Formation of Vesicular Stomatitis Virus Pseudotype
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病菌学杂志 2005年第19期
Department of Virology and Preventive Medicine, Gunma University Graduate School of Medicine, Showa-machi, Maebashi, Gunma 371-8511
Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
ABSTRACT
It has been difficult to propagate and titrate hepatitis B virus (HBV) in tissue culture. We examined whether vesicular stomatitis virus (VSV) pseudotypes bearing HBV surface (HBs) proteins infectious for human cell lines could be prepared. For this, expression plasmids for three surface proteins, L, M, and S, of HBV were made. 293T cells were then transfected with these plasmids either individually or in different combinations. 293T cells expressing HBs proteins were infected with VSVG-G, a recombinant VSV expressing green fluorescent protein (GFP), to make VSV pseudotypes. Culture supernatants together with cells were harvested and sonicated for a short time. The infectivities of freshly harvested supernatants were determined by quantifying the number of cells expressing GFP after neutralization with anti-VSV serum and mouse monoclonal antibodies (MAbs) against HBs protein. Among 14 cell lines tested for susceptibility to HBV pseudotype samples, HepG2, JHH-7, and 293T cells were judged to be the most susceptible. Namely, the infectious units (IU) of the culture supernatant samples neutralized with anti-VSV in the absence and presence of anti-HBs S MAbs and titrated on HepG2 cells ranged from 1,000 to 4,000 IU/ml and 200 to 400 IU/ml, respectively, suggesting the presence of VSVG(HBV) pseudotypes. This infectivity was inhibited by treatment with lactoferrin or dextran sulfate. Pretreatment of the cells with trypsin or tunicamycin inhibited plating of the pseudotype samples. The HBV pseudotypes can be used to analyze early steps of HBV infection, including the entry mechanism of HBV.
INTRODUCTION
Hepatitis B virus (HBV), which belongs to the virus genus Hepadnaviridae, is an important etiological agent for acute and chronic hepatitis as well as liver cirrhosis or primary hepatocellular carcinoma (15, 18). The surface proteins of HBV contain three related HBV-encoded glycoprotein species, termed large (L), middle (M), and small (S) proteins (37). As shown in Fig. 1A, all surface proteins are produced from a single open reading frame (ORF) by the use of three different translational start sites that divide this ORF into three domains: preS1, preS2, and S. Translation from the first ATG yields L protein, comprising 390 amino acids (aa), whereas translation from the ATG at aa positions 109 and 164 generates M and S proteins, respectively. The three proteins are posttranslationally modified: each of them exhibits a glycosylation site in the S domain, and an additional glycosylation occurs in the preS2 region of the M protein (Fig. 1A). Either M or S protein, when expressed alone, can be secreted as virus-like particles (25, 45). In contrast, L protein cannot be secreted when expressed alone; instead, it is mainly retained within the endoplasmic reticulum and pre-Golgi regions in the form of intraluminal particles (27, 45). It has been reported that the sequence between amino acids 3 and 77 of the preS1 region is necessary for infection of primary human hepatocytes in vitro (21). The preS2 region of M protein has been thought to be involved in polymerized albumin-mediated interaction of HBV (48), but no precise function has yet been definitively assigned to the preS2 region of the M protein. Kann et al. (18) proposed that in association with the preS1 domain of L protein, the preS2 domain of M protein might have a supportive function in viral attachment. Thus, it is still necessary to study the roles of all three proteins in the HBV infection process to clarify the process of entry of HBV into cells.
Analysis of HBV infection in vitro has still been difficult due to the lack of a suitable culture system, i.e., lack of susceptible cell lines or of cells producing HBV abundantly. Most in vitro studies have used concentrated, whole virions or human serum samples as virus sources and primary human hepatocytes or human liver cell lines, e.g., HepG2, HuH7, and PH5CH8, as target cells (12, 13, 21, 31, 41). However, their infectivities are not so high, and human serum samples and human hepatocytes are not readily available. Despite these difficulties, infection of cells with viral inocula or transfection of cells with HBV DNA has been employed in order to analyze HBV infection in vitro (12, 14, 46). In vitro infection of human hepatoma (HepG2) cells with hepatitis B virus obtained from the serum of a chronic HBV patient was first reported by Bchini et al. (3).
There seems to be only one report that describes an HBV pseudotype: murine leukemia virus (MLV) pseudotype, expected to bear the L and S proteins of HBV (41). The reported MLV(HBV) pseudotype, which was prepared after cotransfection of 293T cells with plasmids expressing HBV surface (HBs) L and S proteins, infects only primary human hepatocytes, and up to 200 HBV antigen-positive cells were detected among 106 primary human hepatocytes inoculated with it.
Vesicular stomatitis virus (VSV) pseudotypes have been used to analyze early steps of infection with various human viruses, like human T-cell leukemia virus type 1 (HTLV-1) (6), human immunodeficiency virus type 1 (HIV-1) (23), hepatitis C virus (HCV) (19), and Ebola virus (16). These pseudotypes will be especially useful for the study of viruses that do not grow well in tissue culture, like HCV or HTLV-1, or viruses that are highly biohazardous to humans, like Ebola virus. In addition, assays using the VSV pseudotypes can usually be done in a short time compared with those using the original viruses. In this study, we examined whether we could detect VSV pseudotypes bearing various surface proteins of HBV. We have been using VSVG-G to prepare pseudotypes bearing surface proteins of HTLV-1 or HCV and found that the formation of these pseudotypes could be detected after treatment with highly active anti-VSV serum (43). Thus, using similar procedures to detect HTLV-1 and HCV pseudotypes, we tested whether VSV pseudotypes bearing HBs L, M, or S protein were formed and examined their reactivity with HBV-specific antibodies or chemicals. Results thus obtained showed that VSV pseudotypes bearing HBs proteins were formed.
MATERIALS AND METHODS
Cells. All cell lines used in this study are shown in Table 1. The 293T cell line was derived from the human embryonic kidney cell line 293 and contains the simian virus 40 large T antigen (9). The PH5CH8 cell line was cloned from nonneoplastic human hepatocytes, PH5CH cells (30). PH5CH8 cells were maintained as described previously (30). HepG2 cells were derived from human liver tumor biopsies (1). HepG2 and Huh-7 (26) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Nissui Seiyaku, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS). The JHH-7 cell line was derived from HBs antigen (HBsAg)-positive hepatocellular carcinoma with liver cirrhosis (10). PLC/PRF/5 cells (Alexander cells) were derived from a primary carcinoma of the liver of an HBsAg-positive patient (2). NP-2 (39), U251MG (4), and HeLa (36) cells were maintained in Eagle's minimum essential medium (Nissui) supplemented with 10% FBS. HL60, K562, MOLT4 (40), C8166 (35), MT-2, and C91/PL cells (33) were cultured in RPMI 1640 medium (Nissui) supplemented with 10% FBS. The K562 cell line was derived from Ph'-positive chronic myelogenous leukemia in BLAST crisis (22). The HL60 cell line was derived from promyelocytic leukemia (8). MT-2 is a T-cell line persistently infected with HTLV-1 (24).
Plasmids. The genes for three surface proteins of HBV, large (L), middle (M), and small (S), were amplified by PCR from the plasmid pBSadr4x2(R), which contains two copies of the full-length HBV genome (11), using the following sense primers:
L, 5'-TCGCGAATTCATGCAGTTAATCATTACTTC-3'; M, 5'-ATGCAGTGGAACTCCACAACATTCCACCAA-3'; and S, 5'-CACCGAATTCATGGAGAACACAACATCAGG-3'. As an antisense primer, 5'-GCAACCCCGATGAGGGAACTTAAGTACCCT-3' was used. For the convenience of cloning manipulation, the L and S sense primers contained a flanking sequence with EcoRI sites, which are underlined above. The PCR products, using the L and S sense primers, were inserted into the expression plasmid pCAGGS (kindly provided by J. Miyazaki) (29). The PCR product, using the M sense primer, was first inserted into the TA cloning vector and then subcloned into pCAGGS. The sizes and orientations of the inserts in the cloned plasmid DNAs were confirmed by restriction enzyme digestion, and the inserts were subjected to partial sequencing of the plasmid DNAs. The pCAGGS/L, pCAGGS/M, and pCAGGS/S plasmids thus obtained were thereby confirmed to carry the genes for HBs L, M, and S proteins, respectively.
Virus. VSVG is a recombinant VSV derived from a full-length cDNA clone of the VSV genome in which the coding region of the VSV G protein was replaced with that of the green fluorescent protein (GFP) gene (42). VSVG pseudotype-bearing VSV G protein (VSVG-G) was prepared by infecting 293T cells with VSVG-G, which had been tranfected with the G protein expression plasmid DNA pCAGGS/VSV-G 1 day earlier, and culture supernatants were harvested after incubation for 24 h. VSVG-G was kindly provided by M. A. Whitt.
Monoclonal antibodies. Anti-HBs monoclonal antibodies (MAbs) referred to in this paper stand for MAbs against S and preS2 domains of HBs. Different clones of anti-HBs S MAb, e.g., clone 3E7 (DAKO Japan Co., Ltd., Tokyo, Japan), clone ZCH16 (Zymed Laboratories, Inc.), clone 2D1.2 (Chemicon International Inc.), and anti-preS2 MAb (clone S26; Chemicon) were used in this study. In this text, these MAbs are designated DA-3E7, ZY-ZCH16, CH-2D1.2, and CH-S26. The antigenic determinants of all the anti-HBs S MAbs are supposed to locate in the "a" determinant region of the S domain that is specific for the subtype ad/ay. The antigenic determinant for the anti-preS2 MAb (CH-S26) is the 132- to 137-amino-acid region (adw subtype specific) of the preS2 domain. Due to the presence of the S domain in the L and M proteins, three anti-HBs S MAbs used in this experiment are expected to recognize all three surface proteins (7). Rat monoclonal antibody against HTLV-1, LAT-27, was a generous gift of Y. Tanaka of Rykyu University (44).
Expression of HBV surface proteins. The expression of HBV surface (HBs) proteins was detected by indirect immunofluorescense assay (IFA), flow cytometry (FCM), and Western blot (WB) analysis. Subconfluent 293T cells seeded in 6.0-cm plates were transfected with 0.5 to 2.0 μg of one of the L, M, or S expression plasmids or with a combination (total amount of DNA being 1.0 μg for each of these transfections) by using FuGENE 6.0 transfection reagent (Roche Pharmaceuticals, Mannheim, Germany) according to the protocol recommended by the manufacturer. For IFA, 1 day after DNA transfection the cells were detached from dishes and then seeded onto 14-well chamber slides to make cell smears for IFA. After overnight incubation at 37°C, the cells were fixed with acetone or 2.0% paraformaldehyde. Half of the paraformaldehyde-fixed cell samples were further treated with 0.1% Triton X-100 to make them permeabilized. Acetone- or paraformaldehyde-fixed smears of 293T cells were treated with MAb against S protein (DA-3E7) at a 1:40 dilution for 1 h. After the smears were washed with phosphate-buffered saline (PBS) containing 0.1% sodium azide, fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin G (IgG) (DAKO) diluted at 1:100 was added. After 1 h of incubation at 37°C, the cell smears were washed and examined under a fluorescence microscope. Expression of HBs in Alexander cells was determined by seeding the cells in 14-well chamber slides as described above and staining them with MAb against HBsAg (CH-2D1.2).
Flow cytometric analysis. 293T cells were detached from plates by pipetting and suspended in culture medium. These cells were then centrifuged, and the cell pellets were resuspended in PBS. The cells were fixed with 1.0% paraformaldehyde and then either permeabilized by treatment with 0.2% Saponin or left untreated. Permeabilized and nonpermeabilized cells were incubated separately with MAb against S protein (DA-3E7) diluted at 1:40 on ice for 1 h. After washing with wash buffer (PBS containing 1.0% FBS and 0.01% sodium azide), the cells were incubated on ice for 1 h with FITC-conjugated rabbit anti-mouse IgG (DAKO) diluted at 1:100. The cells were again washed with wash buffer and suspended in PBS containing 1.0% paraformaldehyde and finally analyzed using a CytoACE-150 flow cytometer (Japan Spectroscopic Co. Ltd., Tokyo, Japan). Percentages of antigen-positive cells were determined as described previously (38).
Western blot analysis. 293T cells were transfected with the plasmids to express HBV surface antigen HBs S, M, or L protein. Forty-eight hours after transfection, the cells were collected, lysed with lysis buffer (25 mM Tris-Cl, pH 7.4, 250 mM Nacl, 5 mM EDTA, 1.0% NP-40), and centrifuged to collect cell lysates, and finally sample buffer (50 mM Tris-Cl, pH 6.8, 2.0% sodium dodecyl sulfate [SDS], 0.1% bromophenol blue, 10% glycerol) with or without 100 mM dithiothreitol (DTT), abbreviated as DTT(+) or DTT(–), respectively, was added to the lysates. The samples were heated at 100°C for 5 min and subjected to SDS-12% polyacrylamide gel electrophoresis. Separated proteins in gels were blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, Mass.). HBV surface proteins were detected by staining the membranes with MAbs against S protein (DA-3E7, ZY-ZCH16, or CH-2D1.2) and with peroxidase-conjugated secondary antibody. Finally, an ECL kit (Amersham Biosciences Ltd., Buckinghamshire, United Kingdom) was used to detect bands in X-ray films. High-Range Rainbow Molecular Weight Markers (Amersham Biosciences) were used as size markers.
Preparation of pseudotype virus samples harboring HBs surface proteins. Culture plates used for transfection were coated with poly-L-lysine (PLL) (Sigma Aldrich Co. Ltd., St. Louis, Mo.). 293T cells (2.4 x 106 cells per 6.0-cm plate) were seeded into PLL-coated dishes, and the next day the cells were transfected with the HBs-expressing plasmid DNAs. The cells were infected 30 h later with VSVG-G at a multiplicity of infection of 2.0. The virus was allowed to adsorb for 2.5 h at 37°C, and the cells were washed four times with DMEM without FBS and cultured in DMEM containing 2.0% FBS. After incubation for 21 h at 37°C, the cells were detached using cell scrapers (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) and suspended in culture medium. The cell suspensions were sonicated with a Sonifier 250 (Branson Co., Ltd., Danbury, Conn.) for 1.0 s and centrifuged to collect the supernatants. The culture supernatants were then aliquoted and stored at –80°C. Collected VSV samples were designated VSVG(HBs/L), VSVG(HBs/M), and VSVG(HBs/S), which were prepared using 293T cells transfected with pCAGGS/L, pCAGGS/M, and pCAGGS/S, respectively. VSVG(HBs/L+M), VSVG(HBs/M+S), VSVG(HBs/L+S), and VSVG(HBs/L+M+S) samples were similarly prepared after transfection of the two or three expression plasmids as explained below. Namely, pCAGGS/L+M indicates that a DNA mixture of pCAGGS/L and pCAGGS/M in equal amounts was transfected. In each combination, the total amount of DNA transfected was 1.0 μg. In addition, as a control, culture supernatant of the VSVG(–) sample was harvested from mock-transfected 293T cells infected with VSVG-G. VSVG-G was also inoculated onto 293T cells transfected with HBs fragment-free pCAGGS, and the culture supernatant was harvested and used as a control virus, designated VSVG(–). VSVG-G was titrated using 293T cells.
Detection and neutralization of HBs pseudotypes. For detection of a VSV pseudotype bearing HBs proteins (HBs pseudotypes), target cells were seeded into 96-well plates. VSV samples (114 μl) prepared using 293T cells transfected with the HBs-expressing plasmids were mixed with goat anti-VSV serum (6 μl) and anti-HBs S MAbs (DA-3E7, ZY-ZCH16, and CH-2D1.2) or MAb against preS2 (CH-S26) at different dilutions and incubated for 30 min at 37°C. Fifty microliters of VSV mixture per well was then added onto the target cells in duplicate. The presence of the HBs pseudotypes was also tested by treating the VSV samples with commercially available hepatitis B immunoglobulin (HBIG) (Nippon Seiyaku Co., Ltd., Tokyo, Japan) prepared from pooled human anti-HBs antibody-positive sera. The mixture was then inoculated onto target cells in 96-well tissue culture plates. After adsorption for 2 h, the mixture was removed and the cells were washed by DMEM, which was further replaced with fresh medium. The cultures were incubated at 37°C, but HepG2 cells were incubated at 33°C after inoculation of the pseudotype samples. Cells expressing GFP were counted under a fluorescence microscope. In addition, a rat MAb against HTLV-1, LAT-27, was also used to check the specificity of the reaction of anti-HBs MAbs with the VSV pseudotypes.
Effect of bovine lactoferrin and sulfated polysaccharides on the infectivity of the pseudotypes. The VSV pseudotype sample was treated with various concentrations of bovine lactoferrin (bLF) (Sigma), heparin, dextran sulfate (molecular weight, 500,000), and dextran (molecular weight, 7,000) at 37°C for 1 h and then inoculated onto HepG2 cells. The number of GFP-positive cells was determined using a fluorescence microscope as described above.
Chemical modification of HepG2 cells. A glycosylation inhibitor, tunicamycin (Sigma), was added to the target HepG2 cells soon after seeding cells into 96-well plates, and the cells were incubated at 37°C overnight. HepG2 cells, seeded 1 day before infection, were treated with various concentrations of trypsin for 5 min, and an equal volume of fresh medium was added to the cells to stop the reaction. The pseudotype sample was inoculated onto these pretreated HepG2 cells, and the number of GFP-positive cells was determined as described above.
RESULTS
Detection of HBs surface proteins by IFA and FCM. The optimum level of expression of the surface proteins was determined by using different amounts of plasmid DNA for transfection. Staining of acetone-fixed cell smears with a MAb against HBs S protein (DA-3E7) by IFA showed that the expression levels of all three HBV surface (HBs) proteins were optimal at 1.0 μg of DNA (Fig. 1B). The intracellular expression after transfection with 1.0 μg of each HBs expression plasmid individually as detected by IFA is shown in Fig. 1C. HBs S or M protein was detected to be expressed at a similar efficiency with similar localization, while that of L was different. PLC/PRF/5 cells (Alexander cells) were stained positive with only one (CH-2D1.2) of the three MAbs tested. The localization of HBsAg in Alexander cells looked similar to that of 293T cells transfected with either S or M protein (Fig. 1C). JHH-7 cells did not stain positively with any of the anti-HBs MAbs tested (data not shown). In order to differentiate their intracellular expression from surface expression, transfected 293T cells were permeabilized with either Triton X-100 (for IFA) or Saponin (for FCM). About 5 to 10% of the cells were positive for L, M, and S proteins, whereas 10 to 30% of the cells treated with Triton X-100 were positive for L, M, and S proteins (Table 2). Flow cytometric (FCM) analysis results for HBs protein expression were also consistent with those of IFA. In FCM, about 10% of the cells were detected to express S and M proteins on the surface, but L protein was not detected on the surface at a significant level, whereas intracellular expression of L, M, and S proteins was detected in about 25 to 35% of the cells (Fig. 2A, Table 2).
Detection of HBs proteins by Western blot. 293T cells transfected with pCAGGS plasmids containing genes for HBs S, M, and L proteins were lysed, and their cell lysates were subjected to Western blot (WB) analysis in the presence (+) or absence (–) of DTT. The DTT(+) samples gave a positive reaction with none of HBs MAbs tested (data not shown). The DTT(–) samples gave positive reactions with two (CH-2D1.2 and ZY-ZCH16) of the three anti-HBs S MAbs tested. These results suggest that the antigenicity of the surface proteins detected by these MAbs is mostly conformation dependent in nature. Because of the presence of a glycosylation site in the S domain and an additional glycosylation site in the M domain (37), under reducing conditions, DTT(+), S, M, and L proteins could be detected as both major nonglycosylated and glycosylated forms of 24 and 27 kDa, 33 and 36 kDa, and 39 and 42 kDa, respectively, as reported by Lambert and Prange (20). Since we could detect WB bands only under nonreducing conditions, DTT(–), the bands corresponding to expected molecular sizes could not be detected. WB of the DTT(–) samples detected using MAb CH-2D1.2 (Fig. 2B) showed multiple bands, i.e., 33, 42, and 45 kDa in the lysates of cells transfected with pCAGGS/S. Transfection of pCAGGS/M plasmid gave bands with estimated sizes of 33, 42, 45, and 66 kDa, whereas the lysate of the pCAGGS/L-transfected cells gave two major bands of 45 and 88 kDa. After transfection of pCAGGS/M plasmid, several bands with similar sizes observed in the lane of pCAGGS/S were detected. Transfection of pCAGGS/L and pCAGGS/M plasmids gave L-plasmid-specific 88-kDa and M-plasmid-specific 66-kDa bands, respectively, whereas an S-plasmid-specific 45-kDa band was shared by all the transfected cell lysates, namely, cells transfected with pCAGGS/L, pCAGGS/M, or pCAGGS/S. The major bands of about 45, 66, and 88 kDa detected after transfection of 293T cells with pCAGGS/S, pCAGGS/M, and pCAGGS/L plasmids, respectively (Fig. 2B), might be explained by formation of dimer forms of the respective proteins. As shown in Fig. 2B, in addition to these major bands, other bands were also shown to be present in cases of transfection with plasmids containing genes of M and L proteins. These additional bands could be partly due to an internal initiation of translation from the translation site of the S protein. Even in cases of transfection with the HBs S expression plasmid, some of the additional bands were also detected (Fig. 2B, lane 1); partial deglycosylation might produce these bands.
Detection of VSVG(HBV) pseudotypes. HepG2 cells, grown in 96-well plates, were used to detect formation of VSV pseudotypes. The culture samples prepared using 293T cells transfected with the HBs expression plasmids and infected with VSVG-G were incubated with goat anti-VSV serum and anti-HBs S MAb (DA-3E7) and used to infect the HepG2 cells. Infectious units (IU/ml) of the VSVG-G-infected samples were determined by counting the number of GFP-expressing cells. Candidate VSV pseudotype samples prepared by transfection of the plasmids coding for HBs L, M, and S proteins gave infectious titers of about 1,500, 3,800, and 1,000 IU/ml, respectively (Fig. 3A). Transfection of 293T cells with combinations of the expression plasmids did not further increase the infectivities. The infectivities of all these pseudotype samples were inhibited to the background level when treated with the anti-HBs S MAb, i.e., less than 300 IU/ml (Fig. 3A). The neutralization of the pseudotype infectivities by the anti-HBs S MAb suggests the successful formation of VSVG(HBV) pseudotypes.
Neutralization of the pseudotype samples by MAbs. In order to confirm the presence of VSVG(HBV) pseudotypes, neutralization tests with other MAbs against viral surface proteins were done. That is, 293T cells were transfected with pCAGGS/M plasmid and infected with VSVG-G. The VSVG-G-infected sample designated VSVG(HBs/M) was prepared after sonication and tested for neutralization by the goat anti-VSV serum together with MAbs against HBV or HTLV-1. Figure 3B shows that the infectivity was markedly neutralized by the other two mouse anti-HBs S MAbs (ZY-ZCH16 and CH-2D1.2) as well. An anti-HTLV-1 MAb, LAT-27, did not affect its plating onto HepG2 cells, whereas a HTLV-1 pseudotype was well neutralized by LAT-27 but by none of the anti-HBs S MAbs (Fig. 3B). MAb against preS2 (CH-S26) neutralized neither VSVG(HBs/M) nor VSVG(HTLV-1) pseudotype sample. Thus, the infectivity of the VSVG(HBs/M) sample was especially neutralized by the three HBs S protein-specific MAbs but not neutralized by anti-preS2 MAb or anti-HTLV-1 MAb (Fig. 3B). In addition, anti-HBs S MAb (DA-3E7) also neutralized the infectivity of VSVG(HBs/L) and VSVG(HBs/S) pseudotype samples (Fig. 3A), but anti-HTLV-1 MAb could neutralize none of these samples (data not shown).
Infectivity of the VSVG(HBs/M) pseudotype sample for various cell lines. The VSVG(HBs/M) sample prepared as described above was used to infect various target cells in order to study its cell tropism. Target cells were chosen according to the previous reports on their susceptibilities to HBV (13, 31, 41) and HCV (19, 43) infection. The only HBV pseudotype reported so far, MLV(HBV) pseudotype (41), exclusively infects only primary human hepatocytes but not any of the established cell lines tested, such as HepG2 or 293T. We were interested in examining whether any of the cell lines derived from human hepatoma, glioma, kidney, osteosarcoma, etc., could be infected by a VSVG(HBs/M) pseudotype sample. Infectious titers of the VSVG(HBs/M) samples in 14 different human cell lines were determined. As shown in Fig. 3 and Table 1, the VSVG(HBs/M) sample was able to infect HepG2 cells. Among the other 13 cell lines, a hepatocarcinoma cell line, JHH-7, and a human embryonic kidney cell line, 293T, were judged to be clearly susceptible to the VSVG(HBs/M) pseudotype sample. Alexander cells were apparently but less susceptible to the pseudotype sample than HepG2 or JHH-7 cells (data not shown). None of the other cell lines, namely hematopoietic, glioma, or osteosarcoma cell line, tested in this study was apparently, if not at all, susceptible to infection by the pseudotype sample (Table 1). The VSVG-G pseudotype was used as a control to test the susceptibilities of various cell lines to VSV replication. The cell lines used in this study showed different but high degrees of susceptibilities to VSVG-G infection, indicating that a lack of susceptibility to the VSVG(HBs/M) sample was not due to their resistance to VSVG-G.
Effect of chemicals on infection of VSVG(HBs/M) pseudotype samples. Infection of HBV to human hepatocytes was reported to be inhibited by bLF (13). Heparin and other sulfated polysaccharides have also been shown to be strong inhibitors of the binding of HBV to HepG2 cells (49). Thus, we examined effects of bLF, heparin, and dextran sulfate and noticed that infectivity was inhibited by about 90% at 1 mg/ml bLF (Fig. 4A). Sulfated polysaccharides, heparin and dextran sulfate, at 100 μg/ml, also inhibited the infectivity by about 50% and 80%, respectively (Fig. 4B). In contrast, the nonsulfated dextran did not inhibit the infectivity.
We next examined whether a glycosylation of protein(s) on the cell surface is required for HBV infection. For this, HepG2 cells were treated with trypsin just before infection or with tunicamycin 1 day before infection. Treatment with 1.0% trypsin and 10 μg/ml tunicamycin showed about 80% and 70% inhibition of the infectivity, respectively (Fig. 5).
DISCUSSION
In order to prepare VSV pseudotype viruses, the expression of HBs proteins in 293T cells was studied by IFA, FCM, and WB. The S and M proteins are localized mainly in the cytoplasm, and their distribution seemed to be homogeneous inside it, whereas localization of the L protein was granular with its heterogeneous distribution inside the cells (Fig. 1C). This result is consistent with the findings demonstrated by several reporters (32, 47). Localization of S, M, or L protein detected by specific MAbs against HBsAg in hepatocytes of patients with chronic HBV infection reported previously (17) is similar to that observed in 293T cells transfected with corresponding plasmids.
We performed cotransfection experiments using different combinations of the S, M, and L expression plasmids, but the expression levels could not be raised further, as was shown upon individual plasmid transfection (data not shown). This result seems to be different from the result for the recently reported MLV pseudotype. That is, in the report of Sung and Lai (41), the surface expression of L or S protein individually can hardly be detected, whereas about 10% of the surface expression was shown in our experiment when S or M plasmids were transfected independently. We considered that this would be due to a difference between systems employed for the transfection or the use of different expression plasmids to express the HBs proteins. For example, Sung and Lai (41) used retroviral vectors whereas we used expression plasmids containing a ?-actin promoter.
After transfection, the transfected cells were infected with VSVG-G for production of VSVG(HBV) pseudotypes that contain the VSV core protein bearing the surface proteins of HBV. As VSV pseudotype titers preliminarily determined had not been sufficiently high (data not shown), the cells detached and suspended in their culture supernatants were sonicated prior to making stocks of the pseudotype samples as described by Tamura et al. (43) to prepare HCV pseudotypes bearing the native form of HCV surface proteins. The infectious titers of the pseudotype samples made with sonication were about five times as high as those of nonsonicated samples (data not shown).
Although the infectivity of the pseudotype was shown to be efficiently neutralized by the anti-HBs S MAbs, the infectivity of the pseudotype sample could not be well neutralized by concentrated human immunoglobulin positive for HBs antibody and available commercially (HBIG) (data not shown). Low titers of HBIG in the neutralization of HBV infection in vitro have already been reported: the HBIG titer is about 2,000 times as low as that of anti-HBs-humanized MAb produced in Ryu et al.'s laboratory (34). Our finding may be consistent with this finding. It is probable that HBIG contains antibodies reacting with the preS1 antigenic determinant, as this region has been considered one of the receptor-binding sites, and that human antibodies may preferentially neutralize native HBV in the blood.
The inhibitory effects of various chemicals, like bLF, tunicamycin, trypsin, and sulfated polysaccharides, on infection of the pseudotype sample was also studied (Fig. 4 and 5). The infectivity of the VSVG(HBs/M) pseudotype was inhibited by bLF in our experiment. The inhibitory effect of bLF, however, seemed to be different from that reported by Hara et al. (13). They reported that preincubation of target cells with bLF is necessary for the inhibition. In contrast, our result is similar to the effect of bLF in HCV infection reported by several groups (13, 43), including Hara's group: preincubation of the cells with bLF before HBs pseudotype infection could not show any inhibitory effect (data not shown). Our results suggest that the bLF-binding site may locate in the preS2 or the S domain of M protein. It is also probable that bLF binds to a region to which neutralizing anti-HBs MAbs also bind. Further study will be necessary to identify a site to which bLF binds.
One of the interesting findings in our study may be that among three HBs proteins, transfection of the expression plasmid DNA for HBs M protein gave the slightly but clearly highest infectious titer of the pseudotype (Fig. 3A) when titrated in HepG2 cells. This finding suggests that M protein of HBV, especially the preS2 region, has an important role in the infection of HBV. This result looks contradictory to some previous reports about its role. For example, Le Seyec et al. (21) reported that most of the preS2 region is dispensable for HBV infection. Cho et al. (5), however, reported the presence of a binding region in the preS2 region for a cellular HBV receptor(s). Namely, the binding region, which contains the N-terminal 5 aa of preS2, potentially contributes to attachment to and entry into target cells (21). Likewise, our results suggest that the preS2 region of M protein may contain a binding region to target cells. The infectious titer of the VSVG(HBs/S) sample was the lowest among all pseudotype samples tested in this study, although all three anti-HBs MAbs neutralized all types of HBs pseudotypes tested.
WB analysis (Fig. 2B) using anti-HBs S MAb showed that WB bands with similar sizes were detected even after transfection of the HBs expression plasmid for the HBs M gene. Thus, it is probable that S protein also is produced in 293T cells transfected with the plasmid for HBs M protein. S protein together with M protein may lead to an efficient production of HBs pseudotype in the HBs/M pseudotype sample. The 21- to 47-aa sequence of preS1 and 120- to 145-aa sequence of preS2 have been reported to be a crucial and an auxiliary cell-binding sites, respectively (28). The presence of the secondary attachment region in HBs S protein has also been reported by Paran et al. (31). However, a direct interaction of natural HBs S protein with hepatocytes or a role of HBs S protein in the penetration of hepatocytes has not been convincingly demonstrated. MAbs used in this study could recognize S-S bonds in the region of HBs S protein, since bands for the specific proteins could be detected upon WB with DTT(–) samples only but not WB with DTT(+) samples, and this region was recognized by the neutralizing MAbs. This finding suggests that a receptor-binding site as well as a MAb-binding site in HBV surface proteins may be created by the S-S bond. In other words, the S-S bond in the S protein or S domain in M and L proteins may play an important role in entry of HBV into target cells.
Although there seem to be some contradictory results in our study to those reported previously, the discrepancies might be attributable to the difference in systems to detect HBV infection, such as whether inoculating samples are native virus derived from the blood or VSV pseudotypes, whether target cells are primary hepatocytes or established cell lines, or whether titers of inoculating viruses are high or low. Properties of HBs proteins produced in hepatocytes in vivo or those produced in 293T cells in vitro may be different. If titers of inoculating viruses are detected to be different, a region of HBs gene necessary for infection or a susceptibility of cells to HBV can be determined differently.
In conclusion, we considered that VSV pseudotypes bearing the surface proteins of HBV could be prepared. These pseudotypes most efficiently infected the human hepatoma cell lines HepG2 and JHH-7 among establish human cell lines tested. To our knowledge, this is the first report of production of VSV pseudotype virus bearing HBs proteins. The VSVG(HBV) pseudotypes might be useful in a safe and rapid assay system to clarify early steps of HBV infection. This system may also contribute to screening and development of antiviral drugs that interfere with HBV infection.
ACKNOWLEDGMENTS
We thank M. A. Whitt for kindly supplying VSVG-G and J. Miyazaki for kindly supplying the plasmid pCAGGS.
This work was supported by a grant-in-aid from the Japanese Society for the Promotion of Science, grants from the Japan Health Sciences Foundation and CREST, and the 21st century COE program supported by the Ministry of Education, Culture, Sports Science, and Technology of Japan.
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Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
ABSTRACT
It has been difficult to propagate and titrate hepatitis B virus (HBV) in tissue culture. We examined whether vesicular stomatitis virus (VSV) pseudotypes bearing HBV surface (HBs) proteins infectious for human cell lines could be prepared. For this, expression plasmids for three surface proteins, L, M, and S, of HBV were made. 293T cells were then transfected with these plasmids either individually or in different combinations. 293T cells expressing HBs proteins were infected with VSVG-G, a recombinant VSV expressing green fluorescent protein (GFP), to make VSV pseudotypes. Culture supernatants together with cells were harvested and sonicated for a short time. The infectivities of freshly harvested supernatants were determined by quantifying the number of cells expressing GFP after neutralization with anti-VSV serum and mouse monoclonal antibodies (MAbs) against HBs protein. Among 14 cell lines tested for susceptibility to HBV pseudotype samples, HepG2, JHH-7, and 293T cells were judged to be the most susceptible. Namely, the infectious units (IU) of the culture supernatant samples neutralized with anti-VSV in the absence and presence of anti-HBs S MAbs and titrated on HepG2 cells ranged from 1,000 to 4,000 IU/ml and 200 to 400 IU/ml, respectively, suggesting the presence of VSVG(HBV) pseudotypes. This infectivity was inhibited by treatment with lactoferrin or dextran sulfate. Pretreatment of the cells with trypsin or tunicamycin inhibited plating of the pseudotype samples. The HBV pseudotypes can be used to analyze early steps of HBV infection, including the entry mechanism of HBV.
INTRODUCTION
Hepatitis B virus (HBV), which belongs to the virus genus Hepadnaviridae, is an important etiological agent for acute and chronic hepatitis as well as liver cirrhosis or primary hepatocellular carcinoma (15, 18). The surface proteins of HBV contain three related HBV-encoded glycoprotein species, termed large (L), middle (M), and small (S) proteins (37). As shown in Fig. 1A, all surface proteins are produced from a single open reading frame (ORF) by the use of three different translational start sites that divide this ORF into three domains: preS1, preS2, and S. Translation from the first ATG yields L protein, comprising 390 amino acids (aa), whereas translation from the ATG at aa positions 109 and 164 generates M and S proteins, respectively. The three proteins are posttranslationally modified: each of them exhibits a glycosylation site in the S domain, and an additional glycosylation occurs in the preS2 region of the M protein (Fig. 1A). Either M or S protein, when expressed alone, can be secreted as virus-like particles (25, 45). In contrast, L protein cannot be secreted when expressed alone; instead, it is mainly retained within the endoplasmic reticulum and pre-Golgi regions in the form of intraluminal particles (27, 45). It has been reported that the sequence between amino acids 3 and 77 of the preS1 region is necessary for infection of primary human hepatocytes in vitro (21). The preS2 region of M protein has been thought to be involved in polymerized albumin-mediated interaction of HBV (48), but no precise function has yet been definitively assigned to the preS2 region of the M protein. Kann et al. (18) proposed that in association with the preS1 domain of L protein, the preS2 domain of M protein might have a supportive function in viral attachment. Thus, it is still necessary to study the roles of all three proteins in the HBV infection process to clarify the process of entry of HBV into cells.
Analysis of HBV infection in vitro has still been difficult due to the lack of a suitable culture system, i.e., lack of susceptible cell lines or of cells producing HBV abundantly. Most in vitro studies have used concentrated, whole virions or human serum samples as virus sources and primary human hepatocytes or human liver cell lines, e.g., HepG2, HuH7, and PH5CH8, as target cells (12, 13, 21, 31, 41). However, their infectivities are not so high, and human serum samples and human hepatocytes are not readily available. Despite these difficulties, infection of cells with viral inocula or transfection of cells with HBV DNA has been employed in order to analyze HBV infection in vitro (12, 14, 46). In vitro infection of human hepatoma (HepG2) cells with hepatitis B virus obtained from the serum of a chronic HBV patient was first reported by Bchini et al. (3).
There seems to be only one report that describes an HBV pseudotype: murine leukemia virus (MLV) pseudotype, expected to bear the L and S proteins of HBV (41). The reported MLV(HBV) pseudotype, which was prepared after cotransfection of 293T cells with plasmids expressing HBV surface (HBs) L and S proteins, infects only primary human hepatocytes, and up to 200 HBV antigen-positive cells were detected among 106 primary human hepatocytes inoculated with it.
Vesicular stomatitis virus (VSV) pseudotypes have been used to analyze early steps of infection with various human viruses, like human T-cell leukemia virus type 1 (HTLV-1) (6), human immunodeficiency virus type 1 (HIV-1) (23), hepatitis C virus (HCV) (19), and Ebola virus (16). These pseudotypes will be especially useful for the study of viruses that do not grow well in tissue culture, like HCV or HTLV-1, or viruses that are highly biohazardous to humans, like Ebola virus. In addition, assays using the VSV pseudotypes can usually be done in a short time compared with those using the original viruses. In this study, we examined whether we could detect VSV pseudotypes bearing various surface proteins of HBV. We have been using VSVG-G to prepare pseudotypes bearing surface proteins of HTLV-1 or HCV and found that the formation of these pseudotypes could be detected after treatment with highly active anti-VSV serum (43). Thus, using similar procedures to detect HTLV-1 and HCV pseudotypes, we tested whether VSV pseudotypes bearing HBs L, M, or S protein were formed and examined their reactivity with HBV-specific antibodies or chemicals. Results thus obtained showed that VSV pseudotypes bearing HBs proteins were formed.
MATERIALS AND METHODS
Cells. All cell lines used in this study are shown in Table 1. The 293T cell line was derived from the human embryonic kidney cell line 293 and contains the simian virus 40 large T antigen (9). The PH5CH8 cell line was cloned from nonneoplastic human hepatocytes, PH5CH cells (30). PH5CH8 cells were maintained as described previously (30). HepG2 cells were derived from human liver tumor biopsies (1). HepG2 and Huh-7 (26) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Nissui Seiyaku, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS). The JHH-7 cell line was derived from HBs antigen (HBsAg)-positive hepatocellular carcinoma with liver cirrhosis (10). PLC/PRF/5 cells (Alexander cells) were derived from a primary carcinoma of the liver of an HBsAg-positive patient (2). NP-2 (39), U251MG (4), and HeLa (36) cells were maintained in Eagle's minimum essential medium (Nissui) supplemented with 10% FBS. HL60, K562, MOLT4 (40), C8166 (35), MT-2, and C91/PL cells (33) were cultured in RPMI 1640 medium (Nissui) supplemented with 10% FBS. The K562 cell line was derived from Ph'-positive chronic myelogenous leukemia in BLAST crisis (22). The HL60 cell line was derived from promyelocytic leukemia (8). MT-2 is a T-cell line persistently infected with HTLV-1 (24).
Plasmids. The genes for three surface proteins of HBV, large (L), middle (M), and small (S), were amplified by PCR from the plasmid pBSadr4x2(R), which contains two copies of the full-length HBV genome (11), using the following sense primers:
L, 5'-TCGCGAATTCATGCAGTTAATCATTACTTC-3'; M, 5'-ATGCAGTGGAACTCCACAACATTCCACCAA-3'; and S, 5'-CACCGAATTCATGGAGAACACAACATCAGG-3'. As an antisense primer, 5'-GCAACCCCGATGAGGGAACTTAAGTACCCT-3' was used. For the convenience of cloning manipulation, the L and S sense primers contained a flanking sequence with EcoRI sites, which are underlined above. The PCR products, using the L and S sense primers, were inserted into the expression plasmid pCAGGS (kindly provided by J. Miyazaki) (29). The PCR product, using the M sense primer, was first inserted into the TA cloning vector and then subcloned into pCAGGS. The sizes and orientations of the inserts in the cloned plasmid DNAs were confirmed by restriction enzyme digestion, and the inserts were subjected to partial sequencing of the plasmid DNAs. The pCAGGS/L, pCAGGS/M, and pCAGGS/S plasmids thus obtained were thereby confirmed to carry the genes for HBs L, M, and S proteins, respectively.
Virus. VSVG is a recombinant VSV derived from a full-length cDNA clone of the VSV genome in which the coding region of the VSV G protein was replaced with that of the green fluorescent protein (GFP) gene (42). VSVG pseudotype-bearing VSV G protein (VSVG-G) was prepared by infecting 293T cells with VSVG-G, which had been tranfected with the G protein expression plasmid DNA pCAGGS/VSV-G 1 day earlier, and culture supernatants were harvested after incubation for 24 h. VSVG-G was kindly provided by M. A. Whitt.
Monoclonal antibodies. Anti-HBs monoclonal antibodies (MAbs) referred to in this paper stand for MAbs against S and preS2 domains of HBs. Different clones of anti-HBs S MAb, e.g., clone 3E7 (DAKO Japan Co., Ltd., Tokyo, Japan), clone ZCH16 (Zymed Laboratories, Inc.), clone 2D1.2 (Chemicon International Inc.), and anti-preS2 MAb (clone S26; Chemicon) were used in this study. In this text, these MAbs are designated DA-3E7, ZY-ZCH16, CH-2D1.2, and CH-S26. The antigenic determinants of all the anti-HBs S MAbs are supposed to locate in the "a" determinant region of the S domain that is specific for the subtype ad/ay. The antigenic determinant for the anti-preS2 MAb (CH-S26) is the 132- to 137-amino-acid region (adw subtype specific) of the preS2 domain. Due to the presence of the S domain in the L and M proteins, three anti-HBs S MAbs used in this experiment are expected to recognize all three surface proteins (7). Rat monoclonal antibody against HTLV-1, LAT-27, was a generous gift of Y. Tanaka of Rykyu University (44).
Expression of HBV surface proteins. The expression of HBV surface (HBs) proteins was detected by indirect immunofluorescense assay (IFA), flow cytometry (FCM), and Western blot (WB) analysis. Subconfluent 293T cells seeded in 6.0-cm plates were transfected with 0.5 to 2.0 μg of one of the L, M, or S expression plasmids or with a combination (total amount of DNA being 1.0 μg for each of these transfections) by using FuGENE 6.0 transfection reagent (Roche Pharmaceuticals, Mannheim, Germany) according to the protocol recommended by the manufacturer. For IFA, 1 day after DNA transfection the cells were detached from dishes and then seeded onto 14-well chamber slides to make cell smears for IFA. After overnight incubation at 37°C, the cells were fixed with acetone or 2.0% paraformaldehyde. Half of the paraformaldehyde-fixed cell samples were further treated with 0.1% Triton X-100 to make them permeabilized. Acetone- or paraformaldehyde-fixed smears of 293T cells were treated with MAb against S protein (DA-3E7) at a 1:40 dilution for 1 h. After the smears were washed with phosphate-buffered saline (PBS) containing 0.1% sodium azide, fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin G (IgG) (DAKO) diluted at 1:100 was added. After 1 h of incubation at 37°C, the cell smears were washed and examined under a fluorescence microscope. Expression of HBs in Alexander cells was determined by seeding the cells in 14-well chamber slides as described above and staining them with MAb against HBsAg (CH-2D1.2).
Flow cytometric analysis. 293T cells were detached from plates by pipetting and suspended in culture medium. These cells were then centrifuged, and the cell pellets were resuspended in PBS. The cells were fixed with 1.0% paraformaldehyde and then either permeabilized by treatment with 0.2% Saponin or left untreated. Permeabilized and nonpermeabilized cells were incubated separately with MAb against S protein (DA-3E7) diluted at 1:40 on ice for 1 h. After washing with wash buffer (PBS containing 1.0% FBS and 0.01% sodium azide), the cells were incubated on ice for 1 h with FITC-conjugated rabbit anti-mouse IgG (DAKO) diluted at 1:100. The cells were again washed with wash buffer and suspended in PBS containing 1.0% paraformaldehyde and finally analyzed using a CytoACE-150 flow cytometer (Japan Spectroscopic Co. Ltd., Tokyo, Japan). Percentages of antigen-positive cells were determined as described previously (38).
Western blot analysis. 293T cells were transfected with the plasmids to express HBV surface antigen HBs S, M, or L protein. Forty-eight hours after transfection, the cells were collected, lysed with lysis buffer (25 mM Tris-Cl, pH 7.4, 250 mM Nacl, 5 mM EDTA, 1.0% NP-40), and centrifuged to collect cell lysates, and finally sample buffer (50 mM Tris-Cl, pH 6.8, 2.0% sodium dodecyl sulfate [SDS], 0.1% bromophenol blue, 10% glycerol) with or without 100 mM dithiothreitol (DTT), abbreviated as DTT(+) or DTT(–), respectively, was added to the lysates. The samples were heated at 100°C for 5 min and subjected to SDS-12% polyacrylamide gel electrophoresis. Separated proteins in gels were blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, Mass.). HBV surface proteins were detected by staining the membranes with MAbs against S protein (DA-3E7, ZY-ZCH16, or CH-2D1.2) and with peroxidase-conjugated secondary antibody. Finally, an ECL kit (Amersham Biosciences Ltd., Buckinghamshire, United Kingdom) was used to detect bands in X-ray films. High-Range Rainbow Molecular Weight Markers (Amersham Biosciences) were used as size markers.
Preparation of pseudotype virus samples harboring HBs surface proteins. Culture plates used for transfection were coated with poly-L-lysine (PLL) (Sigma Aldrich Co. Ltd., St. Louis, Mo.). 293T cells (2.4 x 106 cells per 6.0-cm plate) were seeded into PLL-coated dishes, and the next day the cells were transfected with the HBs-expressing plasmid DNAs. The cells were infected 30 h later with VSVG-G at a multiplicity of infection of 2.0. The virus was allowed to adsorb for 2.5 h at 37°C, and the cells were washed four times with DMEM without FBS and cultured in DMEM containing 2.0% FBS. After incubation for 21 h at 37°C, the cells were detached using cell scrapers (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) and suspended in culture medium. The cell suspensions were sonicated with a Sonifier 250 (Branson Co., Ltd., Danbury, Conn.) for 1.0 s and centrifuged to collect the supernatants. The culture supernatants were then aliquoted and stored at –80°C. Collected VSV samples were designated VSVG(HBs/L), VSVG(HBs/M), and VSVG(HBs/S), which were prepared using 293T cells transfected with pCAGGS/L, pCAGGS/M, and pCAGGS/S, respectively. VSVG(HBs/L+M), VSVG(HBs/M+S), VSVG(HBs/L+S), and VSVG(HBs/L+M+S) samples were similarly prepared after transfection of the two or three expression plasmids as explained below. Namely, pCAGGS/L+M indicates that a DNA mixture of pCAGGS/L and pCAGGS/M in equal amounts was transfected. In each combination, the total amount of DNA transfected was 1.0 μg. In addition, as a control, culture supernatant of the VSVG(–) sample was harvested from mock-transfected 293T cells infected with VSVG-G. VSVG-G was also inoculated onto 293T cells transfected with HBs fragment-free pCAGGS, and the culture supernatant was harvested and used as a control virus, designated VSVG(–). VSVG-G was titrated using 293T cells.
Detection and neutralization of HBs pseudotypes. For detection of a VSV pseudotype bearing HBs proteins (HBs pseudotypes), target cells were seeded into 96-well plates. VSV samples (114 μl) prepared using 293T cells transfected with the HBs-expressing plasmids were mixed with goat anti-VSV serum (6 μl) and anti-HBs S MAbs (DA-3E7, ZY-ZCH16, and CH-2D1.2) or MAb against preS2 (CH-S26) at different dilutions and incubated for 30 min at 37°C. Fifty microliters of VSV mixture per well was then added onto the target cells in duplicate. The presence of the HBs pseudotypes was also tested by treating the VSV samples with commercially available hepatitis B immunoglobulin (HBIG) (Nippon Seiyaku Co., Ltd., Tokyo, Japan) prepared from pooled human anti-HBs antibody-positive sera. The mixture was then inoculated onto target cells in 96-well tissue culture plates. After adsorption for 2 h, the mixture was removed and the cells were washed by DMEM, which was further replaced with fresh medium. The cultures were incubated at 37°C, but HepG2 cells were incubated at 33°C after inoculation of the pseudotype samples. Cells expressing GFP were counted under a fluorescence microscope. In addition, a rat MAb against HTLV-1, LAT-27, was also used to check the specificity of the reaction of anti-HBs MAbs with the VSV pseudotypes.
Effect of bovine lactoferrin and sulfated polysaccharides on the infectivity of the pseudotypes. The VSV pseudotype sample was treated with various concentrations of bovine lactoferrin (bLF) (Sigma), heparin, dextran sulfate (molecular weight, 500,000), and dextran (molecular weight, 7,000) at 37°C for 1 h and then inoculated onto HepG2 cells. The number of GFP-positive cells was determined using a fluorescence microscope as described above.
Chemical modification of HepG2 cells. A glycosylation inhibitor, tunicamycin (Sigma), was added to the target HepG2 cells soon after seeding cells into 96-well plates, and the cells were incubated at 37°C overnight. HepG2 cells, seeded 1 day before infection, were treated with various concentrations of trypsin for 5 min, and an equal volume of fresh medium was added to the cells to stop the reaction. The pseudotype sample was inoculated onto these pretreated HepG2 cells, and the number of GFP-positive cells was determined as described above.
RESULTS
Detection of HBs surface proteins by IFA and FCM. The optimum level of expression of the surface proteins was determined by using different amounts of plasmid DNA for transfection. Staining of acetone-fixed cell smears with a MAb against HBs S protein (DA-3E7) by IFA showed that the expression levels of all three HBV surface (HBs) proteins were optimal at 1.0 μg of DNA (Fig. 1B). The intracellular expression after transfection with 1.0 μg of each HBs expression plasmid individually as detected by IFA is shown in Fig. 1C. HBs S or M protein was detected to be expressed at a similar efficiency with similar localization, while that of L was different. PLC/PRF/5 cells (Alexander cells) were stained positive with only one (CH-2D1.2) of the three MAbs tested. The localization of HBsAg in Alexander cells looked similar to that of 293T cells transfected with either S or M protein (Fig. 1C). JHH-7 cells did not stain positively with any of the anti-HBs MAbs tested (data not shown). In order to differentiate their intracellular expression from surface expression, transfected 293T cells were permeabilized with either Triton X-100 (for IFA) or Saponin (for FCM). About 5 to 10% of the cells were positive for L, M, and S proteins, whereas 10 to 30% of the cells treated with Triton X-100 were positive for L, M, and S proteins (Table 2). Flow cytometric (FCM) analysis results for HBs protein expression were also consistent with those of IFA. In FCM, about 10% of the cells were detected to express S and M proteins on the surface, but L protein was not detected on the surface at a significant level, whereas intracellular expression of L, M, and S proteins was detected in about 25 to 35% of the cells (Fig. 2A, Table 2).
Detection of HBs proteins by Western blot. 293T cells transfected with pCAGGS plasmids containing genes for HBs S, M, and L proteins were lysed, and their cell lysates were subjected to Western blot (WB) analysis in the presence (+) or absence (–) of DTT. The DTT(+) samples gave a positive reaction with none of HBs MAbs tested (data not shown). The DTT(–) samples gave positive reactions with two (CH-2D1.2 and ZY-ZCH16) of the three anti-HBs S MAbs tested. These results suggest that the antigenicity of the surface proteins detected by these MAbs is mostly conformation dependent in nature. Because of the presence of a glycosylation site in the S domain and an additional glycosylation site in the M domain (37), under reducing conditions, DTT(+), S, M, and L proteins could be detected as both major nonglycosylated and glycosylated forms of 24 and 27 kDa, 33 and 36 kDa, and 39 and 42 kDa, respectively, as reported by Lambert and Prange (20). Since we could detect WB bands only under nonreducing conditions, DTT(–), the bands corresponding to expected molecular sizes could not be detected. WB of the DTT(–) samples detected using MAb CH-2D1.2 (Fig. 2B) showed multiple bands, i.e., 33, 42, and 45 kDa in the lysates of cells transfected with pCAGGS/S. Transfection of pCAGGS/M plasmid gave bands with estimated sizes of 33, 42, 45, and 66 kDa, whereas the lysate of the pCAGGS/L-transfected cells gave two major bands of 45 and 88 kDa. After transfection of pCAGGS/M plasmid, several bands with similar sizes observed in the lane of pCAGGS/S were detected. Transfection of pCAGGS/L and pCAGGS/M plasmids gave L-plasmid-specific 88-kDa and M-plasmid-specific 66-kDa bands, respectively, whereas an S-plasmid-specific 45-kDa band was shared by all the transfected cell lysates, namely, cells transfected with pCAGGS/L, pCAGGS/M, or pCAGGS/S. The major bands of about 45, 66, and 88 kDa detected after transfection of 293T cells with pCAGGS/S, pCAGGS/M, and pCAGGS/L plasmids, respectively (Fig. 2B), might be explained by formation of dimer forms of the respective proteins. As shown in Fig. 2B, in addition to these major bands, other bands were also shown to be present in cases of transfection with plasmids containing genes of M and L proteins. These additional bands could be partly due to an internal initiation of translation from the translation site of the S protein. Even in cases of transfection with the HBs S expression plasmid, some of the additional bands were also detected (Fig. 2B, lane 1); partial deglycosylation might produce these bands.
Detection of VSVG(HBV) pseudotypes. HepG2 cells, grown in 96-well plates, were used to detect formation of VSV pseudotypes. The culture samples prepared using 293T cells transfected with the HBs expression plasmids and infected with VSVG-G were incubated with goat anti-VSV serum and anti-HBs S MAb (DA-3E7) and used to infect the HepG2 cells. Infectious units (IU/ml) of the VSVG-G-infected samples were determined by counting the number of GFP-expressing cells. Candidate VSV pseudotype samples prepared by transfection of the plasmids coding for HBs L, M, and S proteins gave infectious titers of about 1,500, 3,800, and 1,000 IU/ml, respectively (Fig. 3A). Transfection of 293T cells with combinations of the expression plasmids did not further increase the infectivities. The infectivities of all these pseudotype samples were inhibited to the background level when treated with the anti-HBs S MAb, i.e., less than 300 IU/ml (Fig. 3A). The neutralization of the pseudotype infectivities by the anti-HBs S MAb suggests the successful formation of VSVG(HBV) pseudotypes.
Neutralization of the pseudotype samples by MAbs. In order to confirm the presence of VSVG(HBV) pseudotypes, neutralization tests with other MAbs against viral surface proteins were done. That is, 293T cells were transfected with pCAGGS/M plasmid and infected with VSVG-G. The VSVG-G-infected sample designated VSVG(HBs/M) was prepared after sonication and tested for neutralization by the goat anti-VSV serum together with MAbs against HBV or HTLV-1. Figure 3B shows that the infectivity was markedly neutralized by the other two mouse anti-HBs S MAbs (ZY-ZCH16 and CH-2D1.2) as well. An anti-HTLV-1 MAb, LAT-27, did not affect its plating onto HepG2 cells, whereas a HTLV-1 pseudotype was well neutralized by LAT-27 but by none of the anti-HBs S MAbs (Fig. 3B). MAb against preS2 (CH-S26) neutralized neither VSVG(HBs/M) nor VSVG(HTLV-1) pseudotype sample. Thus, the infectivity of the VSVG(HBs/M) sample was especially neutralized by the three HBs S protein-specific MAbs but not neutralized by anti-preS2 MAb or anti-HTLV-1 MAb (Fig. 3B). In addition, anti-HBs S MAb (DA-3E7) also neutralized the infectivity of VSVG(HBs/L) and VSVG(HBs/S) pseudotype samples (Fig. 3A), but anti-HTLV-1 MAb could neutralize none of these samples (data not shown).
Infectivity of the VSVG(HBs/M) pseudotype sample for various cell lines. The VSVG(HBs/M) sample prepared as described above was used to infect various target cells in order to study its cell tropism. Target cells were chosen according to the previous reports on their susceptibilities to HBV (13, 31, 41) and HCV (19, 43) infection. The only HBV pseudotype reported so far, MLV(HBV) pseudotype (41), exclusively infects only primary human hepatocytes but not any of the established cell lines tested, such as HepG2 or 293T. We were interested in examining whether any of the cell lines derived from human hepatoma, glioma, kidney, osteosarcoma, etc., could be infected by a VSVG(HBs/M) pseudotype sample. Infectious titers of the VSVG(HBs/M) samples in 14 different human cell lines were determined. As shown in Fig. 3 and Table 1, the VSVG(HBs/M) sample was able to infect HepG2 cells. Among the other 13 cell lines, a hepatocarcinoma cell line, JHH-7, and a human embryonic kidney cell line, 293T, were judged to be clearly susceptible to the VSVG(HBs/M) pseudotype sample. Alexander cells were apparently but less susceptible to the pseudotype sample than HepG2 or JHH-7 cells (data not shown). None of the other cell lines, namely hematopoietic, glioma, or osteosarcoma cell line, tested in this study was apparently, if not at all, susceptible to infection by the pseudotype sample (Table 1). The VSVG-G pseudotype was used as a control to test the susceptibilities of various cell lines to VSV replication. The cell lines used in this study showed different but high degrees of susceptibilities to VSVG-G infection, indicating that a lack of susceptibility to the VSVG(HBs/M) sample was not due to their resistance to VSVG-G.
Effect of chemicals on infection of VSVG(HBs/M) pseudotype samples. Infection of HBV to human hepatocytes was reported to be inhibited by bLF (13). Heparin and other sulfated polysaccharides have also been shown to be strong inhibitors of the binding of HBV to HepG2 cells (49). Thus, we examined effects of bLF, heparin, and dextran sulfate and noticed that infectivity was inhibited by about 90% at 1 mg/ml bLF (Fig. 4A). Sulfated polysaccharides, heparin and dextran sulfate, at 100 μg/ml, also inhibited the infectivity by about 50% and 80%, respectively (Fig. 4B). In contrast, the nonsulfated dextran did not inhibit the infectivity.
We next examined whether a glycosylation of protein(s) on the cell surface is required for HBV infection. For this, HepG2 cells were treated with trypsin just before infection or with tunicamycin 1 day before infection. Treatment with 1.0% trypsin and 10 μg/ml tunicamycin showed about 80% and 70% inhibition of the infectivity, respectively (Fig. 5).
DISCUSSION
In order to prepare VSV pseudotype viruses, the expression of HBs proteins in 293T cells was studied by IFA, FCM, and WB. The S and M proteins are localized mainly in the cytoplasm, and their distribution seemed to be homogeneous inside it, whereas localization of the L protein was granular with its heterogeneous distribution inside the cells (Fig. 1C). This result is consistent with the findings demonstrated by several reporters (32, 47). Localization of S, M, or L protein detected by specific MAbs against HBsAg in hepatocytes of patients with chronic HBV infection reported previously (17) is similar to that observed in 293T cells transfected with corresponding plasmids.
We performed cotransfection experiments using different combinations of the S, M, and L expression plasmids, but the expression levels could not be raised further, as was shown upon individual plasmid transfection (data not shown). This result seems to be different from the result for the recently reported MLV pseudotype. That is, in the report of Sung and Lai (41), the surface expression of L or S protein individually can hardly be detected, whereas about 10% of the surface expression was shown in our experiment when S or M plasmids were transfected independently. We considered that this would be due to a difference between systems employed for the transfection or the use of different expression plasmids to express the HBs proteins. For example, Sung and Lai (41) used retroviral vectors whereas we used expression plasmids containing a ?-actin promoter.
After transfection, the transfected cells were infected with VSVG-G for production of VSVG(HBV) pseudotypes that contain the VSV core protein bearing the surface proteins of HBV. As VSV pseudotype titers preliminarily determined had not been sufficiently high (data not shown), the cells detached and suspended in their culture supernatants were sonicated prior to making stocks of the pseudotype samples as described by Tamura et al. (43) to prepare HCV pseudotypes bearing the native form of HCV surface proteins. The infectious titers of the pseudotype samples made with sonication were about five times as high as those of nonsonicated samples (data not shown).
Although the infectivity of the pseudotype was shown to be efficiently neutralized by the anti-HBs S MAbs, the infectivity of the pseudotype sample could not be well neutralized by concentrated human immunoglobulin positive for HBs antibody and available commercially (HBIG) (data not shown). Low titers of HBIG in the neutralization of HBV infection in vitro have already been reported: the HBIG titer is about 2,000 times as low as that of anti-HBs-humanized MAb produced in Ryu et al.'s laboratory (34). Our finding may be consistent with this finding. It is probable that HBIG contains antibodies reacting with the preS1 antigenic determinant, as this region has been considered one of the receptor-binding sites, and that human antibodies may preferentially neutralize native HBV in the blood.
The inhibitory effects of various chemicals, like bLF, tunicamycin, trypsin, and sulfated polysaccharides, on infection of the pseudotype sample was also studied (Fig. 4 and 5). The infectivity of the VSVG(HBs/M) pseudotype was inhibited by bLF in our experiment. The inhibitory effect of bLF, however, seemed to be different from that reported by Hara et al. (13). They reported that preincubation of target cells with bLF is necessary for the inhibition. In contrast, our result is similar to the effect of bLF in HCV infection reported by several groups (13, 43), including Hara's group: preincubation of the cells with bLF before HBs pseudotype infection could not show any inhibitory effect (data not shown). Our results suggest that the bLF-binding site may locate in the preS2 or the S domain of M protein. It is also probable that bLF binds to a region to which neutralizing anti-HBs MAbs also bind. Further study will be necessary to identify a site to which bLF binds.
One of the interesting findings in our study may be that among three HBs proteins, transfection of the expression plasmid DNA for HBs M protein gave the slightly but clearly highest infectious titer of the pseudotype (Fig. 3A) when titrated in HepG2 cells. This finding suggests that M protein of HBV, especially the preS2 region, has an important role in the infection of HBV. This result looks contradictory to some previous reports about its role. For example, Le Seyec et al. (21) reported that most of the preS2 region is dispensable for HBV infection. Cho et al. (5), however, reported the presence of a binding region in the preS2 region for a cellular HBV receptor(s). Namely, the binding region, which contains the N-terminal 5 aa of preS2, potentially contributes to attachment to and entry into target cells (21). Likewise, our results suggest that the preS2 region of M protein may contain a binding region to target cells. The infectious titer of the VSVG(HBs/S) sample was the lowest among all pseudotype samples tested in this study, although all three anti-HBs MAbs neutralized all types of HBs pseudotypes tested.
WB analysis (Fig. 2B) using anti-HBs S MAb showed that WB bands with similar sizes were detected even after transfection of the HBs expression plasmid for the HBs M gene. Thus, it is probable that S protein also is produced in 293T cells transfected with the plasmid for HBs M protein. S protein together with M protein may lead to an efficient production of HBs pseudotype in the HBs/M pseudotype sample. The 21- to 47-aa sequence of preS1 and 120- to 145-aa sequence of preS2 have been reported to be a crucial and an auxiliary cell-binding sites, respectively (28). The presence of the secondary attachment region in HBs S protein has also been reported by Paran et al. (31). However, a direct interaction of natural HBs S protein with hepatocytes or a role of HBs S protein in the penetration of hepatocytes has not been convincingly demonstrated. MAbs used in this study could recognize S-S bonds in the region of HBs S protein, since bands for the specific proteins could be detected upon WB with DTT(–) samples only but not WB with DTT(+) samples, and this region was recognized by the neutralizing MAbs. This finding suggests that a receptor-binding site as well as a MAb-binding site in HBV surface proteins may be created by the S-S bond. In other words, the S-S bond in the S protein or S domain in M and L proteins may play an important role in entry of HBV into target cells.
Although there seem to be some contradictory results in our study to those reported previously, the discrepancies might be attributable to the difference in systems to detect HBV infection, such as whether inoculating samples are native virus derived from the blood or VSV pseudotypes, whether target cells are primary hepatocytes or established cell lines, or whether titers of inoculating viruses are high or low. Properties of HBs proteins produced in hepatocytes in vivo or those produced in 293T cells in vitro may be different. If titers of inoculating viruses are detected to be different, a region of HBs gene necessary for infection or a susceptibility of cells to HBV can be determined differently.
In conclusion, we considered that VSV pseudotypes bearing the surface proteins of HBV could be prepared. These pseudotypes most efficiently infected the human hepatoma cell lines HepG2 and JHH-7 among establish human cell lines tested. To our knowledge, this is the first report of production of VSV pseudotype virus bearing HBs proteins. The VSVG(HBV) pseudotypes might be useful in a safe and rapid assay system to clarify early steps of HBV infection. This system may also contribute to screening and development of antiviral drugs that interfere with HBV infection.
ACKNOWLEDGMENTS
We thank M. A. Whitt for kindly supplying VSVG-G and J. Miyazaki for kindly supplying the plasmid pCAGGS.
This work was supported by a grant-in-aid from the Japanese Society for the Promotion of Science, grants from the Japan Health Sciences Foundation and CREST, and the 21st century COE program supported by the Ministry of Education, Culture, Sports Science, and Technology of Japan.
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