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Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 484-491 |
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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,
[email protected]
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 signi�ficance 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 un�reactive 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 chromato�graphy 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 immunohis��to�chemistry. 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 tetra�hydro�chloride and hydrogen peroxide (Boster Biological Technology).
In each case of immunohisto�chemistry 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抯 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.
References
1��� Ganem D, Varmus HE. The molecular biology of the hepatitis B viruses. Annu Rev Biochem 1987, 56: 651-693
2��� Gripon P, Cannie I, Urban S. Efficient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral surface protein. J Virol 2005, 79: 1613-1622
3��� Hong HJ, Ryu CJ, Hur H, Kim S, Oh HK, Oh MS, Park SY. In vivo neutralization of hepatitis B virus infection by an anti-preS1 humanized antibody in chimpanzees. Virology 2004, 318: 134-141
4��� Neurath AR, Strick N, Sproul P, Ralph HE, Valinsky J. Detection of receptors for hepatitis B virus on cells of extrahepatic origin. Virology 1990, 176: 448-457
5��� Neurath AR, Strick N. Antigenic mimicry of an immunoglobulin A epitope by a hepatitis B virus cell attachment site. Virology 1990, 178: 631-634
6��� Pontisso P, Ruvoletto MG, Tiribelli C, Gerlich WH, Ruol A, Alberti A. The preS1 domain of hepatitis B virus and IgA cross-react in their binding to the hepatocyte surface. J Gen Virol 1992, 73: 2041-2045
7��� Neurath AR, Strick N, Sproul P. Search for hepatitis B virus cell receptors reveals binding sites for interleukin 6 on the virus envelope protein. J Exp Med 1992, 175: 461-469
8��� Franco A, Paroli M, Testa U, Benvenuto R, Peschle C, Balsano F, Barnaba V et al. Transferrin receptor mediates uptake and presentation of hepatitis B envelope� antigen by T lymphocytes. J Exp Med 1992, 175: 1195-1205
9��� Budkowska A, Quan C, Groh F, Bedossa P, Dubreuil P, Bouvet JP, Pillot J. Hepatitis B virus (HBV) binding factor in human serum: Candidate for a soluble form of hepatocyte HBV receptor. J Virol 1993, 67: 4316-4322
10�� De Falco S, Ruvoletto MG, Verdoliva A, Ruvo M, Raucci A, Marino M, Senatore S et al. Cloning and expression of a novel hepatitis B virus-binding protein from HepG2 cells. J Biol Chem 2001, 276: 36613-36623
11�� Chen ZY, He CY, Ehrhardt A, Kay MA. Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo. Mol Ther 2003, 8: 495-500
12�� Rettinger SD, Kennedy SC, Wu X, Saylors RL, Hafenrichter DG, Flye MW, Ponder KP. Liver-directed gene therapy: quantitative evaluation of promoter elements by using in vivo retroviral transduction. Proc Natl Acad Sci USA 1994, 91: 1460-1464
13�� Li Q, Kay MA, Finegold M, Stratford-Perricaudet LD, Woo SL. Assessment� of recombinant adenoviral vectors for hepatic gene therapy. Hum Gene Ther 1993, 4: 403-409
14�� Kitajima K, Marchadier DH, Burstein H, Rader DJ. Persistent liver expression� of murine apoA-l using vectors based on adeno-associated viral vectors serotypes 5 and 1. Atherosclerosis 2006, 186: 65-73
15�� Lemken ML, Wybranietz WA, Schmidt U, Graepler F, Armeanu S, Bitzer M, Lauer UM. Expression liver-directed genes by employing synthetic transcriptional control units. World J Gastroenterol 2005, 11: 5295-5302
16�� Uchegbu IF, Sadiq L, Pardakhty A, El-Hammadi M, Gray AI, Tetley L, Wang W et al. Gene transfer with three amphiphilic glycol chitosans��the degree of polymerisation is the main controller of transfection efficiency. J Drug Target 2004, 12: 527-539
17�� Botham KM, Zheng X, Napolitano M, Avella M, Cavallari C, Rivabene R, Bravo E. The effects of dietary n-3 polyunsaturated fatty acids delivered in chylomicron remnants on the transcription of genes regulating synthesis and secretion of very-low-density lipoprotein by the liver: Modulation by cellular oxidative state. Exp Biol Med (Maywood) 2003, 228: 143-151
18�� Moscioni AD, Rozga J, Neuzil DF, Overell RW, Holt JT, Demetriou AA. In vivo regional delivery of retrovirally mediated foreign genes to rat liver cells: Need for partial hepatectomy for successful foreign gene expression. Surgery 1993, 113: 304-311
19�� Barr D, Tubb J, Ferguson D, Scaria A, Lieber A, Wilson C, Perkins J et al. Strain related variations in adenovirally mediated transgene expression from mouse hepatocytes in vivo: Comparisons between immunocompetent and immunodeficient inbred strains. Gene Ther 1995, 2: 151-155
20�� Dong JY, Fan PD, Frizzell RA. Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther 1996, 7: 2101-2112
21 Zhang G, Budker V, Wolff JA. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum Gene Ther 1999, 10: 1735-1737
22 Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther 1999, 6: 1258-1266