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Blood, 15 November 2005, Vol. 106, No. 10, pp. 3449-3456. Prepublished online as a Blood First Edition Paper on August 2, 2005; DOI 10.1182/blood-2005-02-0536.
IMMUNOBIOLOGY Ku80 autoantigen as a cellular coreceptor for human parvovirus B19 infectionFrom the Department of Rheumatology and Hematology, and Department of Virology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Human parvovirus B19 (B19) infects human erythroid cells expressing P antigen. However, some cell lines that were positive for P antigen failed to bind B19, whereas some cell lines had an ability to bind B19 despite undetectable expression of P antigen. We here demonstrate that B19 specifically binds with Ku80 autoantigen on the cell surface. Furthermore, transfection of HeLa cells with the gene of Ku80 enabled the binding of B19 and allowed its entry into cells. Moreover, reduction of cell-surface expression of Ku80 in KU812Ep6 cells, which was a high-sensitive cell line for B19 infection, by short interfering RNA for Ku80 resulted in the marked inhibition of B19 binding in KU812Ep6 cells. Although Ku80 originally has been described as a nuclear protein, human bone marrow erythroid cells with glycophorin A or CD36, B cells with CD20, or T cells with CD3 were all positive for cell-surface expression of Ku80. B19 infection of KU812Ep6 cells and bone marrow cells was inhibited in the presence of anti-Ku80 antibody. Our data suggest that Ku80 functions as a novel coreceptor for B19 infection, and this finding may provide an explanation for the pathologic immunity associated with B19 infection.
Human parvovirus B19 (B19) infects erythroid-lineage cells through P antigen and causes various clinical symptoms such as erythema infectiosum, anemia, polyarthritis, or fetal hydrops in humans.1,2 The cellular receptor for B19 infection has been regarded as blood group P antigen based on the failure of B19 infection in a patient with an hereditary defect of P antigen.3 However, the target cells of B19 may be not be exclusively P-antigen–positive erythroid-lineage cells, as illustrated by the poor relationship between P antigen expression levels and the efficiency of B19 infection4 or the failure of B19 binding to globoside.5 Recently, Weigel-Kelley et al described the role of  5 1 integrin as the cellular coreceptor for B19 infection.6 The notion that B19 receptor is not solely P antigen may be compatible with clinical findings that B19 has been detected in mononuclear cells of blood or tonsils with acute or prolonged B19 infection.7,8 Also, following B19 infection, the numbers of peripheral blood lymphocytes may decrease despite undetectable levels of P antigen on their cell surface.9,10 Finally, autoimmune-like phenomena including antinuclear antibodies, rheumatoid factors, or antiphospholipid antibodies are often associated with B19 infection,8,11 and the levels of tumor necrosis factor (TNF-) and interferon  (IFN-) secreted from macrophages or T cells are elevated during acute or prolonged B19 infection.12 Clinical studies have shown that B19 DNA can be amplified from joint samples by polymerase chain reaction (PCR),13,14 and infective B19 was detected in the articular lesions of patients with rheumatoid arthritis. B19 transcripts and B19 protein viral protein 1 (VP1) were also present in T cells, B cells, macrophages, and follicular dendritic cells.14 The cellular mechanism that may allow B19 binding and its entry into nonerythroid cells has not been elucidated. In the present study, we explored a putative receptor for B19 that was distinct from P antigen.
Cells Macrophage cell lines U937, urinary bladder carcinoma cell line T24, colon cancer cell line SW620, renal adenocarcinoma cell line ACHN, and HeLa cells were provided by the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). Human erythroid cell line KU812Ep615 was provided by E. Miyagawa (Institute of Fuji Rebio, Tokyo, Japan). T-cell line H9 was purchased from American Type Culture Collection (Manassas, VA). Bone marrow samples were obtained from the volunteers who gave informed consent for the use of their samples for our study. Informed consent was provided in accordance with the Declaration of Helsinki. Human parvovirus B19 Serum from patient 1 with acute B19 infection was used as the source for B19 in the in vitro infection study. The serum contained 2.5 x 1014 copies of B19-DNA per milliliter but was negative for IgM and IgG anti-B19 antibodies.16 B19 was purified using cellulose hollow fiber, was provided by Dr. K. Yamaguchi,17 and used as an antigen for enzyme-linked immunosorbent assay (ELISA). Recombinant human parvovirus B19 empty capsid protein Recombinant human parvovirus B19 empty capsid protein (rB19ECP), prepared as described previously,18 was kindly provided by K. Kamata at Denka Seiken (Tokyo, Japan). rB19ECP was composed of VP1 and VP2 at a ratio of 5:95, respectively. Antibodies
Monoclonal anti-Ku80 antibodies that recognized N-terminus (amino acids 3-22) or C-terminus of Ku80 (amino acids 610-705) were purchased from Oncogene (Boston, MA) and BD Biosciences (San Jose, CA), respectively. PAR3 is a mouse monoclonal antibody recognizing VP2, which shared with VP1 of B19.19,20 1F5 is a mouse monoclonal antibody with anti-idiotypic activity to an anti-DNA antibody,21 which was used as a control for flow cytometry analysis. GL4, a rabbit polyclonal antigloboside antibody (IgG and IgM), was purchased from Matreya (State College, PA). Monoclonal anti– In vitro infection of B19 Cells (2 x 106) in 0.5 mL RPMI were infected with B19 containing serum from 1 (diluted at 2 x 1011 copies of B19 DNA/mL) for 30 minutes on ice and washed extensively 3 times with phosphate-buffered saline (PBS), pH 7.2, for evaluation of B19 adsorption. To study B19 replication, the prepared cells in 3 mL RPMI containing 10% fetal bovine serum (FBS) were further incubated at 37°C for 48 hours in a 5% CO2 humidified atmosphere, followed by 3 extensive washes with PBS and then evaluated for B19 protein and B19 DNA. Protein precipitation and purification of precipitated protein Cell-surface protein of H9 cells was labeled with sulfo-NHS esters of biotin (Pierce, Rockford, IL), followed by protein precipitation. H9 cells were treated with Nonidet P-40 lysis buffer (1% Nonidet P-40, 140 mM NaCl, 1 mM phenylmethylsulfonyl fluoride [PMSF], 5 mM EDTA [ethylenediaminetetraacetic acid], 50 mM Tris [tris(hydroxymethyl)aminomethane)–HCl, pH 7.4) and immunoprecipitated with rB19ECP- or bovine serum albumin (BSA)–conjugated cyanogen bromide (CNBr)–Sepharose (Amersham Bioscience, Piscataway, NJ). The precipitated samples were separated under denaturing conditions in a 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel, followed by electrotransfer to a polyvinylidene difluoride (PVDF) membrane. Protein was detected with enhanced chemiluminescence (ECL) Western blotting detection system (Amersham Biosciences) and visualized by LAS-1000 (Fujifilm, Tokyo, Japan). A crude membrane fraction of H9 cells (1 x 1011) was prepared and solubilized in 1% Nonidet P-40 lysis buffer. The fraction was then precipitated with rB19ECP-conjugated CNBr-Sepharose for 16 hours at 4°C, and the precipitated proteins were separated by SDS-PAGE followed by Coomassie blue staining. Protein sequencing was carried out by Toray Research Center (Kamakura, Japan). Flow cytometry analysis Cells were suspended in 100 µL 1% BSA-PBS and incubated with 5 µg/mL test antibodies on ice for 30 minutes. Cells were then washed 3 times with PBS. Cells that required secondary antibodies for detection were further incubated with fluorescein isothiocyanate (FITC)–conjugated goat anti–mouse IgG (or FITC-conjugated anti–rabbit IgG for the GL4 primary antibody; Sigma, St Louis, MO) at 1:200 on ice for 30 minutes. Cells were washed 3 times with PBS before flow cytometry analysis (Becton Dickinson, San Jose, CA). For the detection of Ku80 on the cell surface of bone marrow cells, cells were first reacted with 5 µg/mL anti-Ku80 antibody followed by an incubation with FITC-conjugated anti–mouse IgG antibody. After being washed, cells were reacted with the cell-lineage–specific antibodies (anti–glycophorin A, anti-CD3, anti-CD20, anti-CD56, anti-CD14, or anti-CD36 antibodies) conjugated with phycoerythrin (PE; BD Biosciences) according to the manufacturer's instruction. In the in vitro infection study, B19-infected cells were fixed with 4% paraformaldehyde followed by permeabilization with SAP buffer (0.1% saponin, 0.05% NaN3 in Hanks balanced salt solution). Next, cells were incubated with PAR3 at a concentration of 5 mg/mL on ice for 30 minutes, followed by the same procedure as described. ELISA and quantitative PCR ELISA was carried out by using rB19ECP-fixed microwells (Denka Seiken). The basic protocol for ELISA and quantitative PCR for measuring B19-DNA was performed as described before.15,16 Preparation of cell fraction from B19-infected cells Cells (6 x 105) were infected with B19 for 30 minutes on ice. Following three washes with PBS, pH 7.2, DNA was extracted from 2 x 105 cells to measure adsorbed B19. The remaining 4 x 105 cells were further incubated for 30 minutes at 37°C, followed by 3 washes with PBS, pH 4.5. To obtain cytoplasm fractions of B19-infected cells, cells were treated with lysis buffer A (100 mM Tris-HCl, pH 7.5, 1% Triton X-100, 5 mM EDTA, 50 mM NaCl, and 100 µM PMSF), and centrifuged. Then, DNA was extracted from the supernatant to measure B19-DNA in cytoplasm. The pellets were washed with lysis buffer A 3 times and treated with lysis buffer B (100 mM Tris-HCl, pH 7.5, 1% Triton X-100, 5 mM EDTA, 500 mM NaCl, and 100 µM PMSF). Following centrifugation, DNA was extracted from the supernatant to measure B19-DNA in nuclei. Transfection Five micrograms of expression vector pcD22 containing pKu80 was used for the transfection of 1 x 105 HeLa cells, and pcD was used as a vector-only control. Transfection was done using the lipofectin method (Invitrogen, Carlsbad, CA). Transfected HeLa cells were infected with B19 for 30 minutes at 37°C. After being washed 3 times with PBS, pH 7.2, cells were collected with 5 mM EDTA-PBS, and B19 was detected by confocal microscopy analysis. Transfected HeLa cells were incubated with 1 µg/mL biotinylated rB19ECP in the presence of 5 µg/mL inhibitor antibodies for 30 minutes at 37°C. After being washed 3 times with PBS, pH 7.2, cells were collected with 5 mM EDTA-PBS, and rB19ECP or Ku80 was detected by confocal microscopy analysis. RNA interference of K80 in KU812Ep6 The short interfering RNA (siRNA) for Ku80 was synthesized targeting the sequence between nucleotide numbers 130 and 148: 5'-CAAGCAAAGAAGGUGAUAAdTdT-3' (sense), 3'-dTdTGUUCGUUUCUUCCACUAUU-5' (antisense). Disordered siRNA of scrambled nucleotide sequence, used as negative control, was 5'-GCGCGCUUUGUAGGAUUCGdTdT-3' (sense), 3'-dTdTCGCGCGAAACAUCCUAAGC-5' (antisense). Synthesized siRNA (200 nM) was transfected to 1 x 106 KU812Ep6 cells by Cell Line Nucleofector Kit V (Amaxa, Gaithersburg, MD) according to the manufacturer's instructions. Transfected cells were subjected to flow cytometry analysis and in vitro infection study of B19, after 48 hours of incubation. Detection of B19 in HeLa cells by confocal laser microscopy Cells were grown on glass microslides and fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature. Cells were blocked with PBS containing 10% FBS for 30 minutes at 4°C, followed by incubation with mouse monoclonal anti-B19 antibody PAR3 (10 µg/mL) for 30 minutes at 4°C, then washed with PBS twice, and incubated with FITC-conjugated goat anti–mouse IgG (1:100; Sigma) for 30 minutes at 4°C. To detect localization of Ku80 and rB19ECP, cells on glass microslides were incubated with mouse monoclonal anti-Ku80 antibody (5 µg/mL) for 30 minutes at 4°C, washed with PBS twice, and incubated with tetramethyl isothiocyanate (TRITC)–conjugated goat anti–mouse IgG (1:50; Sigma) for 30 minutes at 4°C; cells were further incubated with avidin-FITC (1:100; Gibco, Carlsbad, CA) for 30 minutes at 4°C for the detection of labeled rB19ECP. Confocal miscroscopy analysis was performed with a D-ECLIPSE CI (Nikon, Kawasaki, Japan) mounted with 20x/0.50 or 40x/0.75 Plan Fluor dry objective lenses. Excitation at 488 nm from an argon laser and at 543 nm from a helium-neon laser was used. Images were acquired with E2-Cl 2.00 software (Nikon) and processed with Adobe Photoshop 7.0.1 (Adobe Systems, San Jose, CA).
Identification of B19-binding protein on the cell surface of nonerythroid cells
To identify a putative receptor for B19, we first checked the expression of P antigen (Figure 1). Flow cytometry analysis revealed that
To determine the cell-surface molecule responsible for B19 binding to H9 cells, a recombinant empty capsid protein of B19 (rB19ECP) was used. Biotinylated rB19ECP bound H9 in a dose-dependent manner (data not shown). We then purified the rB19ECP-binding molecule from the cell surface of H9 using rB19ECP-conjugated Sepharose (rB19ECP-Sepharose). The precipitated 80-kDa protein (Figure 2A) was analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The obtained data were collated and submitted for homology search using the Swiss Prot and NCB Inr databases. The Ku80 autoantigen was identified as the gene product with the highest homology in both databases. As a confirmation, the rB19ECP-binding 80-kDa protein reacted with anti-Ku80 antibody (Figure 2B). Competitive ELISA further confirmed the specific binding between Ku80 and B19. Biotinylated recombinant Ku80 (rKu80) reacted with rB19ECP fixed to microwells (Figure 3A); the binding was selectively inhibited by unlabeled rKu80 but not by recombinant Ku70 (rKu70), globoside, or recombinant soluble CD26 (sCD26)23 (Figure 3B). This binding was also inhibited in the presence of native B19 particles from infected patients (Figure 3C). Two anti-Ku80 antibodies significantly inhibited the binding of biotinylated rKu80 and rB19ECP, whereas anti-Ku70 antibody or anti-CD106 antibody failed to inhibit the binding (Figure 3D). Ku80 participates in B19 binding and subsequent entry
We next investigated whether Ku80 would participate in B19 binding on the cell surface and facilitate B19 entry. KU812Ep6, U937, H9, and ACHN cells efficiently bound B19 (Figure 1A) and all of these cells clearly expressed Ku80 on their surface (Figure 4A). On the other hand, Ku80 was undetectable on T24, SW620, and HeLa cells, and no binding of B19 occurred (Figures 4A and 1A). An in vitro infection experiment demonstrated efficient replication of B19 DNA in KU812Ep6 cells that expressed both Ku80 and P antigen. B19 failed to amplify itself in U937, H9, and ACHN cells, which express Ku80 but no detectable levels of P antigen on the cell surface (Figures 1 and 4A). T24, SW620, and HeLa cells were nonpermissive for B19 infection although they expressed P antigen (Figure 1) and
Ku80 functions as a coreceptor for B19 infection together with P antigen
We then performed an inhibition test for B19 infection of KU812Ep6 cells using antibodies against Ku80, P antigen,
The role of Ku80 as a coreceptor for B19 infection was also supported by a transfection experiment using HeLa cells that were nonpermissive for B19 infection. Figure 5A shows that the surface of Ku80-transfected HeLa cells (HeLa-Ku80) became positive for Ku80 expression and binding of B19 to the cells was significantly enhanced (Figure 5B). Quantitative analysis of B19 DNA (Figure 5B) and confocal laser microscopy (Figure 5C) confirmed that B19 DNA and B19 protein were present in the cytoplasmic fraction of HeLa-Ku80 cells 30 minutes after infection, similar to Ku812Ep6. Furthermore, a coincubation experiment of rB19ECP and HeLa-Ku80 revealed the colocalization of rB19ECP and Ku80 in the cytoplasm or membrane (or both) of HeLa-Ku80 (Figure 5D). Moreover, association of rB19ECP and HeLa-Ku80 was apparently inhibited by the presence of anti-B19 antibody or anti-Ku80 antibody (Figure 5D). Ku80 is expressed on the surface of bone marrow cells Because Ku80 is known as a nuclear protein, it is important to determine whether or not Ku80 is expressed on the cell-surface in vivo. Ku80 was not detected on the cell surface of peripheral blood mononuclear cells (data not shown). We then examined cell-surface expression of Ku80 in bone marrow cells because bone marrow cells are potential targets of B19 infection. Flow cytometry analysis of bone marrow cells demonstrated that Ku80 was highly expressed on the cell surface of erythroid progenitor cells expressing glycophorin A as well as on the surface of immune cells such as CD20+, CD3+, or CD14+ cells in bone marrow (Figure 6A). A small portion (5.6%) of CD36+ bone marrow cells, which may be permissive to B19 infection,23 were also positive for the expression of Ku80 on the cell surface (Figure 6B). B19 binding to bone marrow cells was inhibited in the presence of anti-Ku80 antibody at B19 infection in vitro (data not shown). Figure 7 shows that the replication of B19 in bone marrow cells was significantly inhibited in the presence of anti-Ku80 antibody or GL4. The inhibition rate of B19 replication in the presence of both anti-Ku80 antibody and GL4 was similar to that in the presence of GL4.
The presented data implicate Ku80 as a coreceptor involved in B19 infection. U937, H9, and ACHN cells expressing Ku80 showed B19 binding, but some cells with P antigen failed to bind B19 unless these cells expressed Ku80 on their surface. A marked increase in B19 binding in Ku80-transfected HeLa cells and the inhibition of B19 infectivity by anti-Ku80 antibody or siRNA to Ku80 suggests a Ku80-dependent B19 interaction with the targeted cells. Specific inhibition of B19 binding by anti-Ku80 antibody that recognized the N-terminus of the Ku80 protein suggests that B19 interacts with specific sites of Ku80 on the cell surface. Further, Epstein-Barr virus or hepatitis virus C failed to bind either to Ku80-expressing HeLa or U937 cells (data not shown). These results suggest that Ku80 is one of the specific receptors for B19 infection. Ku is a heterodimeric DNA-binding protein consisting of a 70-kDa (Ku70) and an 80-kDa (Ku80) subunit and was originally identified as a nuclear antigen recognized by autoantibodies in patients with systemic lupus erythematosus and scleroderma.25 Ku has a central role in multiple nuclear processes, including DNA repair, chromosome maintenance, transcription regulation, and V(D)J recombination. Ku is abundant in the nucleus, consistent with its function as a DNA-protein kinase (DNA-PK).26,27 However, recent studies have shown cytoplasm or surface localization of Ku in various types of cells, including of leukemia, multiple myeloma, and tumor cell lines. Ku is a component of the DNA-PK complex in membrane rafts of mammalian cells.26 Although the role of surface Ku80 has not been well clarified,28 signal transduction and Ku80 are coupled in both B and T cells,25,28,29 and localization of the DNA-PK complex in lipid rafts suggests a putative role in the signal transduction pathway following ionizing radiation.26 It was recently reported that Ku interacts with metalloproteinase 9 at the cell surface of highly invasive hematopoietic cells of normal and tumor cell origin, and Ku80/MMP-9 interaction at the cell membrane may result in contribution to the invasion of tumor cells through regulation of extracellular matrix remodelling.30 Further, the membrane form of Ku, whose expression is induced at hypoxia, mediates cell adhesion of plasma cells,30-32 indicating a role for Ku as an adhesion receptor for fibronectin.33 The present study showed that Ku80 is positive on the surface of CD3+ cells, CD20+ cells, CD14+ cells, glycophorin A+ cells, and CD36+ cells from bone marrow where B19 infection is permissive.
We have discovered a novel role of Ku80 as a cellular receptor in B19 infection. Anti-Ku80 antibody, however, did not cause complete inhibition of B19 infection, whereas pretreatment with anti-Ku80 antibody together with GL4 strongly inhibited B19 infectivity in KU812Ep6 cells and human bone marrow cells, showing the necessity of P antigen as a receptor. A recent report showed that
The use of multiple receptors for entry into cells has been observed frequently in virus infection, such as by
Ku80 is not found in circulating mononuclear cells from healthy volunteers but is positive on the surface of B19-binding cells in vivo, such as immune cells in tonsils, erythroblasts, T cells, B cells, macrophages in bone marrow, and immune cells including follicular dendritic cells in rheumatoid joints, indicating the surface expression of Ku antigen may be restricted by environmental conditions. Of interest is that the oxygen levels are markedly low in bone marrow and joints39-41 compared with that in blood, and surface Ku80 is inducible with hypoxia.31,32 A recent study suggests the efficiency of B19 infection increases with hypoxia.42 These studies suggest that surface Ku80 induced with hypoxia may participate in the process of B19 infection of joints and bone marrow.
Ku80 expression on the surface of immune cells in bone marrow in vivo may explain clinical findings associated with B19 infection to nonerythroid cells. Namely, B19 infection often causes a decreased number of leukocytes or lymphocytes in blood during acute B19 infection, as well as increased levels of TNF-
We are grateful to E. Miyagawa for KU812Ep6, K. Kamata for rB19ECP, T. Mimori for rKu80 and rKu70, C. Morimoto for sCD26, K. Yamaguchi for purified B19, and S. Shibahara for the pKu80.
Submitted February 8, 2005; accepted July 5, 2005.
Prepublished online as Blood First Edition Paper, August 2, 2005; DOI 10.1182/blood-2005-02-0536.
Supported by a Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Science, Sports and Culture in Japan.
Y.M. and T.S.-I. designed with research, performed research, and wrote the paper; K.K.-I., J.H., T.K., and T.I. performed research; Y.H. analyzed data; and Y.K. and T.S. designed research.
Y.M. and T.S.-I. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Yasuhiko Munakata, Department of Rheumatology and Hematology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-cho, Aoba-ku, Sendai 980-8574, Japan; e-mail: mnkt{at}mail.tains.tohoku.ac.jp.
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Top
Abstract
Introduction
) is regarded as B19 adsorption, and the right column (
) as B19 replication. The scale for B19 DNA is shown in logarithm. (B) Detection of B19 protein in B19-infected cells. After a 48-hour incubation with B19, the cells were washed 3 times with PBS and they were fixed with 4% paraformaldehyde followed by permeabilization with SAP buffer (0.1% saponin, 0.05% NaN3 in Hanks balanced salt solution). Then, cells were incubated with PAR3 at a concentration of 5 µg/mL on ice for 30 minutes, followed by an incubation with FITC-conjugated goat anti–mouse IgG. The expression of B19 protein in cytoplasm was analyzed by flow cytometry with PAR3 (line) or isotype-matched antibody 1F5 (shadow; left panel), or by immunofluorescence (IF) staining with PAR3 (right panel). Two types of positive patterns were observed in flow cytometry: dull positive (DP) pattern in KU812Ep6, U937, H9, and ACHN; bright positive (BP) pattern in KU812Ep6. (C) Flow cytometry analysis of P antigen expression on the cell surface. Indicated cells were incubated with antigloboside antibody, GL4, followed by PE-labeled anti–rabbit IgG. Shadow represents staining using rabbit IgG as a negative control.
