Journal of General Virology (2006), 87, 1715–1723
DOI 10.1099/vir.0.81718-0
Induction of hepatitis D virus large antigen
translocation to the cytoplasm by hepatitis B virus
surface antigens correlates with endoplasmic
reticulum stress and NF-kB activation
I-Cheng Huang,1 Chia-Ying Chien,1 Chi-Ruei Huang3
and Szecheng J. Lo1,2,3
1,2
Department of Microbiology and Immunology1, Graduate Institute of Biomedical Sciences and
Department of Life Science2, Chang Gung University, Tao-Yuan, Taiwan 333, Republic of
China
Correspondence
Szecheng J. Lo
[email protected]
3
Institute of Microbiology and Immunology, National Yang Ming University, Taipei, Taiwan 112,
Republic of China
Received 28 November 2005
Accepted 30 January 2006
It is known that hepatitis D virus (HDV) requires hepatitis B virus (HBV) for supplying envelope
proteins (HBsAgs) to produce mature virions, and the HDV large antigen (LDAg) is responsible for
interacting with HBsAgs. However, the signal molecules involved in the cross-talk between
HBsAgs and LDAg have never been reported. It has been previously demonstrated that the small
form of HBsAg can facilitate the translocation of HDV large antigen green fluorescent protein
(GFP) fusion protein (GFP–LD) from the nucleus to the cytoplasm. In this study, it was confirmed
that the small form of HBsAg can facilitate both GFP–LD and authentic LDAg for nuclear export. It
was also shown that the three forms of HBsAgs (large, middle and small) induced various rates
(from 35?4 to 57?2 %) of GFP–LD nuclear export. Since HBsAgs are localized inside the
endoplasmic reticulum (ER), this suggests that ER stress possibly initiates the signal for inducing
LDAg translocation. This supposition is supported by results that show that around 9 % of cells
appear with GFP–LD in the cytoplasm after treatment with the ER stress inducers, brefeldin A (BFA)
and tunicamycin, in the absence of HBsAg. Western blot and immunofluorescence microscopy
results further showed that the activation of NF-kB is linked to the ER stress that induces GFP–LD
translocation. Combining this with results showing that tumour necrosis factor alpha (TNF-a)
can also induce GFP–LD translocation, it was concluded that LDAg translocation correlates with
ER stress and activation of NF-kB. Nevertheless, TNF-a-induced GFP–LD translocation was
independent of new protein synthesis, suggesting that a post-translational event occurs to GFP–LD
to allow translocation.
INTRODUCTION
Hepatitis D virus (HDV) is the smallest human RNA virus
and has a genome of 1?7 kb in the form of a single-,
negative-stranded circle (for a review see Lai, 1995).
Although the genome structures of HDV and plant viroids
share a common evolutionary origin, unlike viroids, which
do not encode any proteins, HDV encodes two antigen
isoforms, the small and large forms (SDAg and LDAg,
respectively) during its replication cycle. The SDAg and
LDAg share 195 aa at their N terminus, while the LDAg
contains 19 extra aa at its C terminus (Weiner et al., 1988).
SDAg is required for HDV RNA replication, whereas LDAg
suppresses this process and interacts with hepatitis B virus
A supplementary table showing the distribution pattern of GFP–LD in
HuH-7 cells is available in JGV Online.
0008-1718 G 2006 SGM
(HBV) envelope proteins (HBsAg) to assemble a mature
virion for secretion and infection (Kuo et al., 1989; Chao
et al., 1990; Chang et al., 1991; Ryu et al., 1992).
Replication of the HDV genome and antigenome is dependent on host cell RNA polymerases and occurs via a rollingcircle method, which produces multiple copies of HDV
RNA in a linear form (Modahl et al., 2000; Macnaughton
et al., 2002). Ribozymes in both the genome and antigenome
then self-cleave the linear RNA into single units, which are
then ligated into a circular form by cellular ligases (Lai, 1995;
Reid & Lazinski, 2000; Taylor, 2003). During the HDV replication cycle, host enzymes called ADARs (adenosine deaminases that act on double-stranded RNA) edit a portion of
the HDV RNA to convert the amber stop codon (UAG) of
SDAg to a tryptophan codon (UGG), which results in the
production of LDAg (Casey & Gerin, 1995; Sato et al., 2001;
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I.-C. Huang and others
Jayan & Casey, 2002). Thereafter, LDAg inhibits HDV RNA
replication and then LDAg together with SDAg and the
HDV genome assemble into a ribonucleoprotein (RNP)
complex (Ryu et al., 1993), which is then transported to the
cytoplasm to form a mature virion with the HBsAgs.
In addition to the host RNA polymerases and ADARs, many
other cellular enzymes involved in the post-translational
modifications of HDAgs are important for the execution
of HDAg’s function (Mu et al., 2004; Li et al., 2004; Lai,
2005). For example, farenyl-transferase is required for LDAg
isoprenylation, which is crucial to the interaction with
HBsAg for secretion (Glenn et al., 1992; Hwang & Lai, 1993;
Sheu et al., 1996). Different kinases are also required for
SDAg and LDAg phosphorylation because the SDAg is
phosphorylated at both serine and threonine, while the
LDAg is phosphorylated only at serine (Chang et al., 1988;
Mu et al., 1999), which may account for their distinct functions. Furthermore, the serine residues at positions 2 and
177 of SDAg have been demonstrated to modulate HDV
RNA replication but have no significant role in subviral
particle formation (Yeh et al., 1996; Yeh & Lee, 1998; Mu
et al., 1999, 2001). Recently, methylation at arginine-13 and
acetylation at lysine-72 of HDAg have been reported to
play an important role in virus replication (Mu et al., 2004;
Li et al., 2004). These lines of evidence fully support the
hypothesis that post-translational modifications of HDAgs
can drive HDAgs to specific cellular compartments and
functions (for a review see Lai, 2005). However, signals
and mechanisms involved in HDAg post-translational
modification have not yet been well studied.
Previously, we used a green fluorescent protein fused to
LDAg (GFP–LD) to demonstrate that translocation of GFP–
LD from the nucleolus to SC-35 speckles can be induced by
treatment with the casein kinase II inhibitor, dichlororibofuranosyl benzimidazole (Shih & Lo, 2001), in which
serine-123 of LDAg in the deposphorylated state is responsible for preferentially targeting to the SC-35 speckles (Tan
et al., 2004). How cellular kinases and phosphotases are
regulated in order to modify LDAg for translocation is still
under question. We also demonstrated that the small form
of HBsAg can facilitate the translocation of GFP–LD from
the nucleus to the cytoplasm (Tan et al., 2004), but the
mechanism remains unclear. In this study, we explore what
signal molecules are generated by HBsAgs, which reside in
the endoplasmic reticulum (ER) and can induce GFP–LD
nuclear export.
METHODS
Plasmids used in this study. Based on the encoded proteins, plas-
mids used in this study can be grouped into four series: (i) pMTLD
(Hu et al., 1996) and pN2LD encode authentic LDAg under the metallothionine (MT) and cytomegalovirus (CMV) promoter, respectively;
(ii) pMTS, pMTMMS[S2] and pMTLS encode HBV small, middle and
large (S, M and L) surface antigens, respectively (Sheu & Lo, 1994),
and pN2LS encodes L HBsAg; (iii) those expressing different versions
of HDAg fused to a GFP, pGFP–LD, pGFP–LDM, pGFP–LD(31–214)
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and pSD–GFP (Shih & Lo, 2001; Shih et al., 2004); and (iv) pIKKa,
which encodes the wild-type IkB kinase a subunit; pKA, which
encodes a mutant form of IKKa; and pNF-kB-Luc, which contains the
NF-kB-response element (TGGGGACTTTCCGC)5 fused to a luciferase gene (kindly provided by Dr Y. S. Chang, Chang Gung University,
Republic of China). The characteristics of the GFP fusion proteins
encoded by the series (iii) plasmids are summarized as follows: (a)
pGFP–LD encodes GFP fused to wild-type LDAg; (b) pGFP–LDM
produces GFP fused to a non-isoprenylated mutant of LDAg; (c)
pGFP–LD(31–214) produces GFP fused to an N-terminal deletion (aa
1–30) mutant of LDAg; and (d) pSD–GFP produces wild-type SDAg
fused to GFP (Shih & Lo, 2001; Shih et al., 2004).
Cell culture and plasmid transfection. Two human cell lines
were used in this study; one is a well-differentiated hepatoma cell
line, HuH-7, while the other is a cervical carcinoma cell line, HeLa.
Most of the experiments were carried out using HuH-7 cells and a
few were done with HeLa cells. Both cell lines were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10 % fetal bovine serum, penicillin (100 IU ml21), streptomycin
(100 mg ml21), Fungizone (50 mg ml21) and 2 mM L-glutamine,
and grown at 37 uC under 5 % CO2. Plasmids in a supercoiled form
were obtained using the Qiagen Plasmid Maxi kit and then used for
transfection. Cells at 60–80 % confluence in a 10 cm Petri dish or
six-well plate were transfected with 10–20 mg of the indicated plasmids by the calcium phosphate-DNA precipitation method (Graham
& van der Eb, 1973) or by adding lipofectamine (Invitrogen). At 1
or 2 days post-transfection, cells were treated with various drugs for
different time intervals: (i) tunicamycin (TM; 2–5 mg ml21) or brefeldin A (BFA; 2–5 mg ml21) for 2 h, and (ii) with or without pretreatment with cycloheximide (10 mg ml21) for 30 min and then
followed by treatment with tumour necrosis factor alpha (TNF-a;
10 ng ml21) for 1 h.
Fluorescence microscopy. To visualize the co-expression of
HBsAgs and various forms of GFP fusion proteins, transfected cells
were reseeded on 22622 mm coverslips. After full attachment, cells
were fixed and permeabilized using methanol for 30 min at 220 uC
and then stained with anti-HBs antibody followed by the secondary
antibody conjugated with rhodamine. In parallel, cells were stained
with Hoechst 33258 for 10 min to show the nucleus. Finally, cells
were mounted onto glass slides with mounting solution, and visualized using a fluorescence microscope (Olympus IX71) with a fluorescein isothiocyanate (FITC) or rhodamine filter. For quantification
of the GFP fusion protein, the distribution of the nucleus only (N)
versus the nucleus–cytoplasm (N+C) pattern was observed, and
between 100 and 500 GFP-positive cells were randomly selected and
their patterns classified as described previously (Tan et al., 2004).
For simplicity in this study, type I, II and III distribution patterns
were redesignated ‘N’ pattern, nucleus only, while the type IV was
redesignated ‘N+C’, nucleus and cytoplasm.
Western blot analysis. To detect the amount of GFP fusion proteins in the nucleus and cytoplasm, transfected cells were fractionated
into nuclear and cytoplasmic fractions using a nuclear extraction kit
(Active Motif), according to the manufacturer’s instructions. Protein
samples were separated by SDS-PAGE and then electro-transferred
onto PVDF membranes. The membranes were incubated with 5 %
non-fat milk for 1 h at room temperature for blocking and then
reacted with anti-GFP, anti-HBsAg, anti-GRP78, anti-NF-kB p65 or
anti-tubulin antibodies (depending on the purpose of the experiment) in the presence of 5 % non-fat milk overnight at 4 uC. This
was followed by incubation with the secondary antibody conjugated
with horseradish peroxidase and the blots were then developed by
enhanced chemiluminescence using a commercial kit (Amersham).
Luciferase activity assay. Measurement of NF-kB activity was
carried out as described by Wu et al. (1998). Briefly, transfected cells
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Journal of General Virology 87
Signals for LDAg nuclear export
were lysed using a buffer supplied in a commercial luciferase assay
kit (Promega) according to the manufacturer’s instructions. The
luciferase activity of each cell lysate was determined by using an
Autolumat LB953 luminometer (Berthold). All experiments were
carried out in duplicate and repeated three times.
RESULTS
The small form of HBsAg can induce nuclear
export of both authentic and GFP fusion LDAg
Previously, we have demonstrated that the small form of
HBsAg facilitated GFP–LD translocation from the nucleus
to the cytoplasm (Tan et al., 2004). In this study, we first
confirmed that HBsAg expression correlates with GFP–LD
in the cytoplasm by co-transfection of pMTS and pGFP–LD
into HuH-7 cells. A representative fluorescence microscopy
image shows that three cells that have GFP appearing in an
N+C pattern, while one cell has GFP located in the nucleus
(N pattern) only [Fig. 1a(i) and a(ii)]. This particular N
pattern cell had no HBsAg expression in contrast to the three
N+C cells [Fig. 1a(iii)], indicating clearly that HBsAg can
facilitate GFP–LD translocation to the cytoplasm. Consistently, the presence of authentic LDAg in the cytoplasm
was highly correlated with the expression of HBsAg in HeLa
cells [Fig. 1b(i), (ii) and (iii)], indicating that HBsAg can
affect similarly on both GFP–LD and authentic LDAg. We
then tested whether HBsAg can facilitate on other HDAg
fusion proteins by performing single, double and triple
plasmid transfection experiments, in which each of four
plasmids [pGFP–LD, pGFP–LDM, pGFP–LD(31–214) and
pSD–GFP] expressing a different version of HDAg fused to
GFP was co-transfected with or without pMTS and pMTLD.
As shown in Table 1, the percentage of N+C pattern cells
for expressing GFP–LD (GFP fused to wild-type LDAg),
GFP–LDM (GFP fused to a non-isoprenylated mutant of
(a)
(i)
(ii)
(iii)
N
N+C
(b)
(i)
(ii)
(iii)
N+C
N
Fig. 1. The effect of HBsAg on GFP–LD [a(i)–(iii)] and authentic LDAg [b(i)–(iii)] distribution. [a(i)–(iii)] HuH-7 cells were cotransfected with pGFP–LD and pMTS. After 24 h post-transfection, cells were permeabilized and reacted with anti-HBsAg
and followed by rhodamine-conjugated antibody. The same cells were photographed using different fluorescent filters and
show: (i) cells stained with DNA dye, Hochest 33258, to outline the nucleus, (ii) GFP–LD pattern inside of cells using an FITC
filter, and (iii) HBsAg distribution using a rhodamine filter. [b(i)–(iii)] HeLa cells were co-transfected with pN2LD and pN2LS,
which contain the CMV promoter to express L HBsAg and LDAg, respectively. After 24 h post-transfection, cells were fixed
and stained with antibody conjugated with rodamine to indicate the expression of LDAg and with FITC-conjugated antibody to
indicate the expression of HBsAg. The same cells were photographed using different fluorescent filters and show: (i) cells
stained with DNA dye, Hochest 33258, to outline the nucleus, (ii) LDAg distribution using a rhodamine filter, and (iii) HBsAg
distribution using an FITC filter. ‘N’ indicates the nucleus pattern and ‘N+C’ indicates the cytoplasm and nucleus pattern.
Bars, 10 mm.
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I.-C. Huang and others
Table 1. Effect of S-HBsAg on distribution pattern of various GFP fusion proteins in HuH-7 cells
These distribution patterns of various GFP fusion proteins were observed after 72 h post-transfection. Each number in the N and N+C
column represents the mean value of three independent experiments. Numbers (1, 2 and 3) at the left represent for transfection conditions:
1, single plasmid transfection; 2, double plasmid transfection; and 3, triple plasmid transfection. ‘N’ and ‘N+C’ distribution pattern is
described in the legend of Fig. 1.
Expressed protein
1.
2.
3.
1.
2.
3.
1.
2.
3.
1.
2.
3.
GFP–LD
GFP–LD+S-HBsAg
GFP–LD+S-HBsAg+L-HDAg
GFP–LDM
GFP–LDM+S-HBsAg
GFP–LDM+S-HBsAg+L-HDAg
SD–GFP
SD–GFP+S-HBsAg
SD–GFP+S-HBsAg+L-HDAg
GFP–LD(31–214)
GFP–LD(31–214)+S-HBsAg
GFP–LD(31–214)+S-HBsAg+L-HDAg
Distribution pattern (%)
N+C pattern difference (%)
N
N+C
2-1
94?9
68?4
70?9
98?7
96?2
82?7
97?7
92?6
79?7
98?9
95?9
95?6
5?1
31?6
29?1
1?3
3?8
17?3
2?3
7?4
20?3
1?1
4?1
4?4
LDAg), GFP–LD(31–214) [GFP fused to an N-terminal deletion (aa 1–30) mutant of LDAg] or SD–GFP (wild-type
SDAg fused to GFP) ranged from 1?1 to 5?1 % in single
plasmid transfections. In the presence of HBsAg, the N+C
pattern of GFP–LD cells increased to 26?5 %, while that of
GFP–LDM, SD–GFP and GFP–LD(31–214) expressing cells
increased ranging from 2?5 to 5?1 %, suggesting that HBsAg
only facilitates the full-length of LDAg translocation to the
cytoplasm but not the single point mutation, which cannot
be isoprenylated (GFP–LDM), N-terminal deletion mutation [GFP–LD(31–214)] or SDAg. As compared with the
result of double plasmid transfection, results of triple plasmid transfection showed 0?3 % or no increase of N+C
pattern in cells expressing GFP–LD(31–214) and GFP–LD,
while 13?5 and 12?9 % increase in cells expressing GFP–
LDM and SD–GFP, respectively (see the last column of
Table 1). A higher increasing percentage of N+C pattern in
GFP–LDM and SD–GFP expressing cells as compared with
that of GFP–LD(31–214) cells in the presence of HBsAg and
LDAg suggested that LDAg can form a complex with GFP–
LDM and SD–GFP via the coiled-coil domain and this
allows them export to the cytoplasm, in contrast, the coiledcoil domain of GFP–LD(31–214) is truncated (Chang et al.,
1992; Shih & Lo, 2001). No significant increase of N+C
pattern in GFP–LD cells between double and triple plasmid
transfections suggested that GFP–LD behaves similarly to
LDAg under the effect of HBsAg.
The three forms of HBsAgs facilitate GFP–LD
translocation to the cytoplasm to different
degrees
It is known that there are three different forms (S, M and L)
of HBsAg in various lengths of amino acids and degrees
of glycosylation modification (Sheu & Lo, 1992). We were
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3-2
26?5
22?5
2?5
13?5
5?1
12?9
3?0
0?3
thus interested to see whether or not the three different
forms of HBsAg exert different capability on the translocation of GFP–LD. Co-transfection of pGFP–LD with pMTS,
pMTMMS[S2] or pMTLS, which express S, M and L HBsAg,
respectively, into HuH-7 cells was performed. The N and
N+C pattern of the GFP–LD cells were analysed at 24, 48
and 72 h post-transfection. As shown in Table 2, GFP–LD
cells had an increased percentage of the N+C pattern when
they were co-expressed with S, M and L HBsAg (10?4, 26?4
and 34?1 %, respectively) at 24 h post-transfection. A
similar trend was also observed in the 48 h post-transfected
cells (25?2, 36?3 and 47?0 %, respectively) as well as in the
72 h post-transfected cells (34?7, 47?8 and 56?5 %, respectively). Since the three forms of HBsAgs reside in the ER, it
was speculated that the presence of HBsAg may induce ER
stress and then in turn cause GFP–LD translocation.
Western blot analyses were performed to test this supposition. Results clearly show that the GRP78 protein/BiP, an ER
stress-induced marker, which functions as a protein chaperone at the ER cisternae (Schröder & Kaufman, 2005; Lee,
2005), was increased in the total lysate of cells co-expressing
GFP–LD with S, M and L HBsAg (Fig. 2, lanes 3–5, bottom
line) compared with cells that were mock transfected or
expressed GFP–LD alone (Fig. 2, lanes 1 and 2, bottom line).
For an unknown reason, the increasing folds of GRP78/BiP
induced by different HBsAgs varied and were not reproduced consistently. In contrast, the amount of p65 subunit
of NF-kB consistently increased in the nuclear fraction of
cells co-expressing S, M and L HBsAg (Fig. 2, lanes 3–5, line
2). A decreased amount of GFP–LD in the nuclear fraction
(Fig. 2, lanes 2–5, line 1) and, conversely, an increased
amount of GFP–LD in the cytoplasmic fraction (Fig. 2, lanes
2–5, line 4) were found in cells expressing GFP–LD alone
or co-expressing GFP–LD with S, M, or L HBsAg. These
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Signals for LDAg nuclear export
Table 2. Distribution pattern of GFP–LD in HuH-7 cells
Each number in the N and N+C column represents the mean value of three independent experiments.
‘N’ and ‘N+C’ distribution pattern is described in the legend of Fig. 1.
Time (h)
24
48
72
Expressed protein
GFP–LD
GFP–LD+S-HBsAg
GFP–LD+M-HBsAg
GFP–LD+L-HBsAg
GFP–LD
GFP–LD+S-HBsAg
GFP–LD+M-HBsAg
GFP–LD+L-HBsAg
GFP–LD
GFP–LD+S-HBsAg
GFP–LD+M-HBsAg
GFP–LD+L-HBsAg
Distribution pattern (%)
N
N+C
99?6
89?2
73?2
65?5
99?4
74?2
63?1
52?4
99?3
64?6
51?5
42?8
0?4
10?8
26?8
34?5
0?6
25?8
36?9
47?6
0?7
35?4
48?5
57?2
Western blot results are highly correlated with the results of
the fluorescence microscopic observation (Table 2), indicating that all three forms of HBsAg have the capability to
induce GFP–LD translocation, but to various degrees.
Treatment with TM and BFA increases GFP–LD
in the cytoplasm
Although an increase of GRP78/BiP in the presence of
HBsAgs is shown in Fig. 2, to further correlate with ER stress
Fig. 2. Western blot analyses of GFP–LD and NF-kB distribution
in the presence of three different forms of HBsAgs (L, M and S).
HuH-7 cells were co-transfected by pGFP–LD with plasmids
encoding L, M or S HBsAg. Cells were then fractionated into
nuclear and cytoplasmic portions at 72 h post-transfection.
Nuclear proteins (15 mg) were analysed with anti-GFP, anti-NFkB p65 and anti-tubulin antibodies (the top three lines as indicated by nuclear fraction), while 15 mg cytoplasmic proteins were
analysed with anti-GFP and anti-tubulin (as indicated by cytoplasmic fraction). Total cell lysate, in 20 mg protein, was analysed
with GRP78/BiP and GFP–LD (the last two lines). Tubulin served
as a positive control for the cytoplasmic fraction and as a negative
control for the nuclear fraction.
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N+C pattern difference (%)
in the presence of HBsAgs
–
10?4
26?4
34?1
–
25?2
36?3
47?0
–
34?7
47?8
56?5
and GFP–LD distribution in the cytoplasm, we treated GFP–
LD expression cells with the ER stress inducers, TM and
BFA, for 2 h and then determined the percentage of N and
N+C pattern cells by fluorescence microscopy. Around 8?8
to 9?3 % N+C pattern increase was observed when they
were treated with 5 mg TM and BFA ml21 (see Supplementary Table S1 available in JGV Online). Consistently,
Western blot results showed a decreased amount of GFP–
LD present in the nuclear fraction, while an increased
amount of GFP–LD present in the cytoplasm when cells
were treated with TM (Fig. 3a, lanes 2–5, lines 1 and 3) and
BFA (Fig. 3b, lanes 2–5, lines 1 and 3). In addition, a slightly
increased amount of GRP78/BiP was also observed in a
dose-dependent manner when cells were treated with lower
doses of TM and BFA (2–4 mg ml21) but not at higher doses
(5 mg ml21) (Fig. 3a and b, line 6). The highest dose was
unable to induce the highest amount of GRP78/BiP and
GFP–LD in the cytoplasm (Fig. 3a and b lane 6, line 6),
which could be due to a desensitization effect. Nevertheless, the data showing a good correlation between increasing
GFP–LD in the cytoplasm and expression of GRP78/BiP
indicate that even in the absence of HBsAg, inducing ER
stress by an exogenous drug also causes GFP–LD translocation to the cytoplasm. Therefore, induction of ER stress is
suggested to be an early event when HBsAg is expressed in
cells to induce GFP–LD translocation.
NF-kB plays a role in GFP–LD transportation
from the nucleus to the cytoplasm
Although an increasing amount of the p65 subunit of NF-kB
in the nucleus has been demonstrated by Western blot in
cells co-expressing GFP–LD and S, M or L HBsAg (Fig. 2),
whether the nuclear form of NF-kB can actively transcribe
its target genes remains to be determined. Similar experiments described in Table 2 were therefore conducted, in
which pNF-kB-Luc containing the luciferase gene followed
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I.-C. Huang and others
Fig. 4. NF-kB activity assay in transfected cells. HuH-7 cells
were transfected with various combinations of plasmids as indicated in the key. After 24, 48 and 72 h post-transfection, cells
were lysed and analysed for luciferase activity. Three independent experiments were performed and the mean±SD data are
shown.
Fig. 3. Western blot analyses of GFP–LD distribution after
treatment with the ER stress inducers, TM (a) and BFA (b).
HuH-7 cells were mock transfected (lane 1) or transfected with
pGFP–LD alone (lanes 2–6) and then treated with various
amounts of drugs for 2 h at 72 h post-transfection. The amount
of drug used is indicated above the gel (2–5 mg). Preparation
of nuclear and cytoplasmic fractions was the same as
described in Fig. 2. Both nuclear and cytoplasmic fractions
were analysed by GFP and tubulin as indicated, while the total
cell lysate in 20 mg was analysed by GFP and GRP78/BiP.
by the NF-kB-response element was co-transfected with
pGFP–LD and plasmids encoding S, M or L HBsAg into
HuH-7 cells. After 24, 48 and 72 h post-transfection, cells
were lysed and luciferase activities were determined. Results
clearly show that the luciferase activity (2?7–3?26105
luminescence intensity unit, liu) was higher in cells coexpressing GFP–LD with S, M or L HBsAg as compared with
in those expressing GFP–LD without HBsAgs (1?36105 liu)
or in pNF-kB-Luc transfection cells (8?26104 liu) at 24 h
post-transfection (Fig. 4). A similar trend was also observed
in 48 h post-transfection cells (3?1–4?16105 liu versus 1?4–
1?76105 liu) and in 72 h post-transfection cells (4?3–7?36
105 liu versus 2?16105 liu). Among the cells co-expressing
various HBsAgs at 72 h post-transfection, the L HBsAg
expression showed the highest luciferase activity (7?36
105 liu), while the S HBsAg expression cells showed the
lowest activity (4?36105 liu). These results are consistent
with the Western blot results and indicate that the nuclear
form of NF-kB is induced by three forms of HBsAgs (Fig. 2,
line 2) and this varies to a range of different levels.
To obtain another line of evidence for the role of NF-kB in
HBsAg-induced GFP–LD translocation, we co-transfected
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plasmid pKA, which expresses a dominant negative form of
IKKa that results in inactivation of NF-kB, to examine
whether the functioning of HBsAg will be blocked or not.
Western blot results show that the GFP–LD remaining in the
nucleus was increased proportionally to the amount of pKA
transfected (Fig. 5a, line 1). Unlike the reciprocal change of
GFP–LD between the nucleus and cytoplasm detected and
shown in Fig. 2, a constant and similar amount of GFP–LD
was present in the cytoplasm of cells with or without pKA
transfection (Fig. 5a, line 3). However, the luciferase activity assay showed several fold decrease in cells expressing
various amounts of mutant IKK (data not shown), suggesting that NF-kB was indeed inhibited in those cells
and this resulted in a higher amount of GFP–LD being
retained in the nucleus. When cells co-expressed GFP–LD
and various amounts of active form of IkB kinase a subunit, an increasing amount of GFP–LD, proportional to the
amount of pIKKa that was transfected was observed in the
cytoplasmic fraction of cells (Fig. 5b, line 3). Conversely, a
decreasing amount of GFP–LD was observed in the nuclear
fraction of cells (Fig. 5b, line 1). Cells having the higher
amount GFP–LD in the nucleus were highly correlated
with the higher luciferase activity (data not shown). Taken
together the evidence shown in Figs 4, 5(a) and (b) conclude
that NF-kB indeed plays a significant role in HBsAg-induced
GFP–LD translocation.
TNF-a treatment also induces GFP–LD
translocation to the cytoplasm in the absence
of protein synthesis
Since TNF-a induces cell responses largely through activation of NF-kB, to correlate further with activation of NF-kB
and GFP–LD distribution, GFP–LD expressing cells were
treated with TNF-a for 1 h and the percentage of N+C cells
was determined by fluorescence microscopy. The results
revealed that 1 h after TNF-a treatment induced more than
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Journal of General Virology 87
Signals for LDAg nuclear export
both TNF-a and cycloheximide (data not shown). However,
the Western blot result shows that the amount of NF-kB
in the nucleus was similar after TNF-a treatment with or
without cycloheximide but it was higher than that in TNF-a
non-treated cells (Fig. 6, line 3). Western blot analysis
also shows that the amount of GFP–LD in the nucleus and
cytoplasm (Fig. 6, lines 1 and 4) correlated well with the
fluorescence microscopic observations.
DISCUSSION
Fig. 5. Western blot analyses of GFP–LD distribution under
various degrees of NF-kB activation. (a) HuH-7 cells were cotransfected with pGFP–LD and pMTS (lanes 1–4) with 4, 6 or
8 mg plasmid pKA (lane 2, 3 and 4, respectively), which produces a dominant negative form of IKK. Post-transfected cells
(72 h) were fractioned into nuclear and cytoplsmic portions as
indicated in the legend of Fig. 2 and GFP–LD changes in the
nuclear and cytoplasmic fractions were then analysed by antiGFP. (b) HuH-7 cells were co-transfected with pGFP–LD with
various amounts (4, 6 or 8 mg) of pIKK, which produces an
active form of IkB kinase a subunit. Post-transfected cells
(72 h) were fractionated into nuclear and cytoplsmic portions
and GFP–LD changes in the nuclear and cytoplasmic fractions
were analysed as indicated in (a).
50 % of GFP–LD expressing HuH-7 or HeLa cells appeared
to have the N+C pattern, while treatment with TM and
BFA induced only 8?8 and 9?3 % of the cells to have the
N+C pattern (Supplementary Table S1 available in JGV
Online). The robust effect of TNF-a allowed us to test
whether the induction of GFP–LD translocation into the
cytoplasm requires newly synthesized proteins or not. This
kind of experiment could not be performed in cells expressing HBsAg because the protein translation inhibitor will
also block HBsAg production. After pre-treatment with
cycloheximide for 30 min, cells were treated with TNF-a
for 1 h and the N+C pattern of GFP–LD was examined.
Around 53?3 % of cells appeared to have the N+C pattern
(Supplementary Table S1), indicating that GFP–LD translocation into the cytoplasm does not require newly synthesized proteins. The inhibition effect by cycloheximide was
also shown to occur by the luciferase activity assay, which
showed that activity was greatly reduced in cells treated with
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During the HDV replication cycle, the HDV RNP, which
contains the HDV RNA genome and two isoforms of
antigen (SDAg and LDAg), is known to be exported out of
the nucleus in order to be enveloped by HBsAgs for
production of mature viral particles. This process is very
likely LDAg-mediated since there is a nuclear export signal
(NES) localized at the C terminus of LDAg and a nuclear
factor (NESI) that binds to the NES has been identified
(Lee et al., 2001; Wang et al., 2005). However, signal molecules involved in HDV RNP export and the underlying
mechanisms are largely unknown. In this study, we apply a
previously established system, in which HBsAg can facilitate
GFP–LD translocation into the cytoplasm, to investigate
signals participating in the GFP–LD translocation. Several
lines of evidence have demonstrated that ER stress and
activation of NF-kB, which are induced by HBsAg, play a
significant role (Figs 2, 4 and 5a). It is known that ER stress
induced by many different viral proteins leads to various
cellular effects (Su et al., 2002; Tardif et al., 2002; Dimcheff
et al., 2003), including full-length and truncated forms of the
HBV L and M surface antigens (Wang et al., 2003; Hsieh
et al., 2004; Hung et al., 2004). The current study is the first
Fig. 6. Western blot analyses to show the effect of TNF-a and
cycloheximide on GFP–LD distribution. HuH-7 cells were transfected with pGFP–LD. After 72 h post-transfection, cells were
untreated (lane 1), treated with 10 ng TNF-a ml”1 for 1 h (lane
2) or pre-treated with cycloheximide (10 mg ml”1) for 30 min
followed by co-treatment of cycloheximide (10 mg ml”1) and
TNF-a (10 ng ml”1) for 1 h (lane 3) and then fractionated into
nuclear and cytoplasmic fractions as indicated in the legend
of Fig. 2. GFP–LD and NF-kB (p65) changes in the nuclear
and cytoplasm fractions under various drug treatments were
analysed as indicated in Fig. 2.
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1721
I.-C. Huang and others
report to show that induction of ER stress results in a nuclear
protein translocation into the cytoplasm.
The current study also provides direct evidence that three
different forms of HBsAgs induce various levels of NFkB activities (Fig. 4) as well as 34?7–56?5 % cells having
GFP–LD in the cytoplasm (Table 2). The various capabilities
exhibited by different forms of HBsAgs could be due to their
intrinsic properties, i.e. capability of ER retention (Bruss &
Ganem, 1991; Sheu & Lo, 1994; Chau et al., 2005). The
effects of HBsAg on NF-kB activity and GFP–LD translocation could be explained by either that they are two parallel
events or that the GFP–LD translocation is the downstream
event following the NF-kB activation. We prefer the second
explanation because in the absence of HBsAg, GFP–LD cells
treated with TNF-a, which induces a higher level of NF-kB,
also appear to have a higher percentage of N+C pattern
(Supplementary Table S1). At the present time, no direct
evidence that HBsAg can trap the newly synthesized LDAg in
the cytoplasm has been demonstrated. However, we favour
the hypothesis that LDAg must enter into the nucleus and
then translocate into the cytoplasm after post-translational
modification, in which HBsAgs exert signals to facilitate the
modification. This hypothesis is supported by results that
cells co-expressing GFP–LD(31–214) and HBsAg with or
without LDAg show a low N+C pattern from 1 to 3 days
post-transfection (Table 1), while in 6 days post-transfected
cells expressing GFP–LD(31–214) can be co-secreted with
HBsAg (Shih & Lo, 2001). Therefore, the current study
suggests that the sequence located between aa 1 and 30 may
be important in helping LDAg translocation into the
cytoplasm and post-translational modification of serine-2
and/or arginine-13 (Mu et al., 2004; Li et al., 2004) is likely
to be involved in facilitating LDAg translocation into the
cytoplasm.
farnesylation of LDAg (O’Malley & Lazinski, 2005). Therefore, the farnesyl-transferase and other unidentified molecules that are directly or indirectly modified by the activated
NF-kB are of interest for future exploration.
In conclusion, the current study presents the first report
showing the presence of signals in the cross-talk between
HDV and HBV using a system of GFP fusion proteins. However, to identify the target enzymes downstream to NF-kB
and to understand how they are activated to modify LDAg
are a great challenge for future studies. Moreover, whether the HDV RNP translocation into the cytoplasm can be
facilitated by HBV and TNF-a remains to be tested.
ACKNOWLEDGEMENTS
We thank Dr Y. S. Chang (Chang Gung University) for providing us
with the plasmids and Dr R. Kirby (National Yang Ming University) for
editing the English in this article. Thanks are also due to lab members,
C.-C. Lee and H.-J. Lai for their discussions and Y. L. Chen for figure
preparation. This work was supported by grants from the National
Science Council (NSC93-2320-B-010-096 and 92PS012) to S. J. L.
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