1 Laboratoire d'Immunobiologie Fondamentale et Clinique, INSERM U503 and UCBL1, IFR128 BioSciences Lyon-Gerland, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France
2 Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Universités d'Aix-Marseille I et II, ESIL, 163 Avenue de Luminy, Case 925, 13288 Marseille, France
3 Unité d'Immunologie Cellulaire et Clinique, INSERM U255 and Université Pierre et Marie Curie Paris VI, Centre de Recherche Biomédicales des Cordeliers, 15 rue de l'école de médecine, 75006 Paris, France
Correspondence
H. Valentin
helene.valentin{at}univ-lyon1.fr
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ABSTRACT |
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These authors contributed equally to this work.
Present address: Immunité et Infections Virales, Faculté de Médecine Lyon RTH Laennec, CNRS-UCBL UMR5537, 69372 Lyon Cedex 08, France.
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INTRODUCTION |
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MV infection induces both an efficient specific immune response and transient, but profound, immunosuppression contributing to secondary infections and mortality in humans (Beckford et al., 1985; Griffin, 1995
; Miller, 1964
). Virus clearance is ensured by specific immunity against MV proteins, particularly MV-N, which confers long-life protection against reinfection (Etchart et al., 2001
; Olszewska et al., 2001
) and includes N-specific T lymphocytes (Etchart et al., 2001
; Ilonen et al., 1990
; Jacobson et al., 1989
; Olszewska et al., 2001
; van Binnendijk et al., 1989
). Although MV-N is a cytosolic protein, the most abundant and rapidly produced antibodies during MV infection are N specific (Graves et al., 1984
; Norrby & Gollmar, 1972
). Thus, anti-N antibody synthesis indicates that MV-N is released into the extracellular compartment, where it binds to the B-cell receptor of antigen (BCR). Indeed, we have previously demonstrated that large amounts of MV-N are extracellularly released in the compartment after apoptosis and/or secondary necrosis of MV-infected cells in vitro (Laine et al., 2003
). By this mechanism, MV-N may become accessible to cell-surface receptors expressed on neighbouring uninfected cells, thereby mediating not only specific immune responses but also immunosuppression.
In contrast to BCR, binding of recombinant MV-N to FcRII and/or NR profoundly disturbs the biology of uninfected B, T and dendritic cells (Laine et al., 2003
; Marie et al., 2001
; Ravanel et al., 1997
). Three human Fc
RII isoforms are generated by alternative splicing, differing in their cytoplasmic tails: Fc
RIIA, -IIB and -IIC (Ravetch & Bolland, 2001
; Tsubata, 1999
). While the Fc
RIIA is critical for antigen-presenting-cell activation, Fc
RIIB1 downregulates B-cell functions (Malbec et al., 1999
; Reth, 1989
). Both human and murine Fc
RII isoforms bind the immune complexes (Cohen-Solal et al., 2004
) and MV-N with low avidity (Ravanel et al., 1997
). Consequently, MV-N binding to Fc
RIIB was shown to block inflammatory immune responses in a murine model (Marie et al., 2001
). MV-N also prevents in vitro interleukin 12 production by human CD40-activated monocyte-derived dendritic cells (Servet-Delprat et al., 2003
), probably after Fc
RIIB aggregation (Grazia Cappiello et al., 2001
). In contrast to Fc
RII, NR is expressed on the surface of a large spectrum of normal cells, except human and murine resting T cells (Laine et al., 2003
). NR detection on different cell species favours ubiquitous and conserved NR expression. Alternatively, MV-N may bind to a group of various receptors sharing similar binding properties. MV-N binding to human NR suppresses normal thymic epithelial and mitogen-activated T-cell proliferation by blocking cells in the G0/G1 phases of the cell cycle (Laine et al., 2003
). Finally, in vitro antibody synthesis of activated human B lymphocytes expressing both Fc
RIIB and NR is dramatically reduced in the presence of MV-N (Ravanel et al., 1997
).
In this work, we aimed to map the MV-N domains involved in the interaction with FcRIIB1 and NR and to determine the relative contribution of each receptor to the suppression of cell proliferation and to apoptosis. To this end, we used a melanoma cell line expressing or not expressing Fc
RIIB1 as a model. We showed that MV-N binds to human Fc
RIIB1 and NR through NCORE and NTAIL, respectively. While Fc
RIIB1 interaction with NCORE triggered apoptosis, the aa 401420 region of MV-N acted predominantly by blocking cell proliferation in the G0/G1 phases of the cell cycle after binding to NR. Therefore, MV-N displays different suppressive activities depending on whether NCORE or NTAIL binds to its respective cell-surface receptor.
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METHODS |
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Cell lines.
The human melanoma cell line HT144, which does not express FcRII, was stably transfected with human Fc
RIIB1 cDNA (HT144IIB1) or with human Fc
RIIB1 cDNA lacking the intra-cytoplasmic tail (HT144IIB1/IC) (Cassard et al., 2002
). The murine fibroblast L Orient cells transfected with human Fc
RII cDNA (L-CD32) were kindly provided by S. Lebecque (Schering-Plough, Dardilly, France). Cell lines were grown in RPMI 1640 (Invitrogen) supplemented with 2 mM L-glutamine (Invitrogen), 10 mM HEPES (Invitrogen), 40 µg gentamicin (Schering-Plough) ml1 and 10 % fetal calf serum (Biomedia).
Production of MV-N, NCORE and NTAIL.
Recombinant MV-N (strain Edmonston B) was produced from Escherichia coli as previously described (Karlin et al., 2002). NCORE was obtained by limited proteolysis of purified N as described by Karlin et al. (2002)
, and NTAIL (strain Edmonston B) was purified as described elsewhere (Laine et al., 2003
; Longhi et al., 2003
).
Construction of expression plasmids encoding NTAIL deletion proteins, and their expression and purification.
All NTAIL constructs were obtained by PCR using the plasmid pet21a/N (Karlin et al., 2002) encoding the MV-N protein (strain Edmonston B) as template. Pfu polymerase was purchased from Promega. Primers were purchased from Invitrogen. The E. coli strain DH5
(Stratagene) was used for selection and amplification of DNA constructs.
The NTAIL1 and NTAIL
3 gene constructs encoded aa 421525 and 401516 of MV-N, respectively. The NTAIL
2,3 construct, previously referred to as NTAIL2 (Bourhis et al., 2004
), encoded aa 401488 of MV-N. The sequences of the coding regions were checked by sequencing (MilleGen).
E. coli strain Rosetta (DE3) pLysS (Novagen) was used for the expression of NTAIL deletion constructs. Culture and induction conditions were as described by Longhi et al. (2003), except that chloramphenicol (17 µg ml1) was used instead of kanamycin.
Expression of tagged full-length NTAIL from the pQE32 vector was carried out as described by Longhi et al. (2003).
Purification of NTAIL proteins was carried out as described by Longhi et al. (2003). The proteins were purified by immobilized metal affinity chromatography (IMAC) using Chelating Sepharose Fast Flow Resin preloaded with Ni2+ ions (Amersham Pharmacia Biotech).
Protein concentrations were calculated as described by Longhi et al. (2003).
Detection of FcRII.
Direct immunofluorescence assays were performed in staining buffer (PBS containing 1 % BSA and 0·1 % sodium azide) as described by Laine et al. (2003). After labelling, cells were analysed (Cellquest software) by flow cytometry analysis using a Calibur flow cytometer (Becton Dickinson). Integrated fluorescence was measured and data were collected from at least 10 000 events.
Detection and competition of MV-N binding by flow cytometry.
To determine MV-N binding to cells, 5x105 cells were incubated for 1 h at 4 °C with 5 µg purified N (50 µg ml1) in the presence or absence of anti-FcRII mAb KB61 (10 µg ml1) in staining buffer. After washes, cells were incubated for 30 min at 4 °C with either biotinylated mAbs (C120 and Cl25) or mAb M2 specific for FLAG fusion proteins. Cells were then incubated with either AvPE or PEGAM for 30 min at 4 °C.
Competition experiments were performed using NTAIL deletion proteins as competitors. MV-N (2·5 µg; 25 µg ml1) was incubated with increasing amounts of NTAIL deletion proteins for 1 h at 4 °C. Cells were then incubated with biotinylated anti-N Cl120 mAb, followed by AvPE incubation. The mean fluorescence intensity (MFI) of MV-N binding was measured after analysis by flow cytometry.
Cell proliferation and cell-cycle analyses.
Cells were plated at 3·5x104 cells cm2 for 24 h in a volume of 700 µl cm2. For cell proliferation assays, cells were seeded in triplicate in a 96-well plate and incubated with various amounts of MV-N or domains thereof. After 12 h of treatment, 0·5 µCi (18·5 kBq) [3H]thymidine per well was added for 24 h as described by Laine et al. (2003).
For cell-cycle analysis, cells were stained with 7-amino-actinomycin D (7AAD) and pyronin Y (PY) (Toba et al., 1995). Briefly, 20 µM 7AAD was incubated with 5x105 cells and 1 µM PY was then added. Cells were analysed by flow cytometry. Data were collected from at least 20 000 events.
Apoptosis detection.
Cells were plated at 3·5x104 cells cm2 for 24 h in a volume of 700 µl cm2 prior to the addition of MV-N or domains thereof. Percentages of attached and floating cells with activated pan-caspase were estimated after staining with the in situ marker FITCVAD-FMK (CaspACE), a FITC conjugate of the pan-caspase inhibitor zVAD-FMK, as described by the manufacturer (Promega). Percentages of attached cells with activated caspase-3 were estimated after staining with the in situ marker FITCDEVD-FMK (BioVision), as described by the manufacturer. For the assessment of nuclear features of apoptosis, attached cells were stained with Hoechst 33342 (10 µg ml1) for 30 min at 37 °C, as previously described (Valentin et al., 1999).
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RESULTS |
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Both NCORE and NTAIL domains of MV-N inhibit spontaneous cell proliferation
We next determined the contribution of the NCORE and NTAIL domains to the suppression of cell proliferation through their interactions with FcRIIB1 and NR, respectively. As shown in Fig. 2(a)
, MV-N and NTAIL, but not NCORE, inhibited up to 95 % of HT144 cell proliferation. The apparent higher anti-proliferative activity of NTAIL compared with that of full-length MV-N could be accounted for by differences in the molar amounts of receptor engaged. As the molecular mass of NTAIL is approximately 15 kDa and that of N is approximately 60 kDa, a fourfold excess of N, compared with NTAIL, has to be added to yield the same molar amount and the same biological effect.
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We next analysed the cell-cycle distribution of MV-N-, NCORE- or NTAIL-treated HT144 and HT144IIB1 cells by measuring the DNA/RNA contents. We observed a significant increase in the percentage of both cell types arrested in the G0/G1 phases (up to 35 %) after MV-N treatment compared with untreated cells (Fig. 2c). Subsequently, a decrease in the percentage of MV-N-treated cells in both S and G2/M phases of the cell cycle by day 1 was observed (data not shown). Similar results were obtained with NTAIL but the difference was less marked (Fig. 2c
, and data not shown). As expected, NCORE induced a smaller increase (up to 21 %) in the percentage of G0/G1-arrested HT144IIB1 cells compared with MV-N. Conversely, no significant increase in the percentage of arrested HT144 cells was observed in the presence of NCORE (4 %; Fig. 2c
). In conclusion, these results indicate that the NCORE and NTAIL domains of MV-N are responsible for human cell growth arrest through interaction with Fc
RIIB1 and NR, respectively.
NCOREFcRIIB1 interaction triggers cell apoptosis through caspase-3 activation
HT144IIB1 cells treated with MV-N or NCORE, but not those treated with NTAIL, appeared dispersed and damaged compared with untreated cells, with some detaching and displaying apoptotic morphology (Fig. 3). When MV-N or NTAIL were added to HT144 cells, the monolayers also appeared dispersed, enlarged and displayed a round shape, although they remained attached to the culture dish, while the addition of NCORE did not have this effect (Fig. 3
). These results indicate that both MV-N and NCORE, but not NTAIL, efficiently trigger HT144IIB1 cell death through Fc
RIIB1.
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Binding of conserved Box1 of Morbillivirus NTAIL to NR suppresses spontaneous cell proliferation
In addition to the NTAIL domain of MV-N, three different N proteins derived from various members of the genus Morbillivirus also bind to human NR (Laine et al., 2003). We thus hypothesized that the region(s) involved in NR binding may be located in one of the three conserved regions in the Morbillivirus NTAIL (aa 401420, 489506 and 517525) (Diallo et al., 1994
). To test this possibility, we purified three NTAIL deletion proteins carrying different combinations of such boxes, NTAIL
1, NTAIL
2,3 and NTAIL
3 (Fig. 6a
). These deletion proteins were all found in the soluble fraction of the bacterial lysate (Fig. 6b
) and were purified by IMAC (Fig. 6b
).
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Finally, we investigated whether NR engagement by the deletion proteins affected HT144 cell proliferation. As illustrated in Fig. 6(e), NTAIL
2,3 and NTAIL
3 deletion proteins inhibited human cell proliferation in a dose-dependent manner, although to a lower extent than NTAIL. As expected, NTAIL
1 was unable to inhibit HT144 cell proliferation, even at concentrations as high as 20 µg per well (Fig. 6e
). Similar results were obtained using L and thymic epithelial cell lines (data not shown). Thus, the aa 401420 region of MV-N is required for NR binding and, as a result, is responsible for cell proliferation inhibition.
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DISCUSSION |
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Our results point to an exclusive interaction of NCORE with FcRIIB1 and show that MV-N binds to Fc
RIIB1 more efficiently than NCORE. This latter point is probably related to the increased rigidity of NCORE compared with N (Longhi et al., 2003
), which possibly renders sequential and/or conformational epitopes less accessible to Fc
RIIB1. Fc
RIIB1 is not a receptor responsive to any nucleocapsid-like particles. While the N of MV, canine distemper virus (CDV) and peste-des-petits-ruminants virus (PPRV) bind to Fc
RIIB1, rinderpest virus (RPV) N does not (Laine et al., 2003
), thus supporting specific NCORE binding to Fc
RIIB1. The distinctive behaviour of RPV-N may be due to its unique sequence properties within the putative region of interaction with Fc
RIIB1. Although the amino acid sequence of NCORE is well conserved among Morbillivirus members (overall sequence similarity of 80 %), the similarity drops to 40 % for the aa 122144 region (Diallo et al., 1994
). This variable region, already described as an antigenic region (Giraudon et al., 1988
), is low in hydrophobic clusters and may therefore form a loop exposed to the solvent (Karlin et al., 2003
), possibly involved in binding to Fc
RIIB1. The conserved serine 138, occurring in MV-N, CDV-N and PPRV-N, is replaced by a glycine residue in RPV-N (Diallo et al., 1994
). This substitution may lead to a conformational change in RPV-N, thus resulting in a spatial conformation unsuitable for the proper interaction with Fc
RIIB1. Interestingly, the RPV-N C-terminal domain is more antigenic than its N-terminal counterpart. Among the three highly immunogenic epitopes located at both the C and N terminus of RPV-N, only one (aa 520525) is conserved in MV NTAIL (aa 519523) (Buckland et al., 1989
; Choi et al., 2003
, 2004
). These results suggest that the Morbillivirus N epitopes involved in immune activation are different from the regions involved in binding to cell-surface receptors and thus in immunosuppression.
We showed that NR binds Box1 (aa 401420), which is well conserved among Morbillivirus members (Diallo et al., 1994). Moreover, Box1 is also well conserved among wild-type and vaccine MV strains, as judged by the comparison between the amino acid sequence of NTAIL from the Edmonston B, other vaccine strains and 48 wild-type strains. Four conservative substitutions (T402A, I406T/V, A415S and L420I) occur individually in eight wild-type strains, and the conservative K405R substitution is observed in the majority of the wild-type strains (D. W. Kouomou & F. T. Wild, personal communication). This latter substitution is also observed in other Morbillivirus N capable of interacting with NR (Diallo et al., 1994
; Laine et al., 2003
). This analysis also revealed complete conservation of the sequence in the aa 407414 region between Edmonston B and all of the wild-type strains. Thus, the high conservation of Box1 highlights the biological relevance of studies focused on the interaction between NR and NTAIL from Edmonston MV strain.
We also reported that cell-cycle arrest was mediated predominantly by the NTAILNR interaction in a human melanoma line, whereas apoptosis was mediated primarily by the NCOREFcRIIB1 interaction in melanoma cells expressing Fc
RIIB1. Fc
RIIB1 aggregation by antibodies also results in inhibition of spontaneous and activated B-cell proliferation (Pearse et al., 1999
). Few data are available concerning the role of N from single-stranded RNA viruses in the modulation of cell proliferation. The only available data concern the role of intracellular N from Borna disease virus and hepatitis C virus in blocking cell proliferation through interaction with cell-cycle regulators (Planz et al., 2003
; Yao et al., 2003
). As no inhibition of cell proliferation has been documented so far for intracellular MV-N, our results strongly suggest that the effect of NCOREFc
RIIB1 interaction on cell proliferation can be ascribed to B cells, while the NTAILNR interaction inhibits both B- and T-cell proliferation. Similarly, both wild-type and vaccine MV strains suppress both infected and uninfected B- and T-cell proliferation in vitro (Gerlier et al., 2005
; Hahm et al., 2003
; McChesney et al., 1987
, 1988
; Naniche et al., 1999
; Schlender et al., 1996
). There are a several lines of evidence suggesting that cell growth arrest cannot be ascribed to infectious virus or conventional cytokines (Fujinami et al., 1998
; Sanchez-Lanier et al., 1988
; Sun et al., 1998
). In addition to the role of H/F proteins from MV, RPV and PPRV (Heaney et al., 2002
; Schlender et al., 1996
), soluble anti-proliferative factors produced from dead MV-infected cells arrest uninfected B and T cells in the G0/G1 phases (Fujinami et al., 1998
; Wang et al., 2003
). As MV-N is released from apoptotic MV-infected cells (Gerlier et al., 2005
), it would be interesting to determine whether MV-N is the factor responsible for growth arrest. In addition to the potent anti-proliferative effect of MV-N, aggregation of Fc
RIIB1 by NCORE triggers apoptosis via the intra-cytoplasmic tail of Fc
RIIB1. Previous data have already demonstrated that cross-linking of murine Fc
RIIB by a combination of antibodies is sufficient to induce apoptosis of B cells independent of BCR co-ligation (Pearse et al., 1999
). However, the link between inhibition of cell proliferation and induction of apoptosis mediated by both MV-N and NCORE after binding to Fc
RIIB1 is difficult to establish, and we cannot exclude the possibility that the induction of apoptosis is the consequence of suppression of cell proliferation. To our knowledge, only hepatitis C virus CORE protein has been described to exert a pro-apoptotic effect in transfected cells (Realdon et al., 2004
). Apoptosis of uninfected B and T lymphocytes by MV, CDV and PPRV has been described, and lymphopenia, primarily due to apoptosis of uninfected lymphocytes, seems to arise mainly from indirect effects of the viruses (Gerlier et al., 2005
; Mondal et al., 2001
; Okada et al., 2000
; Schobesberger et al., 2005
). Although RPV-induced apoptosis has been ascribed to direct cytopathogenic RPV infection (Stolte et al., 2002
), we hypothesize that the four Morbillivirus members trigger cell growth arrest via NTAILNR interaction, with MV, CDV and PPRV being also involved in NCOREFc
RIIB1-induced apoptosis. The severe lymphopenia observed with measles patients does not occur with the vaccine strains (Okada et al., 2001
). In the cotton rat model, wild-type MV strains induce a higher anti-proliferative effect than vaccine strains through H/F glycoproteins (Niewiesk et al., 1997
; Pfeuffer et al., 2003
). However, high-titre measles vaccine administration to young infants increased mortality, thus suggesting that vaccine virus may mimic the immunosuppressive effects of wild-type MV (Moss & Polack, 2001
). Thus, further studies are necessary to understand in more detail the contribution of MV-N to the mechanisms of immunosuppression following MV infection. Finally, the potential effect of MV-N on cell death via Fc
RIIB, in addition to its potent and global anti-proliferation effect via NR, may represent a promising approach for the local treatment of cancer cells expressing Fc
RIIB, as well as for cell-cycle manipulation of rapidly proliferating cells expressing NR.
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ACKNOWLEDGEMENTS |
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Received 26 November 2004;
accepted 11 February 2005.