) HBV particles from one ml of transfected Huh-7 cells were
analyzed for envelope proteins by immunoblotting using an anti-HBsAg (anti-S) antibody (R247)
specific for a linear epitope in the cytosolic loop of S-HBsAg. The glycosylated (gp) and
nonglycosylated (p) forms of S-HBsAg (p24 and gp27) M-HBsAg (gp36) and L-HBsAg (p39 and
gp42) proteins are indicated. Particles immunoprecipitated with an anti-pre-S1 antibody (Blanchet
M, Sureau C, 2007 J Virol 81:5841-5849) were analyzed by immunoblotting with a human anti-core
antibody. The core (p21) protein is indicated. B) Twenty µl of a 100-fold concentrate of HBV
particles produced by transfection of Huh-7 cells, were subjected to electrophoresis through a 0.8%
agarose gel, in 20 mM Tris-acetate pH: 8.3, 1 mM EDTA. Duplicate samples were run in parallel in
the same gel. After electrophoresis, virus particles were transferred to two identical PVDF
membranes by blotting in TNE buffer. After transfer, the membranes were incubated in 0.1% SDS,
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washed in 25 mM Tris-HCl pH: 8.3, 200 mM glycine before immunological detection of envelope
proteins (anti-S) or core proteins (anti-core). The position of SVPs and virions (V) is indicated. The
position of non-envelope nucleocapsid (NC) is indicated.
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