- Email: [email protected]
The human bone-marrow-derived B-cell line CE, susceptible to hepatitis C virus infection
C) INSTITUT PASTEUR/ELsEVIER Paris 1993
Res. Virol. 1993, 144, 281-285
The human bone-marrow-derived B-cell line CE, susceptible to hepatitis C virus infection L. Bertolini (1), S. Iacovacci
(2),
A. Ponzetto (3), G. Gorini (4), M. Battaglia (2) and G. Carloni (2)(*)
") Institute of Cell Biology, (2) Institute of Experimental Medicine, CNR, 00137 Rome, t3) Division o f Gastroenterology, Le Molinette Hospital, 10126 Turin, and (4) Dipartimento AMB-Bio, ENEA, CRE Casaccia, 00060 Rome
SUMMARY The euploid-diploid cell line CE, issuing spontaneously from a normal human bone marrow culture, displays B-cell differentiation and activation markers and is positive ( ~> 90 % of cells) for Epstein-Barr nuclear antigens. CE suspensions were inoculated with serum from a patient chronically infected with hepatitis C virus (HCV). After inoculation, the cells were regularly subcultured with a split ratio of 1:2 every 4-6 days. RNA extracted as late as 65 days after infection from the inoculated cells were positive by polymerase chain reaction for the 5' untranslated region of the HCV genome, and viral antigens were detected by immunofluorescence. Virus was also released from the infected cells into the medium. Intracellular HCV could be successfully passaged twice in CE cultures. On the basis of these findings, the CE cell line appears promising as a model for studying HCV replication and persistent infection in vitro.
Key-words: HCV, Replication, B lymphocyte; Bone marrow, CE cell line, Cluster differentiation antigens, Viral antigens.
INTRODUCTION Peripheral blood mononuclear cells of patients infected with hepatitis C virus (HCV) have been shown to be extrahepatic targets for virus replication (Zignego et al., 1992). It has been demonstrated that HCV replicates in vivo in B lymphocytes from chronically infected patients (Muller et aL, 1992). Evidence for in vitro repli-
Received April 15, 1993. (*) Correspondingauthor.
cation of HCV in lymphocytes has been obtained only in a human T-ceU line (Shimizu et ak, 1992). In order to develop an alternative in vitro system of HCV infection for better clarifying viral replication and hepatitis pathogenesis, we inoculated, with an HCV strain from an infected patient's serum, cell line CE, a B-cell line which spontaneously arose from a normal human bone marrow culture (Bertolini et al., 1992).
282
L. B E R T O L I N I E T A L . MATERIALS AND METHODS
Cell culture
The human bone-marrow-derived CE cell line has been described in detail elsewhere (Bertolini et al., 1992). Cells were grown at 37°C in Dulbecco's modified Eagle's medium supplemented with 10 °70 foetal calf serum and 10 % trypticase soy broth, and routinely split 1:2 every 4-6 days. Growth kinetics, proliferative activity, karyotype and tumorigenicity of CE cells for nude mice were determined according to Bertolini et aL, (1992). Live CE cells in suspension were repeatedly tested for ploidy between the 5th and 100th subculture. Cells were phenotyped by indirect immunofluorescence and flow cytometric analysis for leukocyte cluster differentiation (CD) antigens, HLADR antigens of the major histocompatibility complex II (MHC II), FcTII receptors, and surface immunoglobulin (Ig) markers (Bertolini et al., 1992; Gorini et al., 1987). Cytoplasmic Ig were detected by direct immunofluorescence on cytocentrifuged fixed cells.
HCV infection
Approximately I x 107 CE cells in 5 ml of growth medium were infected by adding 1 ml of serum from a patient chronically infected with HCV. The specimen contained approximately 1 × 105 HCV genomes per ml, as determined by polymerase chain reaction (PCR) in endpoint dilution assay. The cells were washed six times and resuspended in fresh growth medium 18 h after inoculation. For three months after inoculation, cells were regularly subcultured with a split ratio of 1:2 every 4-6 days.
RNA extraction and PCR analysis
Samples of cells and fluids from inoculated as well as from uninoculated control cultures were harvested at intervals of up to three months. Cytoplasmic fractions from CE cells were obtained according to Kawasaki et al., (1987). The RNA were extracted from cytoplasmic samples and ultracentrifuged fluids as described by Nelpas et al. (1992), with the following modifications: 150 V.I of cell pellet (107 cells) or culture fluid, incubated for 1 h at 37°C in a lysis buffer consisting of 50 mM Tris-HCl pH 8.0, 0.01 M
CD HCV lg
= = =
cluster differentiation. hepatitis C virus. immunoglobulin.
EDTA, 0.1 M NaCI, 0.5 07o SDS, 100 v.g per sample of polyA (Sigma, St. Louis, MO) and 1 U per sample of "RNasin" (Promega, Madison, WI) and digested with 50 ~g/ml proteinase K, were extracted with phenol/chloroform and precipitated with ethanol. After storage at - 80°C, the RNA was centrifuged for 45 min at 12,000 g at 4°C, washed once in 70 070 ethanol, air-dried, redissolved in 20 i~l of diethyl-pyrocarbonate-treated distilled water and stored at - 20°C. For eDNA synthesis, l0 I-d of RNA solution were added to a mixture containing 1 x reverse transcription buffer (BRL, Gaithersburg, MD) and 0.6 I~M downstream primer, dithiothreitol and 1 mM each of deoxynucleotide triphosphate (dNTP) (Boehringer Mannheim). After incubation at 68°C for l0 min, 200 IU of Moloney murine leukaemia virus reverse transcriptase (BRL) were added. Samples were incubated at 37°C for 60 min in a final volume of 25 lzl, and eDNA was stored at - 20°C. For nested PCR, a target sequence belonging to the highly conserved 5' untranslated region of the HCV genome was chosen (Okamoto et al., 1990). Outer primers were 5'-TGCACGGTCTACGAGACCTC-3' (anti-sense) and 5'GCCATGGCGTTAGTATGAGT-3' (sense). Inner primers were 5'-GGGCACTCGCAAGCACCCTA-3' (antisense) and 5'-CTGCACCTCCAGGACCCCC-3' (sense). The probe was 5'-CCATAGTGGTCTGCGGAAC C G T G A G T A C A C C G G A A T - 3 ' (positions : 122-158). PCR was performed according to Novati et al., (1992): 10 ~l of eDNA were added to 700-1zl tubes containing 40 ~tl of a mixture composed of 125 [~M each of dNTP, 50 mM KCI, 1.5 mM MgCl 2, 0.01 o70gelatin, 5 pmoi of each outer primer and 2.5 IU of cloned Taq polymerase (Promega). The reaction mixture was then covered with 100 ~l of mineral oil. During the first part of the reaction, the annealing temperatures were 37°C for l cycle and 45°C for 25 cycles. For the second round of PCR, 150 V.1of buffer with 100 pmol of both inner primers were added to each tube. The second part of the reaction was done in a final volume of 200 i~l at an annealing temperature of 45°C for 30 cycles. Denaturation and extension steps were always done at 94 and 72°C, respectively. PCR products 05 I.tl were analysed by 2 o70 agarose gel electrophoresis and ethidium bromide staining, followed by Southern blotting on nylon membranes, hybridization with a 32p-labelled probe and autoradiography. To detect contamination at each step of the procedure, PCR was done on HCVnegative control specimens extracted along with test
mAb PCR
= monoclonalantibody. = polymerasechain reaction.
B-CELL L I N E CE, S U S C E P T I B L E TO H E P A T I T I S C VIRUS I N F E C T I O N
samples, serum-free lysis buffer used for RNA extraction, and the PCR mixture alone. Adherence to anti-contamination protocols was strict (Kwok et al., 1989) with particular attention paid to separation of pre- and post-PCR steps. The samples were coded, and all of them were tested at least twice.
lmmunofluorescence for HCV antigens in infected cultures
Cytosmears of infected and uninfected CE cells were fixed for 15 min in absolute ethanol at - 2 0 ° C and then examined for the presence of viral antigens by indirect immunofluorescence. The following mouse monoclonal antibodies (mAb), all of the IgG 1 subclass, were employed: (i) the TORDJI-22 clone (Biosoft, Paris) directed to the C-I00-3 nonstructural HCV protein, (ii) clone no. 1851 (Virostat, Portland, ME) directed to viral capsid protein, and (iii) antibody M5021 (Anogen, Ontario) obtained from mice immunized with HCV-capsid protein. The binding of antibody to infected ceils was revealed by using fluorescein-conjugated affinity-isolated goat antibody specific for mouse IgG (Sigma). The slides were counterstained with Evans blue and mounted in 90 °70 buffered glycerol pH 8.0, containing 0.625 °70 propyl gallate as an antifading agent (Valnes et al., 1985).
283
Table i. CE cell line markers.
Marker
Percent (*) Marker Percent t*) Ig secretion
CD4 CD10 CD19 CD20 CD21 CD22 CD25 CD34 CD71 CD1 la
1.9 0.4 71.9 97.1 49.4 76.0 13.9 2.1 77.1 97.4
MHC-II HLA-DR
89.2
Fc~,RII
> 95
Surface Ig sIgM 68.1 sIgD 57.3 sIgG 24.3 SK ND s), ND
IgM - IgD -IgG + r + ). --
Cytoplasmic ig(°*) cl~ 5 c~ ND cK 8 c)~ 4
(*) Percentage of CE cells positive for the indicated markers, as determined by direct immunofluorescence and flow cytometry analysis. (**) Percent of cells positive for cytoplasmic lg as determined by direct immunofluorescence. ND = not determined.
Detection o f H C V R N A in infected cultures
RESULTS Characterization of the CE cell line
The data on phenotyping o f the CE line are summarized in table I. The cells expressed nonIg and Ig markers associated with B-ceU ontogeny (CD34, CD10, CD19), differentiationmaturation (CD20, CD21, CD22, sIgM, sIgD) and activation (CD23, CD25, CD71, sIgG), and they released IgG. The detection o f cells positive for intracytoplasmic K and ), light chains suggested that the cell population examined was polyclonal. The cells were shown to be nontumorigenic for nude mice (Bertolini et al., 1992). On the basis o f these findings and on •those reported by Bertolini et al. (1992), the CE line appeared to be a diploid-euploid polyclonal line composed o f subpopulations o f B cells at different stages o f differentiation and maturation, carrying endogenous Epstein-Barr virus and expressing Epstein-Barr nuclear antigens.
The cells were harvested at 22 days after infection and examined for the presence o f H C V R N A by PCR. As shown in figure 1, the R N A extracted from the inoculated cultures were positive for the 5' untranslated region o f the H C V genome. Virus was also released from the infected cells into the m e d i u m (fig. 1). CE cells were found positive for HCV R N A as long as 65 days after i n o c u l a t i o n / d a t a not shown). Intracellufar H C V f r o m frozen and thawed infected cells, harvested 22 days after the first inoculation, was serially passaged twice in CE cells ,:-The virus was passaged successfully, as detected by the presence o f H C V R N A as late as 22 days after inoculation. The cells and culture fluids harvested f r o m uninoculated control cultures were consistently negative for H C V R N A by P C R .
D e t e c t i o n of HC.V antigens in infected cells
The cells were examined for H C V c o r e and C-100-3 antigens by indirect immunofluores-
284
L. BERTOLINI E T AL.
R_ m
.Ik m
N
N
N m
N a.
| = ,_
in
m
U:
H
+
U
U
U
cence with m o u s e m A b . At 24 days after infection, a b o u t 1 °70 o f CE cells were positive for both antigens. The positive cells showed the presence o f highly fluorescent cytoplasmic granules with the m A b directed to the C-100-3 nonstructural H C V protein (fig. 2A). However, all three m A b tested gave similar patterns o f immunostaining. The uninoculated cells did not react with the HCV-specific m A b tested (fig. 2B).
ql!bp =-, DISCUSSION
Fig. 1. Southern blot hybridization of PCR products from HCV-infected CE cells (CE-cell 22 days p.i.) and from their culture fluids (CE-sup 22 days p.i.) harvested 22 claysafter inoculation. A serum sample from an HCV-infected patient is also shown as a positive control (C ÷ Serum).
The existence o f extrahepatic targets in H C V infection have been shown by Zignego et al. (1992), who reported the presence o f the H C V genome in the peripheral blood m o n o n u c l e a r cells from chronically infected patients. In addition, H C V R N A has been detected in EpsteinBarr virus-transformed B lymphocytes from patients with chronic H C V infection (Muller et al., 1992). In the present study, we demonstrate the in vitro susceptibility o f the h u m a n B-cell line CE to infection by H C V . In this cell line, composed o f normal B-cell populations at different
A
Fig. 2, Indirect immunofluorescent staining of HCV-infected CE cells with mAb directed to the C-100-3 non-structural HCV protein at 24 days after infection (A). Lack of staining of uninoculated CE cells (B).
B - C E L L L I N E CE, S U S C E P T I B L E TO H E P A T I T I S C VIRUS I N F E C T I O N
stages o f differentiation and m a t u r a t i o n , viral R N A was detected intracellularly after several cell subcultures (more than 13) and after 2 serial H C V subpassages. Viral RNA was also detected by P C R in the infected cell culture fluids. Using m A b from three different sources, we showed, by indirect immunofluorescence, the presence o f antigens related to the viral capsid and to the nonstructural protein C-100-3 in about 1 % o f the inoculated cells. This further supports the hypothesis that the HCV genome replicates and expresses viral gene products in CE cells. It has recently been demonstrated that H C V is able to replicate in M O L T - 4 cells, a h u m a n T-cell line (Shimizu et al., 1992), where about 1 °70 were shown to express viral antigens. However, to our knowledge, evidence for H C V replication in a h u m a n B-cell line has never been reported previously. The susceptibility to H C V infection, as detected by P C R a n d immunofluorescence techniques, might be dependent on the heterogeneous differentiated state for B-cell lineage o f the CE h u m a n cell line. It remains to be investigated whether the viral progeny produced by infected CE cells possess an altered cell or organ tropism in vivo, and whether variations in the HCV genome might be responsible for such changes. In conclusion, the present data show that H C V is able to express its genome and to replicate in a h u m a n B-cell line derived from normal bone marrow. Our data suggest that B cells might be an important H C V reservoir in vivo, and that the bone marrow, where production and early m a t u r a t i o n o f B lymphocytes takes place, might play an important role in establishment and maintenance of HCV infection. On the basis of these preliminary results, the CE cell line might be useful as a model to study H C V replication and persistent infection in vitro.
Acknowledgements During this study, S.I. was a recipient of a CLONIT scholarship and a CNR, BTBS target project fellowship.
285
This work was partially supported by grants from Consiglio Nazionale delle Ricerche (CNR) "Target projects FATMA and BTBS", and a grant from AIRC (Associazione Italiana per la Ricerca sul Cancro).
References Bertolini, L., Falzetti, F., Lanzilli, G., Moscone, O., Longo, M., Bangrazi, C. & Gorini, G. (1992), Lymphocyte receptors as differentiation markers in a cell line autonomously arisen in vitro from a human normal bone marrow, in "Recent advances in cellular and molecular biology" (Wegmann, R.G. & Wegmann, M.A.) voi. 1 (pp. 77-89). Peeters Press, Leuven, Belgium. Gorini, G., Medgyesi, G.A., Garavini, M., Dorrington, K.J. & Down, J. (1987), Isolation from human chronic-lymphocytic-leukemia cells of membrane glycoprotein associated with Fc-receptor functions. Biochem. J., 245, 75-83. Kawasaki, E.S., Clark, S.S., Coyne, M.Y., Smith, S.D., Champlin, R., Witte, O.N. & McCormick, F.P. (1987), Diagnosis of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplifiedin vitro. Proc. natl. Acad. Sci (Wash.), 85, 5698-5702. Kwok, S. & Higuchi, R. (1989), Avoidingfalsepositive with PCR. Nature (Lond.), 339, 237-238. Muller, H.M., Kallinowski, B., Solbach, C., Goeser, T., Theilmann, L. & Pfaff, E. (1992), Peripheral blood mononuclear ieukocytes as potential site for extrahepatic replication of hepatitis C virus (HCV): Predominant role of B-lymphocytes in viral propagation, in "Hepatitis C virus and related viruses, molecular biology and pathogedesis, first annual meeting" (p. 31). Venezia, Italy. Nelpas, B., Thiers, V., Pol, S., Driss, F., Th6pot, V., Berthelot, P. & Br6chot, C. (1992), Hepatitis C viremia and anti-HCV antibodies in alcoholics.J. Hepat., 14, 381-384. Novati, R., Thiers, V., d'Arminio Monforte, A., Maisonneuve, P., Principi, N., Conti, M., Lazzarin, A. & Br6chot, C. (1992), Mother-to-child transmission of hepatitis C virus detected by nested polymerasechain reaction. J. Infect. Dis., 165, 720-723. Okamoto, H., Okada, S., Sugiyama, Y., Yotsumoto, S., Tanaka, T., Yoshizawa, H., Tsuda, F., Miyakawa, Y. & Mayumi, M. (1990), The 5'-terminal sequence of the hepatitis C virus genome. Jap. J. Exp. Med., 60, 167-177. Shimizu, Y.k., Iwamoto, A., Hijikata, M., Purcell, R.H. & Yoshikura, H. (1992), Evidence for in vitro replication of hepatitis C virus genome in a human T-cell line. Proc. natl. Acad. Sci (Wash.), 89, 5477-5481. Valnes, K. & Brandtzaeg, P. (1985), Retardation of immunofluorescence fading during microscopy. J. Histochem. Cytochem., 33, 755-761. Zignego, A.L., Macchia, D., Monti, M., Thiers, V., Mazzetti, M., Foschi, M., Maggi, E., Romagnani, S., Gentilini, P. & Br6chot, C. (1992), Infection of peripheral mononuclear blood cells by hepatitis C virus. J. Hepat., !5, 382-386.
Res. Virol. 1993, 144, 281-285
The human bone-marrow-derived B-cell line CE, susceptible to hepatitis C virus infection L. Bertolini (1), S. Iacovacci
(2),
A. Ponzetto (3), G. Gorini (4), M. Battaglia (2) and G. Carloni (2)(*)
") Institute of Cell Biology, (2) Institute of Experimental Medicine, CNR, 00137 Rome, t3) Division o f Gastroenterology, Le Molinette Hospital, 10126 Turin, and (4) Dipartimento AMB-Bio, ENEA, CRE Casaccia, 00060 Rome
SUMMARY The euploid-diploid cell line CE, issuing spontaneously from a normal human bone marrow culture, displays B-cell differentiation and activation markers and is positive ( ~> 90 % of cells) for Epstein-Barr nuclear antigens. CE suspensions were inoculated with serum from a patient chronically infected with hepatitis C virus (HCV). After inoculation, the cells were regularly subcultured with a split ratio of 1:2 every 4-6 days. RNA extracted as late as 65 days after infection from the inoculated cells were positive by polymerase chain reaction for the 5' untranslated region of the HCV genome, and viral antigens were detected by immunofluorescence. Virus was also released from the infected cells into the medium. Intracellular HCV could be successfully passaged twice in CE cultures. On the basis of these findings, the CE cell line appears promising as a model for studying HCV replication and persistent infection in vitro.
Key-words: HCV, Replication, B lymphocyte; Bone marrow, CE cell line, Cluster differentiation antigens, Viral antigens.
INTRODUCTION Peripheral blood mononuclear cells of patients infected with hepatitis C virus (HCV) have been shown to be extrahepatic targets for virus replication (Zignego et al., 1992). It has been demonstrated that HCV replicates in vivo in B lymphocytes from chronically infected patients (Muller et aL, 1992). Evidence for in vitro repli-
Received April 15, 1993. (*) Correspondingauthor.
cation of HCV in lymphocytes has been obtained only in a human T-ceU line (Shimizu et ak, 1992). In order to develop an alternative in vitro system of HCV infection for better clarifying viral replication and hepatitis pathogenesis, we inoculated, with an HCV strain from an infected patient's serum, cell line CE, a B-cell line which spontaneously arose from a normal human bone marrow culture (Bertolini et al., 1992).
282
L. B E R T O L I N I E T A L . MATERIALS AND METHODS
Cell culture
The human bone-marrow-derived CE cell line has been described in detail elsewhere (Bertolini et al., 1992). Cells were grown at 37°C in Dulbecco's modified Eagle's medium supplemented with 10 °70 foetal calf serum and 10 % trypticase soy broth, and routinely split 1:2 every 4-6 days. Growth kinetics, proliferative activity, karyotype and tumorigenicity of CE cells for nude mice were determined according to Bertolini et aL, (1992). Live CE cells in suspension were repeatedly tested for ploidy between the 5th and 100th subculture. Cells were phenotyped by indirect immunofluorescence and flow cytometric analysis for leukocyte cluster differentiation (CD) antigens, HLADR antigens of the major histocompatibility complex II (MHC II), FcTII receptors, and surface immunoglobulin (Ig) markers (Bertolini et al., 1992; Gorini et al., 1987). Cytoplasmic Ig were detected by direct immunofluorescence on cytocentrifuged fixed cells.
HCV infection
Approximately I x 107 CE cells in 5 ml of growth medium were infected by adding 1 ml of serum from a patient chronically infected with HCV. The specimen contained approximately 1 × 105 HCV genomes per ml, as determined by polymerase chain reaction (PCR) in endpoint dilution assay. The cells were washed six times and resuspended in fresh growth medium 18 h after inoculation. For three months after inoculation, cells were regularly subcultured with a split ratio of 1:2 every 4-6 days.
RNA extraction and PCR analysis
Samples of cells and fluids from inoculated as well as from uninoculated control cultures were harvested at intervals of up to three months. Cytoplasmic fractions from CE cells were obtained according to Kawasaki et al., (1987). The RNA were extracted from cytoplasmic samples and ultracentrifuged fluids as described by Nelpas et al. (1992), with the following modifications: 150 V.I of cell pellet (107 cells) or culture fluid, incubated for 1 h at 37°C in a lysis buffer consisting of 50 mM Tris-HCl pH 8.0, 0.01 M
CD HCV lg
= = =
cluster differentiation. hepatitis C virus. immunoglobulin.
EDTA, 0.1 M NaCI, 0.5 07o SDS, 100 v.g per sample of polyA (Sigma, St. Louis, MO) and 1 U per sample of "RNasin" (Promega, Madison, WI) and digested with 50 ~g/ml proteinase K, were extracted with phenol/chloroform and precipitated with ethanol. After storage at - 80°C, the RNA was centrifuged for 45 min at 12,000 g at 4°C, washed once in 70 070 ethanol, air-dried, redissolved in 20 i~l of diethyl-pyrocarbonate-treated distilled water and stored at - 20°C. For eDNA synthesis, l0 I-d of RNA solution were added to a mixture containing 1 x reverse transcription buffer (BRL, Gaithersburg, MD) and 0.6 I~M downstream primer, dithiothreitol and 1 mM each of deoxynucleotide triphosphate (dNTP) (Boehringer Mannheim). After incubation at 68°C for l0 min, 200 IU of Moloney murine leukaemia virus reverse transcriptase (BRL) were added. Samples were incubated at 37°C for 60 min in a final volume of 25 lzl, and eDNA was stored at - 20°C. For nested PCR, a target sequence belonging to the highly conserved 5' untranslated region of the HCV genome was chosen (Okamoto et al., 1990). Outer primers were 5'-TGCACGGTCTACGAGACCTC-3' (anti-sense) and 5'GCCATGGCGTTAGTATGAGT-3' (sense). Inner primers were 5'-GGGCACTCGCAAGCACCCTA-3' (antisense) and 5'-CTGCACCTCCAGGACCCCC-3' (sense). The probe was 5'-CCATAGTGGTCTGCGGAAC C G T G A G T A C A C C G G A A T - 3 ' (positions : 122-158). PCR was performed according to Novati et al., (1992): 10 ~l of eDNA were added to 700-1zl tubes containing 40 ~tl of a mixture composed of 125 [~M each of dNTP, 50 mM KCI, 1.5 mM MgCl 2, 0.01 o70gelatin, 5 pmoi of each outer primer and 2.5 IU of cloned Taq polymerase (Promega). The reaction mixture was then covered with 100 ~l of mineral oil. During the first part of the reaction, the annealing temperatures were 37°C for l cycle and 45°C for 25 cycles. For the second round of PCR, 150 V.1of buffer with 100 pmol of both inner primers were added to each tube. The second part of the reaction was done in a final volume of 200 i~l at an annealing temperature of 45°C for 30 cycles. Denaturation and extension steps were always done at 94 and 72°C, respectively. PCR products 05 I.tl were analysed by 2 o70 agarose gel electrophoresis and ethidium bromide staining, followed by Southern blotting on nylon membranes, hybridization with a 32p-labelled probe and autoradiography. To detect contamination at each step of the procedure, PCR was done on HCVnegative control specimens extracted along with test
mAb PCR
= monoclonalantibody. = polymerasechain reaction.
B-CELL L I N E CE, S U S C E P T I B L E TO H E P A T I T I S C VIRUS I N F E C T I O N
samples, serum-free lysis buffer used for RNA extraction, and the PCR mixture alone. Adherence to anti-contamination protocols was strict (Kwok et al., 1989) with particular attention paid to separation of pre- and post-PCR steps. The samples were coded, and all of them were tested at least twice.
lmmunofluorescence for HCV antigens in infected cultures
Cytosmears of infected and uninfected CE cells were fixed for 15 min in absolute ethanol at - 2 0 ° C and then examined for the presence of viral antigens by indirect immunofluorescence. The following mouse monoclonal antibodies (mAb), all of the IgG 1 subclass, were employed: (i) the TORDJI-22 clone (Biosoft, Paris) directed to the C-I00-3 nonstructural HCV protein, (ii) clone no. 1851 (Virostat, Portland, ME) directed to viral capsid protein, and (iii) antibody M5021 (Anogen, Ontario) obtained from mice immunized with HCV-capsid protein. The binding of antibody to infected ceils was revealed by using fluorescein-conjugated affinity-isolated goat antibody specific for mouse IgG (Sigma). The slides were counterstained with Evans blue and mounted in 90 °70 buffered glycerol pH 8.0, containing 0.625 °70 propyl gallate as an antifading agent (Valnes et al., 1985).
283
Table i. CE cell line markers.
Marker
Percent (*) Marker Percent t*) Ig secretion
CD4 CD10 CD19 CD20 CD21 CD22 CD25 CD34 CD71 CD1 la
1.9 0.4 71.9 97.1 49.4 76.0 13.9 2.1 77.1 97.4
MHC-II HLA-DR
89.2
Fc~,RII
> 95
Surface Ig sIgM 68.1 sIgD 57.3 sIgG 24.3 SK ND s), ND
IgM - IgD -IgG + r + ). --
Cytoplasmic ig(°*) cl~ 5 c~ ND cK 8 c)~ 4
(*) Percentage of CE cells positive for the indicated markers, as determined by direct immunofluorescence and flow cytometry analysis. (**) Percent of cells positive for cytoplasmic lg as determined by direct immunofluorescence. ND = not determined.
Detection o f H C V R N A in infected cultures
RESULTS Characterization of the CE cell line
The data on phenotyping o f the CE line are summarized in table I. The cells expressed nonIg and Ig markers associated with B-ceU ontogeny (CD34, CD10, CD19), differentiationmaturation (CD20, CD21, CD22, sIgM, sIgD) and activation (CD23, CD25, CD71, sIgG), and they released IgG. The detection o f cells positive for intracytoplasmic K and ), light chains suggested that the cell population examined was polyclonal. The cells were shown to be nontumorigenic for nude mice (Bertolini et al., 1992). On the basis o f these findings and on •those reported by Bertolini et al. (1992), the CE line appeared to be a diploid-euploid polyclonal line composed o f subpopulations o f B cells at different stages o f differentiation and maturation, carrying endogenous Epstein-Barr virus and expressing Epstein-Barr nuclear antigens.
The cells were harvested at 22 days after infection and examined for the presence o f H C V R N A by PCR. As shown in figure 1, the R N A extracted from the inoculated cultures were positive for the 5' untranslated region o f the H C V genome. Virus was also released from the infected cells into the m e d i u m (fig. 1). CE cells were found positive for HCV R N A as long as 65 days after i n o c u l a t i o n / d a t a not shown). Intracellufar H C V f r o m frozen and thawed infected cells, harvested 22 days after the first inoculation, was serially passaged twice in CE cells ,:-The virus was passaged successfully, as detected by the presence o f H C V R N A as late as 22 days after inoculation. The cells and culture fluids harvested f r o m uninoculated control cultures were consistently negative for H C V R N A by P C R .
D e t e c t i o n of HC.V antigens in infected cells
The cells were examined for H C V c o r e and C-100-3 antigens by indirect immunofluores-
284
L. BERTOLINI E T AL.
R_ m
.Ik m
N
N
N m
N a.
| = ,_
in
m
U:
H
+
U
U
U
cence with m o u s e m A b . At 24 days after infection, a b o u t 1 °70 o f CE cells were positive for both antigens. The positive cells showed the presence o f highly fluorescent cytoplasmic granules with the m A b directed to the C-100-3 nonstructural H C V protein (fig. 2A). However, all three m A b tested gave similar patterns o f immunostaining. The uninoculated cells did not react with the HCV-specific m A b tested (fig. 2B).
ql!bp =-, DISCUSSION
Fig. 1. Southern blot hybridization of PCR products from HCV-infected CE cells (CE-cell 22 days p.i.) and from their culture fluids (CE-sup 22 days p.i.) harvested 22 claysafter inoculation. A serum sample from an HCV-infected patient is also shown as a positive control (C ÷ Serum).
The existence o f extrahepatic targets in H C V infection have been shown by Zignego et al. (1992), who reported the presence o f the H C V genome in the peripheral blood m o n o n u c l e a r cells from chronically infected patients. In addition, H C V R N A has been detected in EpsteinBarr virus-transformed B lymphocytes from patients with chronic H C V infection (Muller et al., 1992). In the present study, we demonstrate the in vitro susceptibility o f the h u m a n B-cell line CE to infection by H C V . In this cell line, composed o f normal B-cell populations at different
A
Fig. 2, Indirect immunofluorescent staining of HCV-infected CE cells with mAb directed to the C-100-3 non-structural HCV protein at 24 days after infection (A). Lack of staining of uninoculated CE cells (B).
B - C E L L L I N E CE, S U S C E P T I B L E TO H E P A T I T I S C VIRUS I N F E C T I O N
stages o f differentiation and m a t u r a t i o n , viral R N A was detected intracellularly after several cell subcultures (more than 13) and after 2 serial H C V subpassages. Viral RNA was also detected by P C R in the infected cell culture fluids. Using m A b from three different sources, we showed, by indirect immunofluorescence, the presence o f antigens related to the viral capsid and to the nonstructural protein C-100-3 in about 1 % o f the inoculated cells. This further supports the hypothesis that the HCV genome replicates and expresses viral gene products in CE cells. It has recently been demonstrated that H C V is able to replicate in M O L T - 4 cells, a h u m a n T-cell line (Shimizu et al., 1992), where about 1 °70 were shown to express viral antigens. However, to our knowledge, evidence for H C V replication in a h u m a n B-cell line has never been reported previously. The susceptibility to H C V infection, as detected by P C R a n d immunofluorescence techniques, might be dependent on the heterogeneous differentiated state for B-cell lineage o f the CE h u m a n cell line. It remains to be investigated whether the viral progeny produced by infected CE cells possess an altered cell or organ tropism in vivo, and whether variations in the HCV genome might be responsible for such changes. In conclusion, the present data show that H C V is able to express its genome and to replicate in a h u m a n B-cell line derived from normal bone marrow. Our data suggest that B cells might be an important H C V reservoir in vivo, and that the bone marrow, where production and early m a t u r a t i o n o f B lymphocytes takes place, might play an important role in establishment and maintenance of HCV infection. On the basis of these preliminary results, the CE cell line might be useful as a model to study H C V replication and persistent infection in vitro.
Acknowledgements During this study, S.I. was a recipient of a CLONIT scholarship and a CNR, BTBS target project fellowship.
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This work was partially supported by grants from Consiglio Nazionale delle Ricerche (CNR) "Target projects FATMA and BTBS", and a grant from AIRC (Associazione Italiana per la Ricerca sul Cancro).
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