Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 484-491
doi:10.1111/j.1745-7270.2006.00190.x
Overexpression of hepatitis B virus-binding protein,
squamous cell carcinoma antigen 1, extends retention
of hepatitis B virus in mouse Liver
Hong-Bin XIA and Xi-Gu CHEN*
Center
of Experimental Animals, Sun Yat-Sen University, Guangzhou 510089, China;
Received: December 4,
2005
Accepted: March 27,
2006
This work was
supported by the grants from the National Natural Science Foundation of China
(No. 30271177 and No. 39870676), and the Natural Science Foundation of
Guangdong Province (No. 021903)
*
Corresponding author: Tel, 86-20-87331393; Fax, 86-20-87331230; E-mail,
Abstract How receptors mediate the entry of hepatitis B virus (HBV)
into the target liver cells is poorly understood. Recently, human squamous cell
carcinoma antigen 1 (SCCA1) has been found to mediate binding and
internalization of HBV to liver-derived cell lines in vitro. In this
report, we investigate if SCCA1 is able to function as an HBV receptor and
mediate HBV entry into mouse liver. SCCA1 transgene under the control of
Rous sarcoma virus promoter was constructed in a minicircle DNA vector that was
delivered to NOD/SCID mouse liver using the hydrodynamic technique.
Subsequently, HBV-positive human serum was injected intravenously. We demonstrated
that approximately 30% of the mouse liver cells expressed a high level of
recombined SCCA1 protein for at least 37 d. The HBV surface antigen was found
to persist in mouse liver for up to 17 d. Furthermore, HBV genome also
persisted in mouse liver, as determined by polymerase chain reaction, for up to
17 d, and in mouse circulation for 7 d. These results suggest that SCAA1 might
serve as an HBV receptor or co-receptor and play an important role in mediating
HBV entry into hepatocytes, although its role in human HBV infection remains to
be determined.
Key words HBV receptor; HBV-binding protein; minicircle DNA plasmid;
hydrodynamics-based procedure
Hepatitis B virus (HBV) is a human hepadnavirus
that causes acute and chronic hepatitis and hepatocellular carcinoma [1]. As
with other viral diseases, HBV infection is likely initiated by specific
binding of the virus to cell membrane structures through one or several viral
envelope proteins. HBV has not been propagated in established cell lines, and
only humans and higher apes are susceptible to infection. HBV replication and
cellular injury are largely confined to the liver, and the hepatocytes are
considered the primary target cells for infection, whereas the significance of
extrahepatic replication of HBV is not yet well understood.The HBV envelope consists of three distinct coterminal proteins
encoded by a single env gene. The domains of these proteins encoded by
the pre-S region of the viral genome represent potential attachment sites of
HBV to the hepatocyte, as pre-S1 and pre-S2 antibodies neutralize infectivity
in vitro [2] as well as in experimental models [3].Although the viral structures involved in attachment to the target
cell have been identified, the cellular receptors for HBV have not yet been
determined and the biochemical events leading to intention remain unknown. As
well as hepatocytes, many cells of nonhepatic origin, such as hematopoietic
cells of the B lymphocyte lineage, peripheral blood lymphocytes, and even some
simian virus 40-transformed cell lines, have receptors for the pre-S1 domain
at amino acid 21–47 region [4]. The pre-S1 domain of the large envelope protein has a
partial sequence homology with the Fc moiety of the a chain of immunoglobulin
(Ig) A [5], therefore a common receptor for the attachment of HBV and IgA to
human liver cells has been proposed [6]. Interleukin 6, containing recognition
sites for the pre-S1 domain, could mediate HBV-cell interaction [7]; and the
transferring receptor might also play a role in the binding of HBV to
hepatocytes through the pre-S2 protein sequence, as this domain is involved in
the binding of hepatitis B surface antigen (HBsAg) particles with T cells [8].
Another HBV binding factor (HBV-BF) was identified in normal human serum
interacting with the pre-S1 and pre-S2 epitopes of the viral envelope located
within the protein domain involved in the recognition of hepatocyte receptors.
Monoclonal antibodies to HBV-BF recognized a membrane component of the normal
human liver cells but were unreactive with the hepatocyte membrane of other
species, and with that of the HepG2 cell line. The results suggested that
HBV-BF represents a soluble fragment of the membrane component and might be
related to the HBV receptor mediating the attachment of HBV to human liver
cells [9].
squamous
cell carcinoma antigen 1 (SCCA1), which is a member of the ovalbumin family of
serine protease inhibitors, was found to play a major role in HBV infection and
might be a new candidate for HBV-BF. By using tetravalent derivative chromatography
from detergent-solubilized HepG2 plasma membranes, a 44 kDa HBV binding
protein (HBV-BP) was found to closely correspond to human SCCA1. There are only
three amino acid changes between them. Direct binding experiments confirmed
the interaction of recombinant HBV-BP with the HBV pre-S1 domain. And in
transfected cells, native HBV particle entry was enhanced. For example, HepG2
cells overexpressing HBV-BP after transfection with corresponding cDNA showed
an increased virus binding capacity by two orders of magnitude compared with
normal cells; and Chinese hamster ovary cells, which normally do not bind to
HBV, acquired susceptibility to HBV binding after transfection. Both
recombinant HBV-BP and antibodies to recombinant HBV-BP blocked virus binding
and internalization in transfected cells as well as in primary human hepatocytes
in a dose-dependent manner [10]. Although many candidates for the HBV receptor
have been suggested in studies in vitro, none of them was proved in
vivo that is much closer to the natural HBV infection in the human body.In the present study, we get a special trangene mouse that
expresses the HBV receptor candidate, the 44 kDa HBV-BP, in the liver cells by
using a hydrodynamics-based intravenous injection procedure and a novel
minicircle plasmid as vector. This novel and economical mouse model will be
very useful in HBV receptor research.
Materials and Methods
Materials
Rabbit anti-SCCA1 antibody was purchased from Santa Cruz
Biotechnology (Santa Cruz, USA). Rabbit anti-HBsAg antibody was from ViroStat
(Portland, USA). X-gal was from TaKaRa Bio. (Kyoto, Japan). The SABC kit was
from Boster Biological Technology (Wuhan, China). The infectant human serum
(HBV-DNA+, 106 copies/ml) was provided by Prof. Ling Li (Third Affiliated Hospital, Sun
Yat-Sen University, Guangzhou, China). The HBV-DNA PCR detection kit was
purchased from Da’an gene
Technology (Guangzhou, China). NOD/SCID mice were purchased from the center of experimental animals
(Sun Yat-Sen University). All other chemicals used were of analytical grade.
Plasmid
Minicircle-producing plasmid p2FC31 was a gift from Dr. M.
A. Kay (Stanford University,
Stanford, USA) [11]. SCCA1 cDNA was purchased from Open Biosystems (Huntsville,
USA). p2FC31.SCCA1 was constructed by cloning SCCA1 under the control
of RSV promoter into the XhoI site of plasmid p2FC31. p2FC31.LacZ was
constructed by placing the bacterial b-galactosidase (LacZ) gene under the
control of CMV promoter into the XhoI site of plasmid p2FC31. Minicircles
encoding CMV-LacZ (MC.LacZ) and RSV-SCCA1 (MC.SCCA1) were prepared according to
the procedure described by Chen et al. [11]. Regular plasmid DNA was
purified by using the CsCl-density gradient centrifugation method and kept in
saline at –20 ?C until use. Purity of plasmid DNA was checked by absorbency at
260 and 280 nm and by 2% agarose gel electrophoresis.
X-gal staining
To examine the site of transgene expression, two mice were given 2
ml saline containing 5 mg of pMC-LacZ plasmid DNA through the tail vein, using a 2.5 ml
latex-free syringe with a 0.45?16 RW LB
needle (Shuang Ge, Shanghai, China). The injection rate was kept at 0.4 ml/s.
The location and level of LacZ gene expression in mouse liver was
determined by the X-gal staining method. Tissue sections (5 mm thick) were
made 24 h after plasmid injection, stained with X-gal for 2 h, then
counterstained in Nuclear Fast Red (Boster Biological Technology) for 30 s. The
positive cells showed blue staining in the cytoplasm or nucleus and the
connective tissues were stained pink.
Immunohistochemistry
evaluation for SCCA1
Two mice were killed on day 3, 7, 17, 27 and 37 after injection of
pMC-SCCA1, respectively. Paraffin sections of liver were analyzed for the
presence of SCCA1 by immunohistochemistry. For SCCA1 detection, rabbit
polyclonal antibody SCCA1/2 (H-390) (Santa Cruz Biotechnology) was used at
1:200 dilution. Sections were incubated with primary antibody overnight at 4
?C. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide,
then the slides were heated in 10 mM sodium citrate in a microwave oven to
block nonspecific protein binding in normal goat serum. Biotinylated goat
anti-rabbit IgG (Boster Biological Technology) was then added at room
temperature for 20 min. Samples were incubated with avidin-peroxidase and
stained with a mixture of 3,3‘-diamino-benzidine tetrahydrochloride
and hydrogen peroxide (Boster Biological Technology). In each case of immunohistochemistry experiment, sections incubated
with the appropriate non-immune IgG were used as the negative control. For
antibody specificity confirmation, human skin specimens for SCCA1 were used as
the positive control and the liver samples from a normal mouse were used as
the negative control. In each case, the semiquantitative immunoreactivity of SCCA1 was
independently evaluated by two pathologists. In all immunohistochemical
analyses, necrotic areas and edges of tissue sections were not included in the
counting to avoid possible immunohistochemical false positivity.
In vivo transfection
Animals were divided into four groups. Group 1 (negative control 1, two mice): the mice did not receive
injection.
Group 2
(negative control 2, two mice): the mice were injected through the tail vein
with 5 mg pMC-SCCA1 in 2 ml saline, and killed 3 d later. Group 3 (negative
control 3, 10 mice): the mice were injected through the tail vein with 2 ml
saline. Twenty-four hours later the mice were injected through the tail vein
with 0.3 ml of HBV-DNA+ serum, and 1 d later the same
quantity of serum was given to each mouse through the celiac artery. Two mice
were killed on day 3, 7, 17, 27 and 37, respectively. Group 4 (experimental group, 10 mice): the mice were injected
through the tail vein with 5 mg pMC-SCCA1 in 2 ml saline and 24 h later the mice were injected
through the tail vein with 0.3 ml of HBV-DNA+ serum.
One day later the same quantity of serum was given to the mice through the
celiac artery. Two mice were killed on day 3, 7, 17, 27 and 37, respectively. For each group, the liver and serum samples were collected at
appropriate times.
Immunohistochemistry detection
for HBsAg
Liver sections from the four groups above were analyzed for the
presence of HBsAg by immunohistochemistry. For HBsAg detection, rabbit
anti-HBsAg antibody (ViroStat) was used as the primary antibody at 1:200
dilution. The protocols were the same as for SCCA1 detection.
Enzyme-linked immunosorbent
assay (ELISA) for HBsAg in mouse serum
The collected mouse sera were tested for HBsAg using an ELISA kit
and the AxSYM system (Abbott Diagnostics, Chicago, USA) according to the
manufacturer?
protocols.
PCR detection of HBV-DNA in
mouse serum and liver
The collected mouse sera were tested for HBV-DNA using the HBV-DNA
detection kit (Da’an) according to the protocols. The genomic DNA was extracted from 200–300 mg mouse liver tissue.
Five hundred microliters of 1?lysis
buffer (40 mM NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA) and 20 ml of RNase
solution (10 mg/ml) was added to each pre-cooled tube. The live tissue was
homogenized while keeping the tube in the pre-cooled heating-block and
transferred to a 15 ml F2097 tube, then 3500 ml of 1?lysis buffer, 300 ml of 0.10% sodium dodecylsulfate and 1000 ml of proteinase K solution
(2.0 mg/ml) were added. The mixture was incubated at 55 ?C for 2 h and at 37 ?C
overnight with shaking. Ten microliters of 10 mg/ml RNase was added,
mixed and incubated at 55 ?C for 2 h with shaking. Two milliliters of saturated
NaCl solution was added, mixed by pipetting up and down five times using a 5 ml
pipette, then centrifuged at 3500 g for 30 min using a clinical
centrifuge. the supernatant was
removed to a fresh tube, 1 ml of saturated NaCl solution was added, and the
mixture was centrifuged for 30 min as above. the
supernatant was removed to a fresh tube and two volumes of 100% ethanol was
added. DNA will show up after mixing the solution by reversing the tube
several times. The DNA was taken to a 1.5 ml Eppendorf tube, washed twice with
75% ethanol and centrifuged at 6000 g to remove as much ethanol as
possible. The DNA was air dried at room temperature for 5 min, then 600 ml of TE buffer
(1 mM EDTA, pH 8.0, 10 mM Tris-HCl) was added to dissolve it. Two microliters
of the DNA solution was used as the template of following PCR. The mouse liver
samples were tested for HBV-DNA using the HBV-DNA detection kit (Da’an)
according to the protocols. Twelve microliters of samples was analyzed by 2.0%
agarose gel electrophoresis. One day later the negative control PCR reaction
was carried out with liver samples from a mouse injected with pMC-SCCA1 but not
with infectant human serum. The positive control PCR reaction was carried out
with 2 ml of positive DNA template solution provided with the kit.
Results
We used LacZ gene as the reporter gene to identify the site
of transgene expression. Twenty four hours after infusion of 5 mg of pMC-LacZ
plasmid DNA, the animals were killed and LacZ gene expression was assessed
in the liver tissue. LacZ gene expression in the liver tissue seems
restricted within certain areas as X-gal-positive cells are clustered around
the central vein [Fig. 1(a)].The pattern of SCCA1 transgene expression and the cell types
that express SCCA1 protein were evaluated by immunohistochemical analysis [Fig.
1(b,C)]. Compared with the
negative control, a number of cells in the liver tissue were stained positively
by the SCCA1 antibody after injection of 5 mg pMC-SCCA1 plasmid into
mice. The cells expressing SCCA1 are located around the central vein.Positive cell counting was used to evaluate the persistence of SCCA1
gene expression. Table 1 shows that SCCA1 gene expression was
persistent at a stable level for at least 37 d after transfection with the
positive cell ratio being approximately 30%.It is evident that no cell was detected to be positively
immunoreactive for HBsAg in the liver of the mouse injected with infectant
human serum only (Fig. 2). The cells around the central vein and
hepatic sinusoid, including the Kupffer cells, sinusoidal endothelial cells
and some hepatocytes, were found to be positively immunoreactive up to day 17
of pMC-SCCA1 group. no positive cells
were detected in the mice of day 27 and 37 of pMC-SCCA1 group.The HBsAg qualitative ELISA analysis showed that the mouse serum on
day 3 was positive and the others (day 7, 17, 27 and 37) were negative in the
control group. In the experimental group, the mouse sera of day 3 and 7 were
positive and the others (day 17, 27 and 37) were negative (Table 2).Data in Fig. 3 show that HBV DNA was detected only in the
mouse serum and liver tissue of day 3 of control group injected with infectant
human serum and not injected with pMC-SCCA1 in advance. However, HBV DNA was
found to be positive in the liver of day 3, 7 and 17 and in the mouse serum of
day 3 and 7 injected with pMC-SCCA1 24 h before injection with infectant human
serum. No HBV DNA was detected in the liver or serum of day 27 or 37.
Discussion
Due to its role in synthesizing many proteins and its involvement in
numerous genetic and acquired diseases, the liver is an important target organ for
gene transfer. A variety of vectors have been used for introducing genes into
the liver, including recombinant retrovirus [12], adenovirus [13],
adeno-associated virus [14], and nonvirus vectors such as liposome [15],
cationic polymer [16], and reconstituted chylomicron remnant [17]. Although
significant progress has been made in the successful delivery of genes to the
liver, many problems are associated with each of these methods. Retrovirus
vector can generate long-term transgene expression, but partial hepatectomy or
liver damage is usually required to stimulate cell division [18]. Adenoviral
vector allows for high transferring efficiency, but gene expression lasts for
a short period due to the host’s immune response against viral protein [19].
Adeno-associated viral vectors produce long-term gene expression, but they can
not deliver genes of a size more than 5.2 kb [20]. The current nonviral vectors
suffer from the major limitation of low transfection efficiency. To overcome
these problems, Zhang et al. [21] explored the hydrodynamics-based
procedure. Using this method, liver cells can be transfected with a foreign
gene by rapid injection of a large volume of plasmid DNA solution. It was
demonstrated that the liver was the only organ that expressed the transferred
gene and the expression persisted for at least 6 months [22]. In our present
study, by using this method we transferred the candidate HBV receptor gene SCCA1
into mouse liver, the target organ of HBV.The loss of transgene expression has been a major obstacle to the
development of nonviral vectors for the treatment of human diseases. Chen et
al. [11] previously demonstrated that bacterial DNA linked to a mammalian
expression cassette resulted in transcriptional silencing of the transgene in
vivo. To develop a means to produce a robust DNA vector that is not
silenced in vivo, they developed a phage FC31 integrase-mediated
intramolecular recombination technology to prepare minicircle vector DNA devoid
of the bacterial backbone. The authors reported that minicircle DNAs devoid of
bacterial sequences expressed 45- and 560-fold more serum human factor IX and
a1-antitrypsin,
respectively, compared to standard plasmid DNAs transfected into mouse liver.
Their data suggest that minicircles are capable of expressing high and
persistent levels of therapeutic products in vivo and have a great
potential to serve as episomal vectors for the treatment of a wide variety of
diseases. The present data showed that mouse liver expressed a high level of recombined
SCCA1 protein at a detectable level under our experimental conditions for at
least 37 d after injection. This HBV-BP transgene mouse would be a very useful
animal model for the study of the function of SCCA1 involved in HBV binding and
internalization in vivo.Our data of the HBV study in this mouse model suggested that HBV
can stay longer in the liver by intravenous injection of pMC-SCCA1 using a
hydrodynamics-based procedure. Although the liver tissues of the 3, 7 and 17 d
groups showed positive in the immunohistochemical examination and HBV-DNA PCR
test, there was no HBV-DNA detected in the 27 or 37 d groups [Fig. 2(g–l) and Fig. 3]. These findings suggested that this protein in
vivo might help HBV binding to the mouse liver cells that express this
protein, and that SCCA1 might serve as an HBV receptor or co-receptor and play
an important role in mediating HBV entry to hepatocytes. However, its role in
human HBV infection remains to be further determined.The sinusoidal endothelial and Kupffer cells near the hepatic
sinusoid were found to be strongly positive in the immunohistochemical test [Fig.
2(h–j)]. This suggests that the cells might play an important role in HBV
clearance as the first defence against HBV infection, and this function deficiency
might give HBV more access to hepatocytes.In summary, our data in this report show that the long-term
expression of the suspected HBV receptor gene can be achieved in a mouse model
by simple tail vein injection of the minicircle plasmid using a
hydrodynamics-based procedure. This method gives access to the HBV receptor
research, and our studies proved in vivo that this HBV-BP can extend
its retention of HBV in mouse liver. However, there are still other factors
involved in HBV infection and further quantitive research on this animal model
is needed.
Acknowledgements
We
thank Zhi-Ying CHEN (Departments of Pediatrics and Genetics, Stanford
University School of Medicine, Stanford, California USA), Wen-Ge HUANG and
Feng-Ying CHEN (Center of Experimental Animals at Sun Yat-Sen University) for
technical support.
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