Download Nuclear accumulation of hepatitis B virus preS fragments

RESEARCH ARTICLE
1115
Translocation and accumulation of exogeneous
hepatitis B virus preS surface proteins in the cell
nucleus
Eun-Wie Cho1, Jung-Hyun Park1, Ook-Joon Yoo2 and Kil Lyong Kim1,*
1Protein Research Laboratory, Korea Research Institute of Bioscience and
2Department of Biological Sciences, KAIST, Taejon 305-701, South Korea
Biotechnology, Yusong, P.O. Box 115, Taejon 305-600, South Korea
*Author for correspondence (e-mail: [email protected])
Accepted 4 January 2001
Journal of Cell Science 114, 1115-1123 © The Company of Biologists Ltd
SUMMARY
Recurrent reports about protease-sensitive sites in the
junction of the preS and S region of the hepatitis B virus
large surface protein have raised the question about a
possible biological role of S protein-depleted, independent
preS protein fragments in the virus life cycle. In the
present study, this question was addressed by exogenous
introduction of fluorescence-labeled recombinant preS
proteins into permeabilized HepG2 cells. While maltosebinding proteins (MBP) were evenly distributed
throughout the cytoplasm, MBP-preS fusion proteins
selectively accumulated in the nucleus. Using truncated
preS proteins, the effective domain for this nuclear
accumulation was localized around the preS2 region. The
mode of this action differs from conventional nuclear
translocation mechanism in its energy- and mediatorindependency and in that it is not saturated regardless of
the increase of preS protein concentration. The biological
meaning of this phenomenon has to be further studied.
However, in regard to hepatitis B virus infection, this
observation might provide a clue for unveiling the still
poorly characterized events after initial internalization
of the virus, which might make use of the nuclear
translocation effect of the preS2 region to facilitate the
infection.
INTRODUCTION
have also been assigned to the LHBs that involve various steps
in the viral life cycle, including the regulation of viral
replication (Summers et al., 1990; Summers et al., 1991) and
the transactivation of a variety of promoter elements (Kekule
et al., 1990; Hildt et al., 1996; Kim et al., 1997). Although it
is true that such functions are performed by the LHBs, on
closer inspection, one can easily see that these multiple roles
are mostly, if not solely, performed by the preS region of the
envelope protein. This observation naturally leads to the
question whether preS protein fragments alone might also have
some biological roles in the absence of the S region. Actually,
in vitro studies with protease-treated HBV particles showed
specific cleavage of preS fragments from LHBs, which indicate
the presence of protease-sensitive sites within the junction of
the preS and S proteins (Gerlich et al., 1993; Lu et al., 1996).
So, while the proteolytic cleavage and generation of free preS
fragments are quite evident, their biological meanings remain
obscure.
With respect to the rapid degradation of exogenous proteins
in the cytosol, a putative role for free preS proteins in the viral
infection process can be assumed to be largely restricted to the
early steps of infection. There are many examples that viral
structural proteins have been shown to be associated with
various subcellular structures, including actin filaments or the
Golgi complex and even the nucleus. Depending on their
intracellular localization, further studies have shown that
these proteins are involved either in exerting some essential
Most viral proteins play multiple roles in the viral life cycle,
thereby maximizing the functionality of each individual
protein. The large hepatitis B surface protein (LHBs) of the
hepatitis B virus (HBV) is a typical case of this (for review,
see Ganem, 1996). The LHBs is transcribed from the env gene
using the first of three overlapping reading frames and is
composed of the preS1, preS2 and S domains (Fig. 1). Two
membrane topological isomers have been described for this
envelope protein, which differ in their display, either to the
external or internal side of the viral envelope, of the N-terminal
preS domain (Bruss et al., 1994; Ostapchuck et al., 1994;
Prange and Streeck, 1995). LHBs with preS domains exposed
to the external side has been implicated in binding to the
cellular virus receptor. In particular, the preS1 region has been
reported to serve as the principal binding site for hepatocytes
(Neurath et al., 1986; Pontisso et al., 1989; Theilmann and
Goeser, 1991) and the preS2 region has been independently
reported to contain an auxiliary binding site using polymerized
human serum albumin as an intermediate receptor (Machida
et al., 1983; Sobotta et al., 2000). However, preS domains
displayed to the inner side of the virus membrane are known
to be involved in viral morphogenesis, establishing a physical
interaction of the viral envelope with preformed cytosolic
nucleocapsids (Bruss, 1997; Poisson et al., 1997).
Independent of the membrane topology, other crucial roles
Key words: Hepatitis B virus, PreS2 region, Energy-independent
translocation
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JOURNAL OF CELL SCIENCE 114 (6)
A.
1
120
PreS1
Fig. 1. Functional domains within the large hepatitis B
surface protein (LHBs) and the amino acid sequences
of preS(1-174) region of HBV (adr subtype). (A) The
LHBs consists of the preS and S domains, of which the
S protein contains four membrane-spanning regions.
Depending on the orientation of the first membrane
spanning domain, LHBs adopts two membrane
topologies as described in the Introduction. The preS
region can be further divided into the 119 amino acid
preS1 region and the 55 amino acid preS2 region,
which has a myristylation site at the 2nd amino acid,
glycine (Persing et al., 1987). The LHBs exerts
multiple functions as shown in this figure and these are
concentrated in the preS domain. At the junction of
preS and S domain is a putative proteolytic site (PEST
sequence), that suggests the presence of independent
preS fragments. (B) The amino acid sequence of the
preS(1-174) as determined for the HBV subtype adr.
Basic amino acid residues are marked as square-boxed
letters for comparison with the NLS sequence.
1Neurath et al., 1986; Pontisso et al., 1989. 2Hildt et al.,
1996; Kim et al., 1997; Hildt et al., 1995. 3Bruss et al.,
1997; Poisson et al., 1997; Le Seyec et al., 1998.
4Sobotta et al., 2000. 5Lu et al., 1996. 6Choi. et al.,
1986.
174
400
PreS2
TM
TM
S
TM
TM
21-47: hepatocyte binding site1
21-90, 120-172: transactivator domain2
103-124: core particle interacting site3
Myristylation
122-134: PHSA binding site4
168-181: PEST sequence5
B.
Amino acid sequence of preS(1-174) (adr subtype6 )
1
.
11
.
21
.
31
.
41
.
MGGWSSKPRQGMGTNLSVPNPLGFFPDHQLDPAFGANSNNDWDFNPNKD
51
.
61
.
71
.
81
.
91
.
QWPEANQVGAGAFGPGFTPPHGGLLGWSPQAQGILTTVPAAPPPASTNRQ
.101
111
.
121
.
131
.
141
.
SGRQPTPISPPLRDSHPQAMQWNSTTFHQALLDPRVRGLYFPAGGSSSGT
.151
161
.
171
.
VNPVPTTASPISSIFSRTGDPAPN
functions in the internalization of the virus (Freed, 1998) or in
the transport of viral particles along the cytoskeletal fibers
(Kasamatsu et al., 1983; Elliott and O’Hare, 1997), besides
performing their primary functions as structural components.
Accordingly, we have investigated the putative role of free preS
proteins in the viral life cycle. To achieve this, fluorescently
labeled preS(1-174) proteins of the LHBs were exogenously
introduced into the detergent-permeabilized cells and the
intracellular distribution and the interaction of preS proteins
with cellular components were analyzed by confocal laserscanning microscopy. Unexpectedly, we found that the preS
proteins were specifically translocated into the nucleus in an
energy- and cytosolic factor-independent manner. We will
discuss the mechanism of this nuclear accumulation and its
biological meaning in the viral life cycle.
MATERIALS AND METHODS
Preparation of MBP-preS fusion proteins
The expression vector for the recombinant production of HBV-preS
proteins was constructed by subcloning the coding sequence for the
whole preS region of HBV (subtype adr) in fusion to the maltosebinding protein (MBP), using the pMAL-c2 vector (New England
Biolabs, Beverly, MA). The insert was prepared by PCR amplification
from the plasmid pHBV (Choi et al., 1986) using forward (5′GGAATTCATGGGAGGTTTGTCTTCCAAA-3′) and backward
primers (5′-TGCACTGCAGTTAGTTCGGCGGTGCAGGGTC-3′).
As these primers contain EcoRI and PstI recognition sites on their
respective 5′ ends, the amplified 522 bp PCR fragment was subcloned
into the EcoRI and PstI sites of pMAL-c2. The resulting expression
vector, pMAL-preS, was then introduced into Escherichia coli (strain
BL21) cells. For the expression of MBP-preS fusion protein,
overnight culture of transformed cells was used to inoculate fresh
media and this culture was induced at its log growth phase with 1 mM
isopropyl-thio-β-D-galactopyranoside (IPTG). After incubation for
further 2 hours, cells were harvested and the pellet was resuspended
in ice-cold TBS (10 mM Tris-HCl, pH 7.4, 150 mM NaCl) containing
5 mM EDTA and 2 mM phenylmethylsulfonylfluoride (PMSF). All
subsequent procedures were performed at 4°C. Cells were destroyed
by sonication and the debris removed by centrifugation at 10,000 rpm
for 20 minutes. The supernatant was then treated with 50%
ammonium sulfate and the precipitate was resuspended in distilled
water and mixed with the same volume of 0.1 M sodium citrate (pH
4.6). Denatured proteins were then removed by centrifugation and, to
concentrate the protein solution, the supernatant was precipitated once
again with 50% ammonium sulfate. The final pellet was resuspended
and dialyzed against column buffer (20 mM Tris-HCl, pH 8.5, 25 mM
NaCl, 10% glycerol, 1 mM EDTA). The solution was passed through
a QAE-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden)
column and bound proteins were eluted in 25-300 mM NaCl gradient.
The successful purification was confirmed by SDS-PAGE (see Fig.
2A). To confirm that the preS region was intact, western blot analysis
was performed with preS epitope-specific antibodies (see Fig. 2B):
mAb F35.25, which is specific for the preS1(21-47) region (Petit et al.,
1991) and mAb H8, which specifically recognizes a conformational
epitope covering the preS2(120-145) region (Chung and Kim, 1987).
MBP was identified using the anti-MBP mAb (Fig. 2B), HAM-19
(Park et al., 1998) and these were detected by peroxidase-conjugated
anti-mouse Ig antibodies (Sigma, St Louis, MO) and the
corresponding substrate.
FITC labeling of MBP and MBP-preS protein
For fluorescein-isothiocyanate (FITC) labeling, MBP or MBP-preS
protein solutions were dialyzed against conjugation buffer (0.1 M
sodium carbonate, pH 9.0) and concentrated to 2 mg/ml. Each 10 µl
Nuclear accumulation of hepatitis B virus preS fragments
of freshly prepared FITC solution (10 mg/ml in DMSO) was added
to 1 ml of each protein solution and incubated in the dark for 2 hours
at room temperature. The reaction was stopped by addition of 1/10
volume of 0.1 M glycine (pH 8.0). The reaction mixture was
fractionated using a 1.5×8 cm Sephadex G50 column (Amersham
Pharmacia Biotech) gel filtration chromatography.
Peptide synthesis and purification
Peptides were synthesized by the solid phase method (Merrifield,
1986) using Fmoc-chemistry. Fmoc-Wang-amino acids resins
(Novabiochem, San Diego, CA) were used as support. For the peptide
chain elongation, dicyclohexylcarbodiimide (DCC) and Nhydroxybenzotriazole (HOBt) were used as coupling agent. The side
chains of the amino acids were protected with the following base
(piperidine)-stable protecting groups: Asp (OtBu), Asn (Trt), Trp
(Boc), Arg (Pmc), Ser (tBu) and Tyr (tBu). The final protected
peptide-resins were cleaved and deprotected with TFA-based reagents
(82.5% TFA, 5% phenol, 3% H2O, 5% thioanisole, 2.5% 1,2ethandithiol and 2% triisopropylsialine) for 2 hours, then precipitated
with diethylether and dried in vacuum. The crude peptides were
purified by a preparative reverse-phase (RP)-HPLC on a Waters 15
µm Deltapak C18 column (300 Å, 1.9×30 cm). Purity of the HPLCisolated peptides was checked by an analytical RP-HPLC on an
Ultrasphere C18 column (5 µm, 0.46×25 cm, Beckman, San Ramon,
CA).
Cell lines and culture
Cell lines were obtained from the ATCC (Rockville, MD). The human
hepatoma cell line HepG2 and the human epitheloid carcinoma cell
line HeLa were cultured in Dulbecco’s modified Eagle’s media
(DMEM; GibcoBRL, Grand Island, NY) supplemented with 10%
fetal bovine serum. Cells were cultured at 37°C in a 5% CO2
atmosphere in a humidified incubator.
Intracellular staining of fixed and permeabilized HepG2
and HeLa cells
The day before analysis, HeLa or HepG2 cells were detached from
stationary cultures and plated on 18×18 mm glass coverslips in sixwell plates (NUNC, Roskilde, Denmark) to 50% confluency. The next
day, adherent cells were washed with ice-cold PBS and then fixed with
2% paraformaldehyde in PBS for 20 minutes at 4°C. All subsequent
procedures were performed at 4°C. The fixation reaction was blocked
by incubation of the cells in 0.1 M glycine in PBS for 5 minutes.
Afterwards, the plasma membrane was permeabilized with 0.1%
Triton X-100 in PBS for 5 minutes and the detergent was removed by
extensive washing with PBS. Permeabilized cells were then incubated
with FITC-labeled MBP-preS or MBP to a concentration of 40 µg/ml
for 1 hour. To determine nuclear integrity, permeabilized cells were
incubated with anti-DNA antibodies (Roche Molecular Biochemicals,
Mannheim, Germany) in 0.2% (w/v) bovine serum albumin (BSA) in
PBS for 15 minutes at room temperature, rinsed and then incubated
with FITC-labeled secondary antibodies. As there were no signals
detectable in the nucleus, it was concluded that the nuclear membrane
had been remained intact during the staining procedure (data not
shown). For the competition assay, permeabilized cells were first
incubated with competitor peptides, the preS(120-174) or the HIV
gp41(584-618) peptide (RILAVERYLKDQQLLGIWGCSGKLICTTAVPWNAS) at a concentration of 4 mg/ml in PBS for 30 minutes.
Without washing, cells were incubated directly with FITC-labeled
MBP-preS or MBP at a concentration of 40 µg/ml for 60 minutes.
Finally, cells were washed with ice-cold PBS and analyzed by
confocal laser-scanning microscopy.
In vitro nuclear transport assay
The selective permeabilization of the plasma membrane and
subsequent nuclear transport assays were performed by the method as
described by Adam et al. (Adam et al., 1990). In brief, one day before
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the transport assay, HepG2 or HeLa cells were plated on 18×18 mm
glass coverslips in six-well plates. On the day of analysis, the
coverslips with the attached cells were washed with ice-cold import
buffer (20 mM Hepes, pH 7.3, 110 mM potassium acetate, 5 mM
sodium acetate, 2 mM magnesium acetate, 0.5 mM EGTA) and then
immersed in ice-cold import buffer containing 40 µg/ml digitonin for
the plasma membrane-specific permeabilization. After five minutes,
the digitonin-containing buffer was removed by aspiration and
replaced with ice-cold import buffer. Coverslips were drained and
blotted to remove excess buffer and then placed in the inverted
orientation over 40 µl of a complete import mixture. The complete
import mixture contained the following components: 50% cytosol,
20 mM Hepes, pH 7.3, 110 mM potassium acetate, 5 mM sodium
acetate, 2 mM magnesium acetate, 2 mM DTT, 0.5 mM EGTA, 1 mM
magnesium ATP, 5 mM creatine phosphate (Roche Molecular
Biochemicals), 20 U/ml creatine phosphokinase (Roche Molecular
Biochemicals), substrates for the nuclear transport assay, and 1 µg/ml
each of aprotinin, leupeptin, and pepstatin. As a source of cytosol
fraction, rabbit reticulocyte lysate (Promega, Madison, WI) was used.
The entire chamber was incubated at 37°C for 30 minutes. At the end
of the assay, each coverslip was washed with import buffer and fixed
by immersion in 4% (w/v) paraformaldehyde dissolved in import
buffer prior to image analysis. As a positive control in the nuclear
transport assay, BSA-conjugated with a classical nuclear localization
signal (NLS) sequence was used (Adam et al., 1990). In brief, the
synthetic peptide, CGGGPKKRKVED, which contains a well-known
nuclear transport signal sequence of SV40 T-antigen (PKKKRKVE)
was synthesized and crosslinked with FITC-labeled BSA via Nterminal cysteine residues. The successfully crosslinked conjugate
was fractionated using 1.5×8 cm Sephadex G50 column (Amersham
Pharmacia Biotech) gel filtration chromatography.
Image analysis
Cells treated with FITC-labeled proteins were analyzed by confocal
laser-scanning microscopy. The confocal microscope system
consisted of a Leica TCS 4D connected to a Leica DAS upright
microscope (Leica Lasertech GmbH, Heidelberg, Germany).
Fluorescent intensity of each cellular fraction (nucleus and cytosol)
was quantitated using NIH Image 1.60 public domain software after
the substraction of the fluorescence intensity of the background.
Relative fluorescence intensity was determined using the fluorescence
intensity of the FITC-MBP staining as standard.
Circular dichroism (CD) analysis of the preS peptide
Far-UV circular dichroism spectra of preS(120-174) peptides were
acquired using a Jasco J720 spectropolarimeter. The peptide
concentration was 100 µg/ml in solutions of PBS, pH 7.4, or 50%
(v/v) NMR grade trifluoroethanol (TFE; Sigma-Aldrich) or 30 mM
sodium dodecylsulfate (SDS) dissolved in 10 mM sodium phosphate
buffer, pH 7.4, respectively. All samples were maintained at 25°C
during the analysis. The spectra were measured at 0.2 nm intervals
from 240 to 195 nm at 25°C using a 1 mm path-length cell. The
average of four scans was calculated and used for the graphic analysis.
The mean residue ellipticity (θ), is given in deg cm2 dmol-1.
RESULTS
Nuclear accumulation of exogenous HBV-preS
proteins in fixed and plasma membrane
permeabilized HepG2 and HeLa cells
The intracellular distribution of exogenously introduced preS
proteins was analyzed for HepG2, human hepatoma cells and
HeLa, an HBV non-permissive epithelial-like cell line (Qiao et
al., 1994), for which recombinant preS proteins as expressed in
C-terminal fusion to the maltose-binding protein (MBP),
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JOURNAL OF CELL SCIENCE 114 (6)
Fig. 2. Preparation and gel-electrophoretic analysis of MBP-preS
fusion proteins. The HBV-preS region was expressed as an MBPfusion protein in E. coli and the preparation was analyzed in a 10%
SDS-PAGE (for details, see Materials and Methods). (A) The
expression of MBP-preS was induced by IPTG (lane 1, before
induction; lane 2, 2 hours after induction) and purified by acidprecipitation and QAE-anion exchange chromatography (lane 3). Lane
4 shows MBP without fused proteins. (B) To confirm the purified
proteins were intact, MBP and MBP-preS proteins were western
blotted and detected by region-specific antibodies, mAb F35.25 (antipreS(21-47)), H8 (anti-preS(120-145)) and HAM19 (anti-MBP).
termed MBP-preS(1-174), was used (Fig. 2). In all of the
experiments, MBP without co-expressed preS proteins was
used as negative control. For the introduction of free preS
proteins into the cytoplasm, these cells were first fixed with 2%
paraformaldehyde and then the plasma membranes were
permeabilized with 0.1% Triton X-100 and finally incubated
with FITC-labeled preS proteins. Interestingly, incubation with
FITC-labeled MBP-preS(1-174) fusion proteins resulted in a
specific accumulation of these proteins in the nucleus of HepG2
and HeLa cells, as shown in Fig. 3A and Fig. 3B respectively.
On the contrary, FITC-labeled MBP proteins were distributed
only to a background level throughout the cytoplasm (Fig.
3C,D) but not in the nucleus. To test the accessibility of
exogeneous molecules to nuclear components of Triton X-100
permeabilized cells, these cells were incubated with anti-DNA
antibodies and their intracellular distribution was examined. As
no signals were detectable in the nucleus (data not shown), it
was concluded that the nuclear envelope was neither solubilized
nor disrupted when using this concentration of Triton X-100.
Therefore the possibility of nuclear accumulation of preS
proteins, induced by nonspecific diffusion caused by nuclear
envelope damage or passive leakage, could be excluded.
Another important observation is that there were no
differences in the intracellular distribution of MBP-preS(1174) between HepG2 and HeLa cells. HepG2 cells are known
to be HBV permissive and previous studies have shown that
they are able to bind HBV particles via the viral preS1
domain (Neurath et al., 1986; Pontisso et al., 1989;
Theilmann and Goeser, 1991; Qiao et al., 1994; Lee et al.,
1996) as well as to produce functional viral particles after
transfection of the viral genome. However, the HeLa cell line
is an HBV non-permissive epithelial-like cell line, generally
used as a negative control in HBV infection (Choi et al.,
1996). So it seems that the penetration and accumulation
effects of the preS(1-174) protein on the nucleus are
independent of HBV susceptibility.
Fig. 3. Intracellular distribution of the FITC-labeled MBP-preS
proteins in HepG2 and HeLa cells. (A,B) FITC-labeled MBP-preS
proteins were incubated with HepG2 or HeLa cells which were
previously fixed with 2% paraformaldehyde and permeabilized by
0.1% Triton X-100. (C,D) FITC-labeled MBP protein was used as
control. After extensive washing with PBS, cells were analyzed by
confocal microscopy.
HBV-preS proteins also accumulate within the nuclei
of semi-permeabilized HepG2 cells
The nuclear envelope is composed of two concentric
membranes, the inner and outer nuclear membrane, and is
perforated by nuclear pores. To accumulate within an intact
nucleus, the preS proteins must translocate itself through this
nuclear envelope. In live cells, molecular traffic between the
cytoplasm and nucleus occurs through nuclear pores. These
nuclear pores contain central hydrophilic channels, but most
proteins are too large to cross these channels, and even when
they are small enough to diffuse through the nuclear pore,
specific mechanisms involving nuclear localization signals and
energy supply are required to cross the pore complex (Mattaj
and Engelmeier, 1998). In the case of fixed and permeabilized
cells, as used in the present experiments (Fig. 3), cytoplasmic
factors are thoroughly washed out and no energy source is
available, which is not compatible with the conditions in living
cells. To examine whether the nuclear accumulation effect
of preS protein is also reproducible in intact cells, in vitro
nuclear transport assays (Adam et al., 1990) (using digitoninpermeabilized HepG2 cells) were performed. To compare the
present finding with the characteristics of conventional nuclear
transport mechanisms, SV40 T-antigen nuclear localization
signal (NLS)-conjugated BSA, a representative nuclear
transport substrate, was used as positive control.
As shown in Fig. 4A, the SV40 T-antigen NLS conjugates
were able to enter the nucleus at 37ºC when cytosol and energy
source were supplied. But, when temperature was lowered to
4ºC (Fig. 4B), or when the cytosolic factors were depleted (Fig.
4C), nuclear accumulation of these substrates was inhibited.
Nuclear accumulation of hepatitis B virus preS fragments
Fig. 4. Energy and cytosolic factors-independent nuclear
translocation of FITC-labeled MBP-preS proteins. In vitro nuclear
transport assays for MBP-preS protein were performed using
digitonin-permeabilized HepG2 cells. (A-C) Positive controls: BSAconjugated with a nuclear transport signal sequence of SV40 Tantigen (PKKKRKVE) was used that had been prepared according to
Adam et al. (Adam et al., 1990). (D) HepG2 cells were
permeabilized with 40 µg/ml of digitonin and incubated with FITClabeled MBP-preS protein and import mixture (containing cytosol
and ATP) at 37°C for 30 minutes. (E,F) To determine the dependency
on the cytosolic factors and energy, assays were also performed
without cytosolic fraction (F) or at 4°C (E). At the end of the assay,
cells were extensively washed and fixed with 4% (w/v)
paraformaldehyde prior to image analysis. (G-I) Negative control:
FITC-labeled MBP protein was also used.
However, preS proteins were still able to accumulate within the
nucleus even under energy- and cytosolic factor-depleted
situations (Fig. 4E,F). Nevertheless, when the temperature was
shifted to 37ºC, the nuclear accumulation was more enhanced
(Fig. 4D), perhaps because of the increase of the molecular
motions induced by the increase of temperature. The same
results were obtained for HeLa cells (data not shown).
From these results, it has been concluded that the nuclear
accumulation effect of preS proteins is independent of
cytosolic factors in intact cells, and that this process would not
follow conventional cytosolic factor- and energy-dependent
nuclear translocation mechanisms.
Nuclear accumulation effect is lost by deletion of the
C-terminal region of the preS protein
preS-mediated nuclear accumulation has been not reported
before. To explore the biological meaning and the mechanism
behind this finding, it is necessary to define the effective
domain more precisely. A clue for the determination of
the active domain was found by analysis of the physical
characteristics of the preS protein. After FITC-conjugation, the
labeled preS proteins were stored in PBS at 4ºC. During the
long-term storage, MBP-preS proteins were slowly degraded,
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Fig. 5. Loss of the nuclear accumulation effect by C-terminal
truncated MBP-preS proteins. (A) During prolonged storage, FITClabeled MBP-preS proteins were degraded at their C-termini, which
resulted in the generation of a truncated protein of 52 kDa as
analyzed by 10% SDS-PAGE. (B) The nuclear accumulation effect
of these C-terminal-truncated MBP-preS proteins was compared with
that of intact MBP-preS and MBP alone in HepG2 cells.
which was detected by a mobility change in an SDS-PAGE
(Fig. 5A). Interestingly, it was then observed that these
truncated MBP-preS proteins had lost their ability to
accumulate within the nucleus (Fig. 5B). Judging from their
molecular weight and their reactivity with preS region-specific
antibodies, the degradation of preS proteins was located around
the C-terminal region. This C-terminal truncation was about
the size of 10 kDa, which would cover the entire preS2 region
(preS(120-174); 55 amino acids, corresponding to 6 kDa) and
a part of the preS1 region (about 4 kDa). When these degraded
proteins were analyzed for their reactivity to the preS regionspecific antibodies, the reactivity to the mAb H8, which
specifically detects the preS(120-145) region, was lost, but not
the reactivity to F35.25, an anti-preS(21-47)-specific mAb.
These results clearly show that the C-terminal region covering
the preS2 domain (data not shown) was deleted by degradation.
Accordingly, we conclude that the effective domain for nuclear
accumulation has to be present within this region.
Nuclear accumulation of preS proteins is enhanced
by co-incubation with synthetic preS peptides
For explaining the nuclear translocation and accumulation of
MBP-preS proteins, some kinds of interactions between
nuclear components and preS proteins have been assumed.
As shown above, the nuclear envelope of the detergent
(Triton X-100 or digitonin)-permeabilized cells remained
intact. Therefore, the preS protein itself must actively cross
the nuclear envelope. This might be achieved either by using
pre-formed channels on the nuclear envelope, i.e. the nuclear
pore complex, or by direct pore formation in the nuclear
envelope. In the former case, preS fragments might either
interact with conventional transport mediators (importin
alpha, beta) or otherwise directly bind to the nuclear pore
complex. However, the experimental results of Triton X-100
or digitonin-permeabilized cells (Figs 3, 4) exclude the
possibility of a conventional NLS-mediated transport. If
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JOURNAL OF CELL SCIENCE 114 (6)
Fig. 6. The effects of pre-incubation of synthetic preS(120-174)
peptides on the nuclear accumulation of MBP-preS proteins. To
examine the interaction mode of the preS proteins with the nuclear
components, permeabilized HepG2 cells were pre-incubated without
or with preS(120-174) peptides and then treated with FITC-labeled
MBP-preS (A,B) or FITC-labeled MBP proteins (D,E). As a control,
non-related peptide, HIV gp41(584-618), was pre-incubated as the
same amount of preS(120-174) peptide and the FITC-labeled
proteins were treated (C,F). At the end of the assay, cells were
washed and fixed with 4% (w/v) paraformaldehyde and analyzed by
confocal laser-scanning microscopy. Fluorescent intensity of each
cellular fraction (nucleus or cytosol) was quantitated using NIH
Image 1.60 public domain software after the subtraction of the
fluorescence intensity of the background. Relative fluorescence
intensity was determined using the fluorescence signal of the FITCMBP staining as standard (G).
of the nuclear membrane caused by high concentration of
synthetic peptides, the HIV gp41 (584-618) peptide was used
in the same concentration in an independent experiment as
negative control. In this case, no nuclear accumulation was
observed, as has it been the case with preS peptides (Fig.
6C,F). The nuclear translocation mechanism of preS proteins
could therefore be explained by the second possibility, which
suggest a direct pore-forming activity of preS proteins in
the nuclear envelope by the modulation of the nuclear
envelope itself, even transiently, which results in the nuclear
translocation and accumulation of these proteins. This
mechanism also could explain the energy- and cytosolic
factor-independency of the accumulation of preS proteins
within the nucleus.
there might be direct interactions between nuclear pore
components and the preS protein, the number of binding
sites should be restricted, and the binding should also be
saturable and competitive. To examine this possibility,
excess amounts of preS peptides were added to inhibit the
accumulation of FITC-labeled MBP-preS protein in a
competitive manner. As it was evident that the nuclear
accumulation effect is dependent on the entire preS2 region
(Fig. 5B), synthetic preS(120-174) peptides were used as
competitors. Surprisingly, pre-incubation with excessive
amounts of preS2 peptides (about 1000 molar ratio in excess)
did not inhibit but rather enhanced (about 1.5-fold) the
accumulation of the MBP-preS protein within the nucleus
(Fig. 6B). Furthermore MBP alone, which itself has no
nuclear accumulation effect, was now translocated into the
nucleus, though not to the extent of MBP-preS proteins (Fig.
6E). To exclude the possibility of a nonspecific perturbation
Conformational behavior of the preS2 peptides
under membrane mimicking conditions
Based on the present results, it is possible that the nuclear
translocation of the preS protein is caused by a membrane
perturbation effect. If there were any interaction between the
preS protein and the lipid bilayer of the nuclear envelope
during the nuclear translocation, some changes might occur in
the conformation of the preS protein or in the topological
arrangement of the lipid bilayer. To study the conformational
behaviors of the preS2 peptide upon interaction with the
nuclear envelope, its secondary structure was analyzed by CD
(circular dichroism) under different membrane-mimicking
conditions. Micelles of sodium dodecyl sulfate served as
an experimental model of the heterogeneous amphipatic
environment of a membrane lipid-aqueous interface, whereby
TFE (trifluoroethanol) solution was used to mimic the
homogeneous hydrophilic face of the membrane (Du et al.,
1998). According to the CD analysis, the whole preS2 peptide
in the aqueous solution was unstructured, without any typical
patterns of a α-helix or β-sheet structure in PBS (Fig. 7). But
in an environment that mimics the lipid bilayer (30 mM SDS
or 50% TFE), preS2 peptides exhibited a typical α-helical
pattern, with the minimum at about 208 nm and the shoulder
at 222 nm. The calculated α-helical contents were 19.8% in
30 mM SDS and 20.2% in 50% TFE. Although these values
indicate relatively low α-helical contents, it is evident that the
hydrophobic environments induce conformational changes of
preS2 peptides, which would provide a further clue about the
membrane-penetrating properties of preS2 peptide on the lipid
bilayer.
Nuclear accumulation of hepatitis B virus preS fragments
15
-3
2
-1
[Θ]MRWX10 (deg.cm .dmol )
10
PBS
30mM SDS
50% TFE
5
0
-5
-10
-15
-20
-25
190 200 210 220 230 240 250
wavelength (nm)
Fig. 7. CD spectra of preS(120-174) in a solution of 30 mM SDS,
50% TFE and in PBS.
DISCUSSION
The present study reports a novel characteristic of the HBV
preS envelope protein as a membrane-penetrating polypeptide.
The membrane-permeabilization effect of preS proteins was
shown by the nuclear translocation of exogenously added
recombinant (Figs 3, 4) or synthetic (Fig.6) preS peptides and
further analysis of the active domain determined the preS2
region as the translocation motif (Fig. 5). The mechanism by
which preS proteins can translocate themselves or fused
proteins through the nuclear membrane is not known. However,
mediation over classical NLSs can be largely ruled out as no
cluster of basic amino acids or other defined NLS motifs were
found within the preS region (Fig. 1). It seems rather that the
membrane-penetration effect depends on the conformational
configuration of the preS2 peptide, which might depend on the
presence of α-helical regions (as has been shown in this study
for the preS region (Fig. 7)) as well as has been reported for
other membrane-penetrating peptides (Derossi et al., 1996).
Another remarkable fact in this process is the absolute
independence of preS proteins on energy sources (Fig. 4),
similar in its property to the HSV VP22 or the third helix of
the antennapedia homeodomain, which have been previously
been described to migrate through cells in an energyindependent manner.
In general, the translocation of proteins across biological
membranes is highly selective and restricted and several
cellular factors are involved. However, there is also a growing
family of proteins and peptides that act via a protein
translocation mechanism, known as “nonselective membrane
translocation”, in which proteins cross biological membranes
(plasma membranes as well as nuclear envelopes) by
themselves without any mediators or energy supply. The
antennapedia homeodomain (AntpHD; Derossi et al., 1996),
the HIV-Tat transcription factor (Fawell et al., 1994; Vives et
al., 1997), the herpes virus VP22 protein (Elliot and O’Hare,
1121
1997) and lactoferrin (He and Furmanski, 1995) are some
examples of these. These proteins are produced by neighboring
cells and can diffuse through the plasma membrane and the
nuclear envelope of the target cells. This phenomenon is
known to be temperature independent and to be not saturable
by increasing concentration of the respective proteins.
Furthermore, the reverse sequence of these proteins also
showed translocation activities, indicating that these effects are
not dependent on the specific binding of chiral protein factors
(Derossi et al., 1996) but rather that they are induced by a
nonselective membrane permeabilization.
The nuclear translocation properties of preS are identical to
the characteristics of such membrane-permeable proteins in
terms of their energy independency (Figs 3, 4), mediatorindependent membrane translocation (Figs 3, 4), non-saturable
translocation effect (Fig. 5) and translocation of preS peptideconjugates (Fig. 3). Therefore, preS might also be regarded as
a new member of energy-independent membrane-penetrating
proteins and peptides. In this regard, it is expected that the
membrane-penetrating pathways of other peptides would
be similar to the mechanism of preS-mediated nuclear
translocation. Considering the mechanism of other membranepenetrating proteins, the most promising possibility seems
to be a preS protein-lipid interaction, which might cause
membrane pertubation that would lead to increased
permissibility of biological membranes. Such a hypothesis has
been tested out, for example, in the case of the AntpHD, where
the cellular entry of various mutants was analyzed (Berlose et
al., 1996). The results from this study proposed that the
interactions between positively charged amino acid residues,
negatively charged phospholipids and α-helical conformations
in a membrane environment made these peptides prone to
interactions with membrane lipids, which resulted in
transmembrane conformations. The already known membranepenetrating proteins, however, do not share sequence
homology with each other or with the preS protein.
Nevertheless, the amino acid residues found within preS
consist mostly of those that have the tendency to form an αhelical conformation; indeed, CD analysis (Fig. 7) showed that
preS peptides in membrane-mimetic environments adopt αhelical conformations, which could act as initiator for proteinlipid interactions. The importance of the preS α-helicity was
recently confirmed in another study (Oess and Hildt, 2000),
where the energy-independent membrane-penetration effect of
an preS2(161-172) fragment was reported. Oess and Hildt
made a similar observation to that described in the present
study, which compromised the plasma membrane translocating
effect of the preS2(161-172) region. However, neither the
biological meaning of this effect nor its role in the viral life
cycle could be clarified, which raised questions about the role
of the membrane translocating ability of viral structural
proteins in general.
However, for the HSV-1 VP22 protein, the HIV-Tat protein
and other membrane-permeable proteins, their property of
energy-independent membrane translocation has been already
discussed and described in context of some possible biological
functions. These include activities such as those undertaken by
microtubule-associated proteins during infection (Elliott and
O’Hare, 1998) and by gene regulatory molecules (Rappaport
et al., 1999). Accordingly, it is possible that the unusual
property as exerted by free preS proteins might also have a role
1122
JOURNAL OF CELL SCIENCE 114 (6)
in vivo. The biological roles for preS fragments can be
proposed in two aspects. The first hypothetical role is based
on the nuclear membrane-permeabilization effect of the preS
proteins, while the second relies more on the observation of
nuclear accumulation of the translocated preS/preS2 peptides.
Regarding the first possibility, a functional role for preS
peptides as mediators for transporting the viral genome into the
nucleus can be proposed. For viruses of the hepadna family,
including HBV, the delivery of the virus genome into the host
cell nucleus is a prerequisite for further progress in the virus
life cycle (Summers and Mason, 1982). However, the exact
mechanism for uncoating and nuclear delivery of HBV is
poorly characterized. Based on cases of other viruses, some
hypotheses have been suggested. One is that the viral genome
is imported by direct attachment of viral particles to the nuclear
membrane and that it is subsequently released through the
channel of the nuclear complex, as it is the case in adenoviruses
(Greber et al., 1996). Recent reports on the direct binding of
HBV core particles to the nuclear core complex support such
view (Kann et al., 1999). Alternatively, it is also assumed that
the virus capsid is disassembled in the cytosol and that the
genome is translocated into the nucleus with the help of some
viral proteins, such as the covalently associated HBV DNA
polymerase (Foster et al., 1991; Kann et al., 1997) or core
proteins (Yeh et al., 1990). Although, so far, none of these
hypotheses has been definitively proved, relying on the results
from the present study, a further scheme can be suggested
where the preS protein acts as a putative mediator in targeting
the core particle or the viral DNA into the nucleus.
This idea is not so far-fetched when regarding the reports
that state the nuclear membrane is impermeable into both
directions to core particles (Guidotti et al., 1994), which makes
it necessary to disassemble the viral capsid in the cytosol for
delivering the virus genome into the nucleus. In this case,
the preS proteins would either directly carry the HBVdisassembled complex into the nucleus or facilitate the nuclear
translocation of the HBV genome by permeabilization of the
nuclear membrane (as has been described in this study; Fig. 4).
Naturally, such hypothetical views need more supporting data,
and further studies with HBV core particles or viral genome
and free preS peptides are necessary to prove the validity of
this hypothesis.
Regarding the second possibility on the in vivo function of
preS-mediated nuclear translocation, another interesting aspect
of free preS fragments must be considered. A large body
of evidences has confirmed that the truncated form of
MHBs (Kekule et al., 1990) and preS/S2 fragments (Hildt
et al., 1995), as well as preS1 proteins (Kim et al., 1997),
are novel transcriptional transactivators. The mode of
transcativating promoters of cellular genes, including c-myc
and c-fos, by these viral transactivators has been extensively
studied (Lauer et al., 1994; Hildt and Hofschneider, 1998). It
was shown that they may act in the cytoplasm by the activation
of the protein kinase C (PKC). However, the PKC signal
triggers the c-raf-1/MAP2-kinase signal transduction pathway,
which finally results in the activation of transcription factors
such as AP-1 and NF-κB (Hildt and Hofschneider, 1998).
While such studies were exclusively based on the assumption
of a cytosolic orientation of preS/S2 domains, the current
finding opens a new possibility for preS proteins in exerting
transactivator functions by acting directly in the nucleus.
Regarding this observation, it is assumable that truncated preS
fragments would freely enter the nucleus and might interact
with nuclear components (including other transactivation
factors), resulting in the accumulation and perhaps direct
transactivation of cellular genes.
While the novel functions for the HBV preS region, as
suggested in the present study, might fill in the missing links
in the HBV infection pathway, it is unlikely that such preSmediated nuclear translocation or a possible nuclear
transactivator function would be the sole mechanism for the
HBV replication after the HBV infection. Le Seyec et al., for
example, showed that the entire preS2 region was dispensable
for a successful HBV infection (Le Seyec et al., 1998). The
preS(3-77) region, however, was shown in the same study to
be essential for infection, which is possibly due to the presence
of a binding region for the cellular HBV receptors. As other
reports have also observed that the preS2 region is not
necessarily a major component in the HBV entry (Fernholz et
al., 1993; Sureau et al., 1994) and infection pathway, the preS2mediated membrane translocation might be not necessarily
represent a major step for HBV infection or replication. Rather,
it could describe just one of many redundant pathways that
HBV can follow in the infection of the host cells.
In conclusion, the unveiling of the exact biological role of
free preS proteins, either in serving as a mediator for nuclear
translocation or as a nuclear transactivator, will greatly broaden
our current understanding about the early events in HBV
infection.
The authors thank Dr M.K. Lee for synthesis of the preS(1-174)
peptide. This study was supported in part by a grant (BG630M) of the
Ministry of Health and Welfare and a grant (HS2470, NL1010) from
the Ministry of Science and Technology, Korea.
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