Supplementary material
Cultivation of HepaRG cells. HepaRG cells were grown in William´s E medium supplemented with
10% of a selected batch of fetal calf serum (FCS), 100 units/ml penicillin, 100 µg/ml streptomycin, 5
µg/ml insulin and 5x10-5 M hydrocortisone hemisuccinate (13). Cells were split 1:5 every two weeks
by trypsination. Two to 3 weeks before infection, cell differentiation was induced by 2% DMSO into
the maintenance medium of confluent cells. Medium was exchanged every 2-3 days.
Preparation of HDV. The plasmid JC126 encoding the HDV genomic sequence was transfected into the constitutively HBV-producing cell line, HepG2.2.15 (14). Cells were grown in
15 cm dishes to 50 % confluency. 35 µg (in 14 µl) JC126 was mixed with 105 µl Fugene 6
(Roche, Mannheim) and 1760 µl Williams E medium without serum or antibiotics and applied
to the cells. 48 hours after transfection, the medium was replaced by Williams E medium
supplemented with 10% FCS, non-heat inactivated), 100 units/ml penicillin, 100 µg/ml
streptomycin, 5 µg/ml insulin and 5x10-5 M hydrocortisone hemisuccinate) and the cells were
grown until confluence. The medium was supplemented with 2% DMSO and the culture
supernatants were collected on days 9, 12, 15 and 18. After removal of cell debris by centrifugation (3,300 x g, 10 min, 4°C), HDV and HBV particles were precipitated with PEG 8000
(12 h, 4°C, 7% final concentration) and pelleted using a JA10 rotor for 1 hour at 8000 rpm.
The pellets were resuspended in PBS and stored at -80°C. HDV titers were quantified by
reverse transcription followed by HDV-specific real-time PCR.
Determination of HDV RNA concentrations by Real time PCR. Total RNA from PEG
precipitated virus stocks was purified using the RNeasy Mini Kit (Qiagen) and subjected to
reverse transcription. 1/50-1/25 of tolal RNA of one twelve well was incubated at 95°C for 10
minutes to denature secondary structures within HDV (3). RNA was then incubated with 10
pmol of the DNA primer HDV-Taqman-F (5´-TATCCTATGGAAATCCCTGGTTTC-3´) in
a total volume of 10.5 µl for 10 minutes at 65oC. Reverse transcription was performed
according to the Expand-RT protocol (Roche Biochemicals, Mannheim, Germany). After
addition of 4 µl 5 x RT-buffer, 2 µl 100mM DTT, 2 µl 10 mM Na-dNTPs and 0,5 U RNasin
in a total volume of 19 µl, 1 µl Expand-RT was added and the reaction was allowed to
proceed for 1 h at 42°C. 4 µl of this reaction mix was directly used for SybrGreen (Invitrogen,
Karlsruhe, Germany) real-time PCR. 12,5 µl Mastermix, 0,5 µl QPCR-ROX (Abgene), 1,5 µl
10 µM HDV-Taqman-F and HDV-Taqman-R (5´-CCCGGAGTCCCCCTTCT-3´) were
mixed and brought to a total volume of 25 µl. The reaction process was performed using an
initial activation step at 95°C for 15 minutes, followed by 45 cycles at 95°C for 15 seconds
and by 60°C for 1 minute.
Cloning procedure of HBV mutants. For the introduction of single nucleotide exchanges into the Lprotein in the context of the HBV-expression plasmid pCH-9/3091 by primer mutagenesis, the primers
were designed without changing the amino acid sequence of the overlapping polymerase open reading
frame. The 3´-PCR-fragments for the overlap extension PCR (16) were obtained using the flanking
primer
HBV-EcoRI-rev
(5`-TTGTGGAATTCCACTGCATGGCCTG-3`)
and
the
particular
mutagenic primers HBVpreS/L11R/uni (5`-TCCACCAGCAATCCTCGGGGATTCTTTCCCGAC3`),
HBVpreS/G12E/uni
HBVpreS/F13S/uni
(5`-ACCAGCAATCCTCTGGAATTCTTTCCCGACCAC-3`),
(5`-AGCAATCCTCTGGGATCCTTTCCCGACCACCAG-3`)
and
HBVpreS/FF13,14SS/uni (5`-AGCAATCCTCTGGGATCCTCTCCCGACCACCAGTTG-3`). The
5`-PCR fragments were generated with the flanking primer HBV-MfeI-uni (5`-GATTGCAATTGATTATGCCTGCC-3`) and the respective anti-sense oligonucleotides complementary to the mutagenic
primers described above. The full-length products resulting from the second PCR with the matching
5`- and 3`-fragments and the flanking primers were cleaved with MfeI and EcoRI and ligated into the
vector fragment derived from pCH-9/3091. The G12E-fragment was obtained by a partial digest
because of the additional EcoRI site created by the mutagenesis procedure. The correct orientation of
the insert was tested by cleavability with MfeI/EcoRI; introduction of intended mutations were
verified by sequencing. For the production of mutant viral particles Huh7 cells on 10cm culture dishes
were transfected with 10µg of the particular plasmid DNA by the calcium phosphate precipitation
method.
Generation and subcloning of preSXaGST fusions. To generate the DNA sequence encoding
HBVpreS/1-48XaGST we first produced an HBVpreS/1-48Xa fragment by polymerase chain reaction
(PCR)
using the
forward
primer
HBVpreS/BglII/uni
(5`-TACATCAGATCTATGGGGCA-
GAATCTTTCCACC-3`), introducing a BglII cleavage site at the 5`-end, and the reverse primer
HBVpreS/48Xa/EcoRI/rev (5`- GATCGAATTCACGACCTTCGATTCCTACCTTGTTGGCGTCTGGCCAGG-3`), encoding the factor Xa recognition sequence IEGR, upstream of an EcoRI cleavage
site. As a template we used a plasmid encoding the HBVpreS sequence of Genotype D. The PCR
fragment was digested with BglII/EcoRI and ligated into pVL1392, resulting in the transfer vector
pVLHBVpreS/1-48Xa. The GST coding sequence was obtained by PCR from pGEX2T (AmershamPharmacia) with a blunt end generating polymerase, using the oligonucleotides GST/EcoRI/uni (5`GATCGAATTCATGTCCCCTATACTAGGTTATTGG-3`)
and
GST/BamHI/rev
(5`-GATCG-
GATCCTCAACGCGGAACCAGATCCGATTTTGGAGG-3`). The PCR fragment was hydrolyzed
with EcoRI and ligated into the pVLHBVpreS/1-48Xa vector, cleaved by EcoRI/SmaI. The resulting
vector pVLHBVpreS/1-48XaGST served as a template for PCR using HBVpreS/NdeI/uni (5`GGAATTCCATATGGGGCAGAATCTTTCCACCAGC-3´)
and
GST/KpnI/rev
(5`-
CGGGGTACCTCAACGCGGAACCAGATCCGATTTTGGAGG-3`) to introduce the NdeI and
Kpn1 restriction sites required for the ligation into the vector pBB132Cα (17). To introduce internal
deletions into the preS1 sequence, PCRs with the respective oligonucleotides HBVpreS/∆5-9/NdeI/uni
(5`-GGAATTCCATATGGGGCAGAATCCTCTGGGATTCTTC-3`) and HBVpreS/∆11-15/NdeI/uni
(5`-GGAATTCCATATGGGGCAGAATCTTTCCACCAGCAATCCCGACCACCAGT-3`)
as
forward primers and GST/KpnI/rev as a reverse primer were performed. For the introduction of the
other deletions two pairs of primers were used: the flanking primer HBVpreS/NdeI/uni and
GST/KpnI/rev and two complementary primers covering the region within the HBVpreS1 sequence
that was to be mutated. Three PCRs using plasmid pBB132 HBVpreS/1-48XaGST as template were
carried out to obtain the final insert: (i) HBVpreS/NdeI/uni was combined with the mutagenic reverse
primer within the preS1 sequence; (ii) GST/KpnI/rev was combined with the mutagenic forward
primer within the preS1 sequence; (iii) the two resulting PCR products were used as templates for a
PCR with the primers HBVpreS/NdeI/uni and GST/KpnI/rev (16). The PCR products were hydrolyzed
with NdeI/KpnI and ligated into the pBB132. To introduce the remaining deletions the following
primers were used: ∆17-21: HBVpreS/∆17-21/uni (5`-CTGGGATTCTTTCCCGACGCCTTCAGAGCAAACACC-3`) and HBVpreS/∆17-21/rev (5`-GGTGTTTGCTCTGAAGGCGTCGGGAAAGAATCCCAG-3`), ∆23-27: HBVpreS/∆23-27/uni (5`-CACCAGTTGGATCCAGCCGCAAATCCAGATTGGGAC-3`) and HBVpreS/∆23-27/rev (5`-GTCCCAATCTGGATTTGCGGCTGGATCCAACTGGTG-3`),
∆29-33:
CAATCCCAACAAGGAC-3`)
HBVpreS/∆29-33/uni
and
GAATGCGGTTGGTGCTCTGAA-3`),
(5`-TTCAGAGCAAACACCGCATT-
HBVpreS/∆29-33/rev
∆35-39:
GATTGGGACTTCACCTGGCCAGACGCCAAC-3`)
(5`-GTCCTTGTTGGGATT-
HBVpreS/∆35-39/uni
and
(5`-AATCCA-
HBVpreS/∆35-39/rev
(5`-
GTTGGCGTCTGGCCAGGTAGAGTCCCAATCTGGATT-3`), ∆41-45: HBVpreS/∆41-45/uni (5`AATCCCAACAAGGACACCAAGGTAGGAATCGAAGGT-3`) and HBVpreS/∆41-45/rev (5`ACCTTCGATTCCTACCTTGGTGTCCTTGTTGGGATT-3`). Single amino acid exchanges were
obtained in the same way using the following oligonucletides: L11R: HBVpreS/L11R/uni (5`TCCACCAGCAATCCTCGGGGATTCTTTCCCGAC-3`)
and
HBVpreS/L11R/rev
(5`-
GTCGGGAAAGAATCCCCGAGGATTGCTGGTGGA-3`), G12E: HBVpreS/G12E/uni (5`-ACCAGCAATCCTCTGGAATTCTTTCCCGACCAC-3`)
and
GTGGTCGGGAAAGAATTCCAGAGGATTGCTGGT-3`),
CAATCCTCTGGGATCCTTTCCCGACCACCAG-3`)
F13S:
and
HBVpreS/G12E/rev
(5`-
HBVpreS/F13S/uni
(5`-AG-
HBVpreS/F13S/rev
(5`-
CTGGTGGTCGGGAAAGGATCCCAGAGGATTGCT-3`), FF13/14SS: HBVpreS/FF13/14SS/uni
(5`-AGCAATCCTCTGGGATCCTCTCCCGACCACCAGTTG-3`) and HBVpreS/ FF1314SS/rev
(5`-CAACTGGTGGTCGGGAGAGGATCCCAGAGGATTGCT-3`), R24G: HBVpreS/R24G/uni (5`TTGGATCCAGCCTTCGGAGCAAACACCGCAAAT-3`)
ATTTGCGGTGTTTGCTCCGAAGGCTGGATCCAA-3`).
15/HBVpreS/16-48XaGST
coding
sequence
the
and
To
HBVpreS/R24G/rev
generate
oligonucleotides
the
(5`-
DHBVpreS/1-
DHBVpreS/NdeI/uni
and
GST/KpnI/rev were used. The two complementary primers consisted of sequences corresponding to
amino acids 11-15 of DHBVpreS and amino acids 16-20 of HBVpreS1. For the first PCR using the
DHBV encoding vector pCD0 as a template DHBVpreS/NdeI/uni and HBV/DHBV/rev (5`TGGATCCAACTGGTGGTCTTCTATCCGTCTGACGTC-3`) were applied. For the second PCR the
oligonucleotides
DHBV/HBV/uni
(5`-ATGGACGTCAGACGGATAGAAGAC
CAC-
CAGTTGGATCCA-3`) and GST/KpnI/rev were combined using the pBB132HBVpreS/1-48XaGST
vector as a template. The resulting PCR products served as a template for the third PCR with the oligonucleotides DHBVpreS/NdeI/uni and GST/KpnI/rev. To produce the DpreS/1-44XaGST the same
flanking
primers
were
applied
GACATCGAAGGTCGTGAATTC-3`)
but
DpreS/44XaGST/uni
and
(5`-CTAGATCACGTGTTA-
GST/Xa44/DpreS
(5`-GAATT-
CACGACCTTCGATGTCTAACACGTGATCTAG-3`) served as complementary primers and pCD0
as a template. The WHVpreS/1-87XaGST construct was produced using the flanking primers
WHVpreS/NdeI/uni
(5`-GGAATTCCATATGGGCAACAACATAAAAGTCACC-3`)
and
GST/EcoRI/rev (5`-GACATGAATTCACGACCTTCGATAGGATCCCTGTTGACCAAT-3`) and the
complementary
primers
TATCGAAGGTCGTGAATTC-3`)
WHV/XaGST/uni
and
(5`-TTGGTCAACAGGGATCC-
GSTXa/WHVrev
(5`-GAATTCAG-
CACCTTCGATGGATCCCTGTTGACCAA-3`) fusing the WHVpreS coding sequence with the Xa
cleavage site and GST. All constructs were controlled by sequencing with the primer GSTseq/rev (5´CCTTCATCGCGCTCATAC-3´) starting within the GST.
Mass spectrometric analysis of HBV-preS-GST fusion proteins. For MALDI-TOF MS analysis,
0.3 µl of a saturated solution of sinapic acid in ethanol were deposited onto individual spots of a
MALDI target plate. Subsequently, 0.5 µl of the protein solution and 0.5 µl of a saturated solution of
sinapic acid in 0.1%TFA/30% acetonitrile were loaded on top of the thin film spots and allowed to cocrystallize slowly at ambient temperature. MALDI mass spectra within a m/z range of 700-13000 were
recorded in the positive ion reflector mode with delayed extraction on a Reflex II™ time-of-flight
(TOF) instrument (Bruker-Daltonik, Bremen, Germany) equipped with a SCOUT-26 inlet and a 337
nm nitrogen laser. Ion acceleration voltage was set to 26.5 kV, the reflector voltage was set to 30.0 kV
and the first extraction plate was set 20.6 kV. Mass spectra were obtained by averaging 50 to 200
individual laser shots. For masses higher than 4500 Da, spectra were calibrated externally using the
average masses of singly-charged insulin at m/z 5734.56 and cytochrom C at m/z 12361.09. For
masses below 4500 Da, calibration was performed with the singly-charged monoisotopic ions of
angiotensin I at m/z 1296.69 and the oxidized B-chain of insulin at m/z 3494.65.