From the Institutes of Biochemistry,
§ Microbiology, and ¶ Molecular Medicine, College of
Medicine, National Taiwan University, No. 1, Jen-Ai Rd., First
Section, Taipei, Taiwan
Received for publication, May 24, 2000, and in revised form, October 30, 2000
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ABSTRACT |
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Hepatitis delta virus (HDV) is a satellite
virus of hepatitis B virus, as it requires hepatitis B virus for
virion production and transmission. We have previously demonstrated
that sequences within the C-terminal 19-amino acid domain flanking the
isoprenylation motif of the large hepatitis delta antigen
(HDAg-L) are important for virion assembly. In this study,
site-directed mutagenesis and immunofluorescence staining demonstrated
that in the absence of hepatitis B virus surface antigen (HBsAg), the
wild-type HDAg-L was localized in the nuclei of transfected COS7 cells.
Nevertheless, in the presence of HBsAg, the HDAg-L became both nuclei-
and cytoplasm-distributed in about half of the cells. An HDAg-L mutant
with a substitution of Pro-205 to alanine could neither form HDV-like
particles nor shift the subcellular localization in the presence of
HBsAg. In addition, nuclear trafficking of HDAg-L in heterokaryons
indicated that HDAg-L is a nucleocytoplasmic shuttling protein. A
proline-rich HDAg peptide spanning amino acid residues 198 to 210, designated NES(HDAg-L), can function as a nuclear export signal (NES)
in Xenopus oocytes. Pro-205 is critical for the NES
function. Furthermore, assembly of HDV is insensitive to
leptomycin B, indicating that the NES(HDAg-L) directs nuclear export of
HDAg-L to the cytoplasm via a chromosome region maintenance
1-independent pathway.
Hepatitis delta virus
(HDV)1 consists of a
single-stranded circular RNA genome of ~1.7 kilobases and the
only known HDV-encoded protein, hepatitis delta antigen (HDAg),
enveloped by the hepatitis B virus surface antigen (HBsAg) (1-3). In
the livers and sera of HDV-infected patients, there are two forms of
HDAg, small HDAg (HDAg-S) and large HDAg (HDAg-L). The two HDAgs are
identical in sequence except for an additional 19-amino acid extension
at the C terminus of the HDAg-L (1, 2, 4). The HDAgs share identical
functional domains within the common region, but exhibit very distinct
functions in viral multiplication. The HDAg-S is required for the
replication of HDV RNA, whereas the HDAg-L is essential for virion
assembly and functions as a potent trans-dominant inhibitor
in viral replication (5-10).
Previous studies have demonstrated that the HDAg-L is capable of
copackaging with the small HBsAg to form virus-like particles in both
transfected Huh-7 and COS7 cells (7-11). HDAg-L is a nuclear phosphoprotein (12), whereas HBsAg confines to the cytoplasm. Extensive
deletion analysis of the HDAg-L revealed several functional motifs,
including nuclear localization signals and oligomerization domains that
are dispensable for the formation of virus-like particles (10, 13). In
addition, the unique domain spanning amino acid residues 198-214 of
the HDAg-L was demonstrated to contain signals sufficient for HDV
assembly (10, 13). These results implied that the HDV assembly occurs
in the cytoplasm. Analysis within the isoprenylation motif of the
HDAg-L (211-CRPQ-214) revealed a good correlation between package
activity and the status of isoprenylation (14), but attachment of an
isoprenylation motif did not render the HDAg-S a structure capable of
copackaging with HBsAg (10). These results further suggested that the
13-amino acid stretch proceeding the isoprenylation motif of the HDAg-L plays a critical role on HDV assembly. In this study, we have further
elucidated the molecular mechanisms by which the 13-amino acid stretch
contributes to the assembly of HDV. Site-directed mutagenesis within
the 13-amino acid stretch indicated that Pro-205 is critical for the
relocalization of HDAg-L from the nucleus to the cytoplasm and is
involved in the assembly of HDV. An interspecies heterokaryon assay
clearly demonstrated that the HDAg-L is a nucleocytoplasmic shuttling
protein. In addition, the C-terminal domain spanning amino acid
residues 198-210 of the HDAg-L could function as a nuclear export
signal, which directs nuclear export of the HDAg-L to the
HBsAg-localized cytoplasm via a CRM1-independent pathway.
Plasmids
Plasmids pECE-d-BE, pECE-d-SM, pECE-C-ES, and
pCsrev-GFP--
Plasmids pECE-d-BE and pECE-d-SM contain cDNAs
encoding the HDAg-L and HDAg-S, respectively (12, 15). Plasmid
pECE-C-ES encodes the small forms of the HBsAg, p24 and gp27 (10).
Plasmid pCsrev-GFP (a gift from G. N. Pavlakis, NCI-Frederick
Cancer Research and Development Center) encodes a fusion protein of the
human immunodeficiency virus (HIV) Rev and the green fluorescent
protein (GFP) (16).2 These
plasmids were used in transfection experiments.
Plasmid pB1-3(NdeI)--
Plasmid pB1-3(NdeI) was
derived from pT7-d-BP (12) by removing the cDNA domain representing
the 5' untranslated region of the HDAg-L. This plasmid was generated
for further construction of HDAg-L mutant plasmids.
HDAg-L Mutant Plasmids--
HDAg-L mutant plasmids encode large
HDAgs with point mutations in the region spanning amino acid residues
198-210. The strategy that we used, with modifications, for
constructing the mutant plasmids has been described previously (17,
18). In brief, a degenerate 65-mer oligonucleotide C244 was chemically
synthesized (5'-TCACTGGGGTCGACAAcTCtGGGgAGaGaAGGgAGgGTcGGcTGgGaAGaGTaTATCCCATGGGA-3'; lowercase letters represent degenerate nucleotides consisting of
91% of the indicated wild-type nucleotide sequences of the corresponding cDNA of the HDAg and 3% each of the other three nucleotides; underlining indicates the extra sequences, including SalI and NcoI recognition sites for cloning
purpose). The C244 oligonucleotide was converted into a double-stranded
DNA by base pairing two molecules around the NcoI site and
mutually primed synthesis with the Klenow fragment of DNA polymerase I. The annealing reaction was performed in a buffer containing 140 mM Tris-HCl, pH 7.6, 15 mM MgCl2,
25 mM dithiothreitol at 65 °C for 30 min and cooled to
room temperature over a period of 2 h. The extension reaction was
carried out in the presence of 10 mM each of the dNTP for
at least 3 h at 37 °C. Following digestions with
SalI and NcoI restriction endonucleases, the
resultant 49-base pair SalI-NcoI fragments were
purified from a 2% agarose gel and used to replace the cognate
fragment of plasmid pB1-3(NdeI). Mutations were identified
by DNA sequencing. Plasmids with desired mutations were isolated and
treated with SmaI and SalI restriction
endonucleases. The resultant SmaI-Sal I fragments were used
to replace the cognate fragment of pECE-d-SM. These generated plasmids
that encode HDAg-L mutant proteins with amino acid substitutions
spanning amino acid residues 198-210. An overview of the amino acid
substitutions in the HDAg-L mutant proteins is given in Fig.
1A.
Cell Lines, DNA Transfection, and Indirect Immunofluorescence
Staining
Monkey kidney cells (COS7), human cervical carcinoma cells
(HeLa), and mouse embryonic fibroblast cells (NIH3T3) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum plus 100 units of penicillin and 100 µg of
streptomycin/ml. DNA transfection (19) and indirect immunofluorescence
staining (20) was performed as described previously, except that cells were seeded at 15-20% confluency 16 h prior to transfection.
Harvest of Virus-like Particles and Determination of Package
Activity
To determine the package activity of HDAg-L mutants, virus-like
particles were collected from culture media as described previously (10), except that the media were harvested 4 days posttransfection. In
addition, the viral pellets were resuspended in 2× sample buffer (12.5 mM Tris-HCl, pH 6.8, 2% SDS, 20% glycerol, 0.25%
bromphenol blue, 5% Immunoblot Analysis
To perform immunoblot analysis, protein lysates separated by
SDS-polyacrylamide gel electrophoresis were electrotransferred onto an
Immobilon-P membrane (Millipore) at 800 V for 2 h at 4 °C. The
membrane was blocked with 5% nonfat dried milk in phosphate-buffered saline (PBS) for 1 h at room temperature in a shaker bath and then
incubated at 4 °C overnight with specific antibodies as follows. For
determination of the package activity of HDAg-L mutants, three-fourths of viral pellet collected from the culture media was analyzed with
rabbit polyclonal antibodies specific to HDAg (21) at 1:2000 dilution
in PBS containing 1% nonfat dried milk, and one-fourth of the viral
pellet was analyzed with goat polyclonal antibodies specific to HBsAg
(Dako) at 1:2000 dilution in the same buffer. The membrane was then
washed at room temperature three times for 10 min each with PBS
containing 0.5% Tween 20, followed by an incubation with
peroxidase-conjugated IgG (Pierce) and staining with
3,3'-diaminobenzidine tetrahydrochloride as described previously (22),
except that instead of the SIB (140 mM NaCl, 8 mM Na2HPO4, 2.7 mM KCl,
1.5 mM KH2PO4, pH 7.2), Tris-HCl
(50 mM, pH 7.4) was used as the buffer system. In nuclear
export assay, the nuclear and cytoplasmic fractions of microinjected
oocytes of Xenopus laevis were analyzed for the presence of
rabbit IgG-conjugated HDAg peptides with horseradish peroxidase-coupled
goat anti-rabbit IgG (Jackson) at a 1:5000 dilution in PBS containing
1% nonfat dried milk and 0.05% Tween 20. Mouse IgG that was used as
an internal control was detected by horseradish peroxidase-coupled goat
anti-mouse IgG (Jackson) at a 1:10,000 dilution in the same buffer. The
specific interactions between antigens and antibodies were detected by the enhanced chemiluminescence system (Amersham Pharmacia
Biotech.).
Interspecies Heterokaryon Assay
The heterokaryon assay was performed as described previously
(23, 24) with modifications. Briefly, HeLa cells were seeded onto
coverslips at 20% confluency and transfected with
HDAg-L-encoding plasmid pECE-d-BE on the next day. At
9 h posttransfection, NIH3T3 cells were added to yield 40%
confluency, and cell fusion was carried out at 30 h
posttransfection to promote interspecies heterokaryon formation.
Cycloheximide (100 µg/ml) (Sigma) was added 1 h prior to cell
fusion, and cell fusion was performed for 2 min with 50% polyethylene
glycol 6000 (Merck) in PBS containing 0.1% glucose. Following an
extensive wash and an additional incubation for 5 h in the culture
medium containing cycloheximide, the cells were fixed and
immunofluorescence staining was performed as described earlier.
Cell nuclei were detected by Hoechst 33258 (1 µg/ml) (Sigma).
The dye gives differential staining patterns of the nuclei of mouse
NIH3T3 cells and human HeLa cells. Cells were viewed using a Zeiss
Axiophot 2 fluorescence microscope.
Conjugation of Synthetic Peptides to Rabbit Immunoglobulin
Peptides NES(HDAg-L) and NES*(HDAg-L) that represent HDAg-L from
amino acid residues 198-210 (ILFPADPPFSPQS) and the peptide with
Pro-205 replaced by Ala (ILFPADPAFSPQS, underlining
indicates the mutation), respectively, were synthesized by peptide
synthesizer (model 431, Applied Biosystems Inc.). Both peptides possess
an additional cysteine residue at the NH2 termini for
conjugation to affinity-purified rabbit IgG (IgG(r)) (Sigma).
Activation of IgG(r) with the bifunctional cross-linking reagent
sulfosuccinimidyl 4-(N-maleimidomethyl)
cyclohexane-1-carboxylate and conjugation to the synthetic peptides
were carried out as described previously (25) with modifications. In
brief, 8 mg of sulfosuccinimidyl 4-(N-maleimidomethyl)
cyclohexane-1-carboxylate were added to 8 mg of IgG(r) in 1 ml of PBS,
pH 7.4, and incubated for 1 h at 20 °C. The activated IgG(r)
was subsequently separated from excess sulfosuccinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate by passing
through a Sephadex P-6 column (Amersham Pharmacia Biotech) equilibrated
with PBS, pH 7.4, and conjugated to equal amount of the synthetic
peptides NES(HDAg-L) and NES*(HDAg-L). The reaction was carried out in
PBS, pH 6.5, at room temperature for 3 h. Free peptides were
removed by passing the mixture through a Sephadex P-6 column.
The coupling ratio of both conjugates, IgG(r)-NES(HDAg-L) and
IgG(r)-NES*(HDAg-L), were estimated to be 10 peptides per IgG(r)
molecule as analyzed by SDS-10% polyacrylamide gel electrophoresis (data not shown). For microinjection, the conjugates were concentrated to 20 mg/ml using a Centricon 30 (Amicon).
Microinjection and Nuclear Export Assay
For the study of nuclear export of HDAg peptides, stage VI
oocytes from ovaries of X. laevis females were manually
defolliculated, maintained at 20 °C in OR2 medium (825 mM NaCl, 2.5 mM KCl, 1.0 mM
CaCl2, 1.0 mM MgCl2, 1.0 mM Na2HPO4, 5.0 mM
HEPES, pH 7.8), and used for microinjection within 3 days. For direct
injection of protein samples into nuclei, the oocytes were carefully
spun at 3,000 rpm for 10 min in an HS-4 rotor (Sorvall) prior to
injection. Microinjection needles were prepared on a Flaming/Brown
micropipette puller (model P-97, Sutter Instrument Co.) using
borosilicate glass capillaries (0.75-1.0 mm, Sutter Instrument Co.).
Rabbit IgG conjugated peptides IgG(r)-NES(HDAg-L) and
IgG(r)-NES*(HDAg-L) were injected at a final concentration of 10 mg/ml,
and an affinity-purified mouse IgG was coinjected as an internal
control. The protein samples were centrifuged at 15,000 × g for 10 min at 4 °C immediately before injection.
Following injections, the oocytes were incubated at room temperature
for 1.5 h and then dissected manually in OR2 medium to separate
the nuclei from cytoplasmic fraction. The cytoplasmic and nuclear
fractions were homogenized by sonication four times for 15 s each
and centrifuged at 12,000 × g for 10 min to remove insoluble pellets. The resulting nuclear and cytoplasmic fractions were
subjected to SDS-10% polyacrylamide gel electrophoresis and immunoblot
analysis as described earlier.
Leptomycin B Treatment
For analysis of the effect of leptomycin B on the subcellular
distribution of Rev-GFP fusion protein, leptomycin B (provided by
Norvatis) was added to the culture media at a final concentration of 2 nM at 24 h posttransfection, and actinomycin D (1 µg/ml) was added 2 h prior to cell fixation. For evaluation of
the effect of leptomycin B on HDV assembly, leptomycin B was added at
24 h posttransfection at a final concentration of 2 or 20 nM for various time periods prior to immunoblot analysis.
A Mutation at Pro-205 of the HDAg-L Affects the Assembly of
HDV--
Previous studies have demonstrated that the unique C-terminal
domain of the HDAg-L, encompassing a Pro-rich region and an isoprenylation motif 211-CRPQ-214, possesses signals sufficient for the
assembly of HDV (10, 13, 14). To determine specific amino acid residues
that contribute to HDV assembly, site-directed mutagenesis in the
domain composed of amino acid residues 198-210 of the HDAg-L was
performed as described under "Experimental Procedures." Following
transformation, 120 recombinant DNA clones were picked for DNA
sequencing, from which 41 substitution mutants of the HDAg-L were
identified (data not shown). Eight mutant constructs harboring single
amino acid substitution were selected (Fig.
1A) for virion package assay.
As shown in Fig. 1B, all of the eight constructs expressed
mutant proteins in COS7 cells at levels similar to that of the
wild-type HDAg-L (top panel). When virus-like particles in
the culture media were harvested and analyzed for the presence of HBsAg
and HDAg-L, both forms of the small HBsAg, p24 and gp27, were detected
at comparable levels among the wild-type control and mutants (Fig.
1B, bottom panel). Nevertheless, the HDAg-L with
single substitution of Pro-205 to Ala, designated HDAg(P205A), was not
detected in the culture medium (Fig. 1B, middle
panel). These results indicated that Pro-205 is critical for the
package activity of HDAg-L and the assembly of HDV. It is noteworthy
that the amino acid substitution at the adjacent Pro-204 residue had little effect on the package activity. The variation of the package activity of the various HDAg-L mutants should reflect the intrinsic properties of individual amino acid residues. As expected, the HDAg-S
that lacks the unique C-terminal sequence of HDAg-L failed to form
virus-like particles with the small HBsAg (Fig. 1B,
lane HDAg-S).
Cytoplasmic Localization of HDAg-L in the Presence of Small
HBsAg--
A previous study demonstrated that isoprenylation of HDAg-L
mediates the interaction between HDAg-L and HBsAg in vitro
and proposed that the interaction is the molecular basis of HDV
assembly (26, 27). However, this hypothesis does not explain where and
how these two proteins come to direct contact in vivo
because HBsAg is localized in the cytoplasm and HDAg-L is a nuclear
phosphoprotein (12). To understand how the interaction occurs, COS7
cells were transfected with plasmids encoding HDAg-L and HBsAg, and
immunofluorescence staining was performed at 12 h intervals 2 days
posttransfection. At 48 h posttransfection, HDAg-L was detected
exclusively in the nucleolus of transfected cells as the Type I pattern
shown in Fig. 2A. At 60 h
posttransfection, about 25-35% of the transfected cells retained the
Type I nucleolus staining pattern, but 65-75% of the cells exhibited
both nucleolus and nucleoplasm staining pattern (Type II pattern) (Fig.
2A). The relocalization of HDAg-L from the Type I to the
Type II pattern at 60 h posttransfection was also observed in the
absence of HBsAg (Fig. 2B). A striking change was observed
at 72 h posttransfection; the HDAg-L became both nuclei- and
cytoplasm-distributed (Fig. 2A, Type III) in about 43-47%
of the transfected cells in the presence of small HBsAg but was
restricted to the nucleolus and nucleoplasm in up to 92-95% of the
cells in the absence of HBsAg (Fig. 2B). In addition, when
COS7 cells were cotransfected with plasmids encoding HDAg-S and small
HBsAg, the HDAg-S confined to the nuclei similar to that of cells
transfected with HDAg-L-encoding plasmid alone (data not
shown).
The Mutation at Pro-205 Abolished the Cytoplasmic Relocalization of
HDAg-L in the Presence of Small HBsAg--
The unique property of
HDAg-L to relocalize to the cytoplasm in the presence of small HBsAg
implied that the intracellular interaction between HDAg-L and HBsAg
occurs in the cytoplasm and is mediated by the unique C terminus
of the HDAg-L. We therefore made an assumption that the relocalization
of HDAg-L to the cytoplasm is an essential step for HDV assembly. To
examine this hypothesis, immunofluorescence staining was performed at
72 h posttransfection with the package-defective HDAg(P205A)
and the package-competent HDAg(P204A) mutant proteins in the presence
of small HBsAg. Interestingly, we observed a similar subcellular
distribution pattern for the wild-type HDAg-L and HDAg(P204A) mutant
protein, but the package-defective HDAg(P205A) mutant protein failed to
relocalize from the nucleus to the cytoplasm. The statistical results
are summarized in Fig. 3. The correlation
between relocalization and package activity of the HDAg-L suggested
that the cytoplasmic relocalization of HDAg-L is facilitated by the
unique C terminus of the HDAg-L and is essential for the viral
assembly; the mutation at Pro-205 abolished cytoplasmic relocalization
and rendered HDAg-L package-defective.
HDAg-L Is a Nucleocytoplasmic Shuttling Protein--
The
cytoplasmic relocalization of HDAg-L and its assembly with the
cytoplasm-localized HBsAg suggested that HDAg-L bears an NES located in
the unique C-terminal domain to facilitate the translocation. To
examine this possibility, an interspecies heterokaryon assay was
performed in the absence of HBsAg. HeLa cells were transfected with
HDAg-L-encoding plasmid, fused with NIH3T3 cells in the
presence of cycloheximide, and analyzed by fluorescence microscopy.
Nuclei in the heterokaryons were readily distinguished by DNA stain
Hoechst 33258; the NIH3T3 nuclei have a speckled staining pattern,
whereas the HeLa cell nuclei were evenly stained (Fig.
4, B and D). When the interspecies heterokaryons were analyzed with antibodies to HDAg-L,
the HDAg-L was detected in both nuclei of the transfected HeLa cells
and the untransfected NIH3T3 cells (Fig. 4, A and
C). Because the new protein synthesis was blocked by
cycloheximide, the nuclear trafficking of HDAg-L in heterokaryons
indicated that HDAg-L possesses an NES and is a nucleocytoplasmic
shuttling protein.
The Subdomain Composed of Amino Acid Residues 198-210 of
the HDAg-L Can Function as a Nuclear Export Signal in a Heterologous
Context--
To further examine whether the NES of HDAg-L is located
in the unique C-terminal domain, an HDAg peptide, NES(HDAg-L),
representing amino acid residues 198-210 of HDAg-L, was synthesized
and chemically conjugated to rabbit IgG (Fig.
5A). The resultant
IgG(r)-NES(HDAg-L) was coinjected with a control mouse IgG (IgG(m))
into the nuclei of Xenopus oocytes. At 1.5 h
postinjection, the oocytes were dissected manually into nuclear and
cytoplasmic fractions, and immunoblot analysis was performed to detect
the subcellular localization of the IgG(r)-NES(HDAg-L) and IgG(m).
Results demonstrated that a majority of the IgG(r)-NES(HDAg-L)
transported from the nucleus to the cytoplasm, whereas the control
IgG(m) retained to be nucleus-localized (Fig. 5B, left
panel). These results indicated that the HDAg-L peptide spanning
amino acid residues 198-210 acted as an NES to escort IgG(r) to the
cytoplasm. To correlate the nuclear export function of HDAg-L to the
assembly of HDV, an HDAg-L mutant peptide with Pro-205 replaced by Ala
was synthesized and conjugated to IgG(r) (Fig. 5A). The
resultant IgG(r)-NES*(HDAg-L) was coinjected with IgG(m) into the
nuclei of Xenopus oocytes. Immunoblot analysis demonstrated
a retention of IgG(r)-NES*(HDAg-L) in the nucleus (Fig. 5B, right
panel). Because HDAg(P205A) was shown to be package-defective (Fig. 1B), these results indicated that Pro-205 is critical
for both the nuclear export of HDAg-L and the assembly of HDV.
Leptomycin B Has Little Effect on the Assembly of HDV--
HDAg-L
has previously been demonstrated to be important for the assembly of
HDAg-S and HDV RNA (28, 29). Our current data strongly suggest that the
NES(HDAg-L) spanning amino acid residues 198-210 of the HDAg-L plays
an essential role in the assembly of HDV by triggering the nuclear
export of HDAg-L. Unlike the prototype leucine-rich NES of the Rev
protein of HIV-1 (Fig. 6), the amino acid
sequences of the NES(HDAg-L),
198ILFPADPPFSPQS210, is rich in proline
residues. Previous studies have identified CRM1 as a receptor for the
leucine-rich NES (30-32). In addition, CRM1-dependent
nuclear export could be specifically blocked by leptomycin B (33, 34).
To investigate whether CRM1 is involved in the nuclear export of HDAg-L
and the assembly of HDV, a system in which leptomycin B blocked the
nuclear export of the HIV Rev protein was first established. The
nucleolus-localized Rev protein exported to the cytoplasm of
transfected COS7 cells when RNA synthesis was inhibited by actinomycin
D. Such a translocation was abolished when leptomycin B was added to
the culture media at 24 h posttransfection (Fig.
7A). This system was used to
study the effect of leptomycin B on HDV assembly. COS7 cells were
transfected with plasmids encoding HDAg-L and small HBsAg, and
leptomycin B was added at 24 h posttransfection. At 48 and 72 h after the addition of leptomycin B, culture media were harvested and
analyzed for the package activity of HDAg-L. Interestingly, we found
that leptomycin B had no significant effect on the package activity of
HDAg-L (Fig. 7B), suggesting that the nuclear export of
HDAg-L is mediated by a CRM1-independent pathway distinct from that of
Rev-like leucine-rich NES.
In this study, we have demonstrated the nuclear trafficking of
HDAg-L in heterokaryons and determined that amino acid residues 198-210 within the unique C-terminal domain of the HDAg-L can function
as a nuclear export signal. In addition, Pro-205 is critical for the
package activity and nuclear export of HDAg-L, but a mutation at the
adjacent residue Pro-204 has little effect on the package and
subcellular distribution. The cytoplasm targeting of HDAg-L is a
prerequisite for the assembly of HDV.
Nuclear export signals have been identified in both viral and cellular
proteins (Fig. 6) (35-37). The Rev protein of HIV-1 binds to Rev
response element and escorts unspliced and partially spliced viral RNAs
to the cytoplasm; the export is mediated by a leucine-rich NES
(35-38). In addition, leptomycin B specifically abrogated the export
of the Rev protein and viral RNA by binding to the cysteine residue in
the middle domain of CRM1, a receptor for the leucine-rich NES (Fig.
7A) (33, 34, 39). These results indicate that the nuclear
export of the Rev protein plays an essential role in the life cycle of
HIV-1. In the present study, we have identified an NES of HDAg-L
spanning amino acid residues 198-210 (Fig. 5). The NES(HDAg-L)
represents an atypical NES that is rich in proline (Fig. 6). In
addition, the feature of insensitive to leptomycin B (Fig. 7) suggest
that the NES(HDAg-L) drives nuclear export of the HDAg-L via a
CRM1-independent pathway different from that of the Rev-like NES.
Interestingly, a recent study also identified a CRM1-independent
nuclear export of heterogeneous RNP K (40). However, the NES of
heterogeneous RNP K is rich in serine and acidic residues (Fig. 6).
These indicate that different mechanisms mediated by a variety of
nuclear export signals are involved in the nuclear export pathway. The
molecular mechanism of HDV assembly is not fully understood, and
cellular receptors participate in the nuclear export remain to be
elucidated. However, the present study has provided some information.
Our current working hypothesis is that HDV genomic RNA forms RNP
complexes with the HDAgs that translocate from nucleoplasm to the
nuclear membrane via an interaction between NES(HDAg-L) and an
unidentified NES receptor. The RNP complexes then move further to the
endoplasmic reticulum for assembly with HBsAg. The isoprenylation motif
that is known to be important for the package activity of HDAg-L (10, 14, 26, 27) and additional cellular factors, including nucleophorins, may also participate in the translocation process. To complete the life
cycle of HDV, progeny viral particles transport to the plasma membrane
and exocytosis occurs to release mature viral particles into the
extracellular milieu.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol), boiled for 3 min, and
subjected to SDS-12% polyacrylamide gel electrophoresis and immunoblot
analysis to examine the presence of HBsAg and HDAg mutants.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Package activity of the HDAg-L mutants with
amino acid substitutions in the unique C-terminal domain.
A, summary of the amino acid substitutions and the package
activity of the HDAg-L mutants. HDAg-L mutants with single
substitutions in the domain spanning amino acid residues 198-210 are
indicated. The activity of each HDAg-L mutant to form virus-like
particles with small HBsAg is indicated by a plus or
minus sign. B, immunoblot analysis. COS7 cells
cultured on 10-cm dishes were cotransfected with 10 µg each of
plasmid pECE-C-ES encoding the small HBsAg and a plasmid encoding the
wild-type HDAg-S or HDAg-L, or a mutant form of the HDAg-L as
indicated. At 4 days posttransfection, immunoblot analysis was
performed with the cell lysates (top panel) and viral
pellets collected from the culture media (middle and
bottom panels) with antibodies specific to HDAg and HBsAg,
as indicated.
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Fig. 2.
Cytoplasmic relocalization of HDAg-L in the
presence of small HBsAg. COS7 cells were transfected with the
HDAg-L-encoding plasmid pECE-d-BE alone or together with
the HBsAg-encoding plasmid pECE-C-ES. At 48, 60, and 72 h
posttransfection, indirect immunofluorescence staining was performed
with rabbit antibodies specific to HDAg. A, representative
patterns of the subcellular distribution of HDAg-L. The staining
patterns were classified into three types. Type I, nucleoli
staining; Type II, both nucleoli and nucleoplasm staining;
Type III, nucleoli, nucleoplasm, and cytoplasm staining.
B, distribution of HDAg-L in the absence or presence of
small HBsAg. Following immunofluorescence
staining at various time points posttransfection, fields
each containing at least 150 HDAg-positive cells were randomly
selected. Cell numbers bearing each type of the defined staining
patterns of HDAg-L were counted and plotted as the percentage of the
total number of the HDAg-positive cells in the same field. The
statistical results represent the average of two independent
experiments.
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Fig. 3.
The mutation at Pro-205 abolished the
cytoplasmic relocalization of HDAg-L. COS7 cells were
cotransfected with the HBsAg-encoding plasmid and a plasmid
encoding the wild-type (WT) or mutant HDAg-L as indicated.
Indirect immunofluorescence staining was performed 72 h
posttransfection. Representative cells with the classified distribution
patterns of the HDAg-L are essentially as those shown in Fig.
2A. The results are presented statistically as described in
Fig. 2B.
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Fig. 4.
Nuclear trafficking of HDAg-L in
heterokaryons. HeLa cells were transfected with the
HDAg-L-encoding plasmid pECE-d-BE and cocultivated with
mouse NIH3T3 cells 9 h posttransfection. Cell fusion was performed
in the presence of cycloheximide on the next day. Five hours after
heterokaryon formation, cells were fixed and analyzed by fluorescence
microscopy with antibodies to HDAg-L ( HDAg-L) (A and
C) or with the DNA stain Hoechst 33258 (B and
D). The images were captured with a charge-coupled device
camera (Kodak). B and D show cells of the same
fields as A and C, respectively.
Arrows indicate the NIH3T3 nuclei in the heterokaryons.
Nuclear trafficking of HDAg-L occurred from the transfected HeLa cells
into the cocultivated NIH3T3 cells.
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Fig. 5.
A subdomain spanning amino acid
residues 198-210 of the HDAg-L can function as a nuclear export signal
in the oocytes of X. laevis. A,
rabbit IgG conjugates used in this study. IgG(r)-NES(HDAg-L) represents
a rabbit IgG conjugate of the HDAg-L peptide spanning amino acid
residues 198-210, whereas IgG(r)-NES*(HDAg-L) represents a conjugate
in which the HDAg peptide harbors an amino acid substitution of Pro-205
to Ala. B, the HDAg-L peptide spanning amino acid residues
198-210 possesses an NES activity. The rabbit IgG conjugates of HDAg-L
peptides as indicated were coinjected with a control IgG(m) into the
nuclei of Xenopus oocytes. At 1.5 h after injection,
two oocytes from each of the injected groups were randomly selected and
dissected manually into nuclear (N) and cytoplasmic
(C) fractions. Immunoblot analysis was performed with goat
anti-rabbit IgG and goat anti-mouse IgG as indicated.
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Fig. 6.
Comparison of nuclear export signals.
The amino acid sequences of the nuclear export signals of HIV-1 Rev
protein (Rev) (35), protein kinase inhibitor
(PKI) (41), hnRNP K (40), and HDAg-L are listed for
comparison. Numbers refer to the positions of the amino acid
sequences in each protein. The rich amino acid residues in each of the
nuclear export signals are shown in boldface.
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Fig. 7.
Leptomycin B has little effect on HDV
assembly. A, leptomycin B inhibits the nuclear export
of HIV Rev protein in the presence of actinomycin D. COS7 cells were
transfected with plasmid pCsrev-GFP encoding a Rev-GFP fusion protein
and incubated for 48 h in the culture medium alone
(left) or in the presence of 1 µg of actinomycin D (AD)/ml
during the last 2 h of incubation without (middle) or
with (right) the addition of 2 nM leptomycin B
(LMB) at 24 h posttransfection. The cells were fixed 48 h
posttransfection. Fluorescence micrographs are shown. B,
leptomycin B has little effect on HDV assembly. COS7 cells were
cotransfected with plasmids encoding HDAg-L and small HBsAg. Leptomycin
B was added 24 h posttransfection at various concentrations, as
indicated, and incubated for various time periods. Immunoblot analysis
was performed with the cell lysates (top panel) and the
viral pellets collected from the culture media (middle and
bottom panels) with antibodies specific to HDAg-L and HBsAg
as indicated. Quantitation was performed with densitometer (ImageMaster
VDS, Amersham Pharmacia Biotech), and efficiencies of HDV
assembly are indicated as relative package activity (%).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Cheng-Ching Wang and Ta-Hsiu Liao for synthesizing HDAg peptides, Chih-Hsin Chen for constructing plasmid pB1-3(NdeI), George N. Pavlakis for providing plasmid pCsrev-GFP, and Norvatis (Vienna, Austria) for providing leptomycin B. We also thank Wei-Kung Wang, Woan-Yuh Tarn, and Y. Henry Sun for helpful discussions and Ho-Ting Su for technical assistance.
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FOOTNOTES |
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* This work was supported by Research Grants NSC 89-2320-B-002-217 and NSC 89-2320-B-002-246 from the National Science Council of the Republic of China.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
886-2-23123456, ext. 8217; Fax: 886-2-23915295; E-mail:
mfchang@ha.mc.ntu.edu.tw.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M004477200
2 G. N. Pavlakis, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: HDV, hepatitis delta virus; CRM, chromosome region maintenance; GFP, green fluorescent protein; HBsAg, hepatitis B virus surface antigen; HDAg, hepatitis delta antigen; HDAg-L, large HDAg; HDAg-S, small HDAg; HIV, human immunodeficiency virus; IgG(m), mouse IgG; IgG(r), rabbit IgG; NES, nuclear export signal; PBS, phosphate-buffered saline; RNP, ribonucleoprotein.
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