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Laboratory of Virology and Vaccinology, Division of Biomedical Research, National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan
Correspondence
Yasuko Mori
ymori{at}nibio.go.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Seroepidemiological studies have indicated that primary infection with HHV-7 occurs early in childhood, although the age of infection with HHV-7 appears to be somewhat later than that with human herpesvirus 6 (HHV-6), and that HHV-7 is another causative agent of exanthem subitum, a common childhood disease (Tanaka et al., 1994; Tanaka-Taya et al., 1996; Wyatt et al., 1991). Molecular biological and immunological studies have demonstrated that HHV-7 belongs to the subfamily Betaherpesvirinae and that HHV-6 is, of the members of this subfamily, related most closely to HHV-7, but nevertheless, they differ from each other significantly (Berneman et al., 1992; Foa-Tomasi et al., 1994; Nakagawa et al., 1997; Nicholas, 1996; Wyatt et al., 1991).
Viral mechanisms of immune-system evasion are numerous. Complement, part of the innate immune defence system, is composed of serum proteins that interact in an amplification cascade. Herpesviruses have many strategies for replication in host cells that allow them to circumvent the humoral immunity mediated by the complement system. In human cytomegalovirus (HCMV) infections, CD55 and CD46 have been reported to be increased severalfold following infection, thus protecting HCMV-infected cells from complement (Spiller et al., 1996). Mouse CD46 has also been shown to be upregulated during murine cytomegalovirus (MCMV) infection (Nomura et al., 2002) and to participate in protection of infected cells from complement-dependent cytolysis. Herpes simplex virus (HSV) glycoprotein C (gC)-1 and gC-2 consist of virions, where gCs bind C3b and block complement-mediated lysis of virions (Friedman et al., 1984; McNearney et al., 1987). Murine gammaherpesvirus 68 (Kapadia et al., 1999), herpesvirus saimiri (Fodor et al., 1995) and Kaposi’s sarcoma-associated herpesvirus (Spiller et al., 2003) encode functional regulators of complement activation. These recent studies suggest that viruses have several strategies for immune evasion from a complement attack.
Like CD55 and CD59, CD46 is a complement-regulatory protein (CRP) that suppresses self-cytolysis via complement by cleaving C3b and C4b in the presence of factor I. Human CD46 (hCD46) also serves as a receptor for several human pathogens, including measles virus (Dorig et al., 1993; Naniche et al., 1993), HHV-6 (Santoro et al., 1999), adenovirus of different serotypes (Gaggar et al., 2003; Segerman et al., 2003), group A Streptococcus pyogenes (Okada et al., 1995) and pathogenic Neisseria (Kallstrom et al., 1997). hCD46 has been reported to be downregulated from the HHV-6-infected cell surface by internalization (Santoro et al., 1999).
Here, we report that HHV-7 infection induces upregulation of hCD46 expression in infected cells at both the transcriptional and translational levels. The increase of CD46 mRNA and protein was confirmed by Northern blot and Western blot analyses, respectively, and flow-cytometric analysis demonstrated that upregulation of CD46 occurred at the cell surface at a late stage of infection. In addition, we found that another CRP, CD59, was increased at a late stage of infection, as well as CD46. Furthermore, our results suggest that HHV-7-infected cells were more resistant to complement-dependent cytotoxicity (CDC) than mock-infected cells, which may be due to the upregulation of CRPs, including CD46 and CD59.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Northern blotting.
Total RNAs isolated from KHR-infected SupT1 cells by TRIzol reagent (Invitrogen) were subjected to purification of poly A+ RNA by using Oligotex-dT30<Super> (TaKaRa). Samples were fractionated by electrophoresis and blotted onto Hybond-N+ membrane (GE Healthcare) as described previously (Takemoto et al., 2001). Template fragments for probes were prepared as described below. CD46 cDNA was excised from pME18S-CD46 STc/CYT2, which was kindly provided by Dr Tsukasa Seya (Hokkaido University, Sapporo, Japan) (Kojima et al., 1993), at EcoRI and PstI sites. HHV-7 gB and GAPDH fragments were generated by PCR using following the primer pairs: 7gB2119bamF (5'-ACCGGATCCCATAAACGATTAGCACAAACACCG-3') and 7gB2469salR (5'-ACCGTCGACTCACAGTTCTTCTGTTGAAAG-3'), GapdhF (5'-GAAGGTGAAGGTCGGAGTC-3') and GapdhR (5'-GAAGATGGTGATGGGATTTC-3'), respectively. The probes were labelled by using ECL direct nucleic acid labelling and detection systems (GE Healthcare) and the following steps of hybridization, washing membrane and detection of the signal were performed according to the manufacturer’s instructions.
Western blotting.
Primary CD4+ T cells were resuspended in 1x SDS sample buffer [50 mM Tris/HCl (pH 6.8), 2 % SDS, 0.1 % glycerol, 0.02 % bromophenol blue] with or without 0.1 M dithiothreitol (DTT) and boiled for 5 min. SupT1 cells were lysed in radioimmunoprecipitation assay (RIPA) buffer [0.01 M Tris/HCl (pH 7.4), 0.15 M NaCl, 1 % sodium deoxycholate, 1 % Nonidet P-40, 0.1 % SDS, 1 mM EDTA, 1 mM PMSF] and centrifuged at 70 000 g for 1 h at 4 °C. An equal volume of 2x SDS sample buffer with or without 0.1 M DTT was added to the supernatant and boiled for 5 min. Samples were loaded onto SDS–polyacrylamide gels, fractionated by electrophoresis and Western-blotted onto PVDF membranes (Bio-Rad). The blots, blocked with 3 % skimmed milk in TBS, were incubated with the following primary monoclonal antibodies (mAbs): anti-CD46 mAb (M75; kindly provided by Dr Tsukasa Seya, Hokkaido University, Sapporo, Japan) (Seya et al., 1990) diluted 1 : 200; anti-CD55 mAb (BRIC 216; Chemicon), diluted 1 : 200; anti-CD59 mAb (MEM-43; Serotec), diluted 1 : 500; anti-
-tubulin (B-5-1-2; Sigma), diluted 1 : 10 000. Detection was performed by using an ECL detection kit (GE Healthcare).
Flow cytometry.
Cells were incubated with mAbs against the following cell-surface markers: CD3 (UCHT1; Ancell), CD4 (34930; R&D Systems), CD46 (J4.48; Beckman Coulter), CD55 and CD59, for 30 min at room temperature and then washed twice. Normal mouse monoclonal IgG (Santa Cruz) was used as a control. Cells, labelled with secondary antibody conjugated with fluorescein isothiocyanate (FITC) (DAKO) for 30 min at room temperature, were washed twice and then analysed by FACS Canto (BD Bioscience). In the case of detection of intracellular antigens U89 (IE1), U41 (single-stranded DNA-binding protein) and U39 (glycoprotein B) (Sadaoka et al., 2006), fixation and permeablization of cells with cold acetone for 20 min at –20 °C (Loor, 1984) and, subsequently, blocking with 3 % FCS for 30 min at room temperature were done prior to incubation with primary antibodies. mAbs against HHV-7 antigens were produced in our laboratory as described previously (Takeda et al., 2000).
CDC assay.
At 5 days post-infection, living cells were sorted by FACS Aria (BD Biosciences) prior to CDC assay to eliminate background derived from dead cells. Cells (4x105 per 100 µl) were incubated with either normal mouse monoclonal IgM (TEPC 183; Ancell) or anti-T-cell receptor (TCR) mAb (T10B9; Chemicon), which is of the IgM subtype and recommended for CDC assay, at 2 µg ml–1 for 1 h at 4 °C, washed twice and then incubated with baby rabbit serum (BRS) (Cedarlane) as a source of complement at the indicated ratios for 1 h at 37 °C. After washing twice, the dead cells were labelled by incubation with 1 µg propidium iodide (PI) ml–1 for 10 min at room temperature and detected by flow cytometry (FACS Canto). To identify the complement pathways, BRS was pretreated for 10 min at 4 °C with 10 mM EDTA to inhibit the classical, mannan-binding lectin and alternative pathways, and 8 mM EGTA and 2 mM Mg2+ to inhibit the classical and mannan-binding lectin pathways.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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To confirm the elevated expression of CD46 mRNA, we performed Northern blot analysis. As shown in Fig. 1(a), it was demonstrated that the level of CD46 mRNA increased in HHV-7-infected cells, supporting the result of our DNA microarray analysis. Glycoprotein B (gB) mRNA, which is expressed at a late stage in HHV-7 infection, was also detected in the infected cells, indicating that HHV-7 infection occurred in the cells. The data demonstrated that HHV-7 infection upregulates CD46 expression at a transcriptional level. The protein level of CD46 was also examined by Western blot analysis using whole-cell lysates of mock- or HHV-7-infected SupT1 cells and primary CD4+ T cells. Infection with HHV-7 resulted in high levels of CD46 protein in SupT1 cells infected with HHV-7, supporting the results of Northern blot analysis. The results indicate that the elevation of CD46 in HHV-7-infected cells occurred at the translational level, as well as at the transcriptional level. The level of CD46 also increased in HHV-7-infected primary CD4+ T cells, although it showed a slight level of increase in CD46 protein compared with that in infected SupT1 cells (Fig. 1b).
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, the staining of intracellular viral antigens IE1, ssDNABP and gB showed a typical cascade of viral gene expression. The immediate-early protein, IE1, was expressed maximally, the early protein, ssDNABP, was first detected at 1 day post-infection and the late protein, gB, was detected at 3 days post-infection. At 3 and 6 days post-infection, the expression of CD46 was elevated on the cell surface, as seen in Fig. 2(a). The upregulation of CD46 appeared to occur at nearly the same time as that of gB, because a positive shift of the CD46 histogram was firstly observed at 3 days post-infection. In contrast, CD4, which is a cellular receptor for HHV-7, was downregulated at 1 day post-infection, as reported by Lusso et al. (1994).
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, in the absence of PFA, the CD46 level was upregulated at 5 days post-infection, as seen in SupT1 cells, whereas CD4 was downregulated. In the presence of PFA, the upregulation of CD46 was eradicated completely, thus indicating that the expression of some late genes encoded by HHV-7 is required for changes of CD46 on the cell surface. These data demonstrated that the increase of CD46 on the cell surface occurred at a late stage of infection. The expression level of CD3 on the cell surface was constant during HHV-7 infection (Fig. 3).
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and 4, the expression level of CD59, as well as CD46, was increased in SupT1 cells infected with HHV-7 (Figs 2b, c, 4), whilst the level was increased slightly in primary CD4+ T cells (Figs 3a, b, 4), although the elevation of CD55 by HHV-7 infection was hardly found in either cell. As shown in Fig. 3, in the presence of PFA, the upregulation of CD59 was eradicated completely, thus demonstrating that the increase of CD59 on the cell surface, as well as that of CD46, occurred at a late stage of infection. Following the result of Western blot, the level of CD59 was highly increased in HHV-7-infected SupT1 cells, but little increased in HHV-7-infected primary CD4+ T cells (Fig. 4). These data give rise to a possibility that there is a cooperative role of CD46 and CD59 in complement regulation.
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), to induce the classical pathway, cells were incubated with BRS as a source of complement. By flow cytometry, cytotoxicity was evaluated by counting living and damaged cells, which were stained negatively and positively with PI, respectively. As shown in Fig. 5(a), the cells labelled with anti-TCR mAb were damaged by the addition of BRS, whereas mock- and HHV-7-infected cells reacted with mouse IgM showed little sensitivity to BRS. This result indicates that antibody-sensitized CD4+ T cells were sensitive to rabbit complement, whereas non-sensitized cells were less sensitive. In the presence of BRS at 10 and 25 %, approximately 65 % of mock-infected cells were lysed, whereas the percentage cytolysis of HHV-7-infected cells was suppressed by about 50 %, demonstrating that sensitivity to CDC is reduced in HHV-7-infected cells. To determine which pathway is actually involved in this cytotoxicity, EGTA and EDTA were added to BRS. EDTA inhibits all three pathways, i.e. the classical, mannan-binding lectin and alternative pathways, and EGTA inhibits only the classical and mannan-binding lectin pathways. The cytotoxicity was suppressed to the basal level in the presence of either EGTA or EDTA, demonstrating that the cells were damaged by the classical pathway (Fig. 5b). These results suggest that CD46 upregulation, in cooperation with CD59 upregulation, may contribute to the protection of infected cells from complement-associated lysis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), whereas only a single band was detected by Northern blotting (Fig. 1a). It is possible that only one transcript in CD46 may be detected by the probe used in this study for Northern blot or that CD46 may be modified in the step of protein synthesis; therefore, two bands may be detected by Western blot. The level of increase of CD46 was different between SupT1 cells and primary CD4+ T cells isolated from PBMCs when infected with HHV-7 (Fig. 1b), because the basal level of CD46 expression may have been higher in primary CD4+ T cells than in SupT1 cells. In fact, uninfected SupT1 cells were more sensitive to complement in CDC assay than primary CD4+ T cells (data not shown). Flow cytometry of the HHV-7-infected cells indicated that CD46 elevation on the cell surface occurred at a late stage of infection (Fig. 2, 3). As shown in Fig. 3, viral DNA synthesis was required for CD46 elevation in HHV-7 infection, supporting the data that CD46 elevation on the cell surface occurred at a late stage of infection. In addition, the data showed that CD59 elevation on the cell surface also occurred at a late stage of infection (Fig. 3). The expression of the viral glycoprotein gB was clearly downmodulated at day 6 post-infection. One possibility is that viral protein synthesis, particularly of gB, is not done in HHV-7-infected cells at 6 days post-infection and that gB proteins are not accumulated, whereas IE1 and U41 proteins are accumulated.
Because CD59 is one of the CRPs that inhibits formation of the membrane attack complex by binding C8 and C9, CD46 and CD59 may be involved cooperatively in the protection of HHV-7-infected cells from complement attack. Although it has been reported that CD46 is mainly involved in the regulation of complement in the alternative pathway (Devaux et al., 1999; Kojima et al., 1993; Seya et al., 1991), it has also been reported that CD46 protects cells from cytolysis mediated through the classical pathway (Liszewski & Atkinson, 1996; Loveland et al., 1993; Miyagawa et al., 1994; Oglesby et al., 1992; Seya et al., 1991). We therefore designed a CDC assay focusing on the regulation of the classical pathway and showed that HHV-7-infected cells were in fact more resistant to CDC than were mock-infected cells (Fig. 5).
In HHV-6A-infected cells, the cellular receptor CD46 has been reported to be downregulated (Santoro et al., 1999). HHV-6 may increase the other cellular CRPs or encode a viral complement receptor (CR) homologue, such as HSV gC. There still remains a possibility that HHV-7 also encodes a viral CR homologue to evade complement attack, which has not been discovered so far among betaherpesviruses, including cytomegaloviruses.
In addition to the conventional function of CD46 as a CRP and a pathogen receptor, another role of CD46 in cellular immunity has recently been discovered. Antibody ligation of CD46 and CD3 triggers induction of T cells showing characteristics of T regulatory 1-type cells (Tr1 cells), which secrete a large amount of IL-10 and inhibit proliferation of conventional CD4+ T cells (Kemper et al., 2003). This phenomenon was also observed when a pathogen was used to challenge cells expressing CD46 (Price et al., 2005). These new findings gave rise to an intriguing possibility that elevated expression of CD46 in HHV-7-infected cells may contribute to the suppression of the cellular immune response against HHV-7 infection by enhancing the induction of Tr1 cells.
In conclusion, this study is the first report of the increase of CRPs and the reduction of CDC by HHV-7 infection, although it remains to be elucidated whether increased CD46 and CD59 levels really contribute to the protection of infected cells from CDC.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 20 July 2006;
accepted 22 December 2006.
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