Measles virus nucleoprotein induces cell-proliferation arrest and apoptosis through NTAIL–NR and NCORE–Fc{gamma}RIIB1 interactions, respectively

D. Laine1,{dagger}, J. M. Bourhis2,{dagger}, S. Longhi2, M. Flacher1, L. Cassard3, B. Canard2, C. Sautès-Fridman3, C. Rabourdin-Combe1 and H. Valentin1,{ddagger}

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


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Measles virus (MV) nucleoprotein (N) is a cytosolic protein that is released into the extracellular compartment after apoptosis and/or secondary necrosis of MV-infected cells in vitro. Thus, MV-N becomes accessible to inhibitory cell-surface receptors: Fc{gamma}RIIB and an uncharacterized nucleoprotein receptor (NR). MV-N is composed of two domains: NCORE (aa 1–400) and NTAIL (aa 401–525). To assess the contribution of MV-N domains and of these two receptors in suppression of cell proliferation, a human melanoma HT144 cell line expressing (HT144IIB1) or lacking Fc{gamma}RIIB1 was used as a model. Specific and exclusive NCORE–Fc{gamma}RIIB1 and NTAIL–NR interactions were shown. Moreover, NTAIL binding to human NR predominantly led to suppression of cell proliferation by arresting cells in the G0/G1 phases of the cell cycle, rather than to apoptosis. NCORE binding to HT144IIB1 cells primarily triggered caspase-3 activation, in contrast to HT144IIB1/IC cells lacking the Fc{gamma}RIIB1 intra-cytoplasmic tail, thus demonstrating the specific inhibitory effect of the NCORE–Fc{gamma}RIIB1 interaction. MV-N- and NCORE-mediated apoptosis through Fc{gamma}RIIB1 was inhibited by the pan-caspase inhibitor zVAD-FMK, indicating that apoptosis was dependent on caspase activation. By using NTAIL deletion proteins, it was also shown that the region of NTAIL responsible for binding to human NR and for cell growth arrest maps to one of the three conserved boxes (Box1, aa 401–420) found in N of Morbilliviruses. This work unveils novel mechanisms by which distinct domains of MV-N may display different immunosuppressive activities, thus contributing to our comprehension of the immunosuppressive state associated with MV infection. Finally, MV-N domains may be good tools to target tumour cell proliferation and/or apoptosis.

{dagger}These authors contributed equally to this work.

{ddagger}Present address: Immunité et Infections Virales, Faculté de Médecine Lyon RTH Laennec, CNRS-UCBL UMR5537, 69372 Lyon Cedex 08, France.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Measles virus (MV), belonging to the genus Morbillivirus of the family Paramyxoviridae, is an enveloped virus with a non-segmented single-stranded negative-sense RNA genome. It encodes two envelope glycoproteins, the haemagglutinin (H) and the fusion (F) proteins required for MV cell entry, a matrix protein (M) and three structural proteins involved in viral genome transcription and replication: the nucleoprotein (N), the phosphoprotein (P) and the large protein (L). The MV genome is encapsidated by N to form the helical nucleocapsid. The N–RNA complex binds to the viral polymerase complex, which consists of the P and L proteins (Curran & Kolakofsky, 1999). Morbillivirus N consists of two domains: a highly conserved N-terminal moiety (NCORE, aa 1–400) and a poorly conserved C-terminal moiety (NTAIL, aa 401–525) (Diallo et al., 1994). NCORE is globular and carries regions required for N self-assembly and RNA binding (Bankamp et al., 1996; Curran et al., 1993; Karlin et al., 2002; Liston et al., 1997). NTAIL is an intrinsically disordered monomeric domain (Longhi et al., 2003) and protrudes from the viral nucleocapsid surface, thus ideally favouring the interaction with viral and cellular partners (Longhi et al., 2003). NTAIL contains the regions responsible for binding to P and to the polymerase complex (Bankamp et al., 1996; Bourhis et al., 2004; Harty & Palese, 1995; Johansson et al., 2003; Liston et al., 1997; Longhi et al., 2003). NTAIL also binds intracellular partners such as the 70 kDa heat-shock protein (Hsp72) and the interferon responsive factor-3 (IRF-3), which modulate viral RNA synthesis and interferon response, respectively (tenOever et al., 2002; Zhang et al., 2002). In addition, MV-N interacts with two extracellular cell-surface receptors: the type II IgG Fc receptor (Fc{gamma}RII/CD32) and an as yet uncharacterized receptor referred to as N receptor (NR) (Laine et al., 2003; Ravanel et al., 1997).

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 Fc{gamma}RII 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{gamma}RII isoforms are generated by alternative splicing, differing in their cytoplasmic tails: Fc{gamma}RIIA, -IIB and -IIC (Ravetch & Bolland, 2001; Tsubata, 1999). While the Fc{gamma}RIIA is critical for antigen-presenting-cell activation, Fc{gamma}RIIB1 downregulates B-cell functions (Malbec et al., 1999; Reth, 1989). Both human and murine Fc{gamma}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{gamma}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{gamma}RIIB aggregation (Grazia Cappiello et al., 2001). In contrast to Fc{gamma}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{gamma}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 Fc{gamma}RIIB1 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{gamma}RIIB1 as a model. We showed that MV-N binds to human Fc{gamma}RIIB1 and NR through NCORE and NTAIL, respectively. While Fc{gamma}RIIB1 interaction with NCORE triggered apoptosis, the aa 401–420 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.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Antibodies.
The monoclonal antibodies (mAbs) used were: biotinylated anti-MV-N Cl25 (Giraudon & Wild, 1981), biotinylated anti-MV-N Cl120 (Giraudon & Wild, 1981) and anti-FLAG (clone M2; Sigma-Aldrich). The blocking mouse mAb KB61 recognizing all human Fc{gamma}RII isoforms was kindly provided by D. Y. Mason (Pulford et al., 1986). Phycoerythrin (PE)-conjugated anti-human Fc{gamma}RII (C1KM5 clone; Caltag Laboratories) mAb was also used. The secondary antibody used was PE-conjugated goat F(ab')2 fragment anti-mouse IgG (PE–GAM) from Immunotech. Streptavidin–PE (Av–PE; Caltag Laboratories) and mouse IgG1 and IgG2 isotypic controls (Immunotech) were also used.

Cell lines.
The human melanoma cell line HT144, which does not express Fc{gamma}RII, was stably transfected with human Fc{gamma}RIIB1 cDNA (HT144IIB1) or with human Fc{gamma}RIIB1 cDNA lacking the intra-cytoplasmic tail (HT144IIB1/IC) (Cassard et al., 2002). The murine fibroblast L Orient cells transfected with human Fc{gamma}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) ml–1 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{alpha} (Stratagene) was used for selection and amplification of DNA constructs.

The NTAIL{Delta}1 and NTAIL{Delta}3 gene constructs encoded aa 421–525 and 401–516 of MV-N, respectively. The NTAIL{Delta}2,3 construct, previously referred to as NTAIL2 (Bourhis et al., 2004), encoded aa 401–488 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 ml–1) 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 Fc{gamma}RII.
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 ml–1) in the presence or absence of anti-Fc{gamma}RII mAb KB61 (10 µg ml–1) 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 Av–PE or PE–GAM for 30 min at 4 °C.

Competition experiments were performed using NTAIL deletion proteins as competitors. MV-N (2·5 µg; 25 µg ml–1) 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 Av–PE 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 cm–2 for 24 h in a volume of 700 µl cm–2. 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 cm–2 for 24 h in a volume of 700 µl cm–2 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 FITC–VAD-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 FITC–DEVD-FMK (BioVision), as described by the manufacturer. For the assessment of nuclear features of apoptosis, attached cells were stained with Hoechst 33342 (10 µg ml–1) for 30 min at 37 °C, as previously described (Valentin et al., 1999).


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
NCORE and NTAIL domains of MV-N bind to human Fc{gamma}RIIB1 and NR, respectively
MV-N is composed of NCORE and NTAIL domains (Fig. 1a). To investigate the MV-N domains responsible for binding to cell-surface receptors, we used either human melanoma HT144 cells expressing only NR or HT144IIB1 cells expressing both Fc{gamma}RIIB1 and NR as a model (Fig. 1b). As anti-MV-N mAb Cl120 reacted strongly with the cell surface of HT144IIB1 cells, anti-FLAG antibody was used for specific detection of MV-N and NCORE binding, while biotinylated Cl25 mAb was used to detect NTAIL (Fig. 1a). HT144 cells efficiently bound MV-N and NTAIL, but not NCORE, whatever amount was used (Fig. 1c, and data not shown), confirming that binding of NTAIL, but not of NCORE, occurs through human NR. These results indicated that NR expression is not restricted to normal cells but that it is also expressed on malignant cell lines (Laine et al., 2003). In addition, NTAIL binding to NR on HT144IIB1 cells did not affect Fc{gamma}RII expression, as judged by the superimposition of the histogram compared with the controls (data not shown). Conversely, HT144IIB1 cells bound MV-N, NTAIL and also NCORE, strongly suggesting that NCORE binding occurs through human Fc{gamma}RIIB1 (Fig. 1c). In most experiments, NCORE bound to HT144IIB1 to a lower extent than MV-N, even if large amounts of NCORE were used (68 µg per well; data not shown).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1. Binding of NCORE and NTAIL to human cell-surface Fc{gamma}RIIB1 and NR, respectively. (a) MV-N domain organization. N is divided into two domains, NCORE (aa 1–400) and NTAIL (aa 401–525). The anti-FLAG mAb (M2 clone) recognizes the FLAG sequence (black bar) at the N terminus of the FLAG fusion N proteins. The epitopes recognized by the anti-MV-N mAbs Cl120 (aa 133–140) and Cl25 (aa 466–474) are indicated. (b) Fc{gamma}RII expression on human HT144 cells, HT144 cells expressing human Fc{gamma}RIIB1 (HT144IIB1) and murine L cells expressing human Fc{gamma}RII (L-CD32) was determined by flow cytometry analysis. (c, d) MV-N, NCORE and NTAIL binding experiments were performed using HT144 (c, upper panels), HT144IIB1 (c, lower panels) and L-CD32 (d). Cells were incubated with 5 µg purified recombinant MV-N per well in the absence (heavy lines) or in the presence (thin lines) of mouse blocking mAb KB61. MV-N and NCORE binding were detected by either anti-FLAG (M2 clone) on HT144IIB1 (c) or by biotinylated (Biot) Cl120 on L-CD32 (d). Biotinylated Cl25 was used to detect NTAIL binding on all tested cell lines (c and d). Prior to flow cytometry analysis, M2 and biotinylated mAbs (Cl120 and Cl25) were revealed with PE–GAM and Av–PE, respectively. As a negative control, cells were incubated without MV-N in the presence of mAbs and secondary antibodies or conjugates (dashed lines). The results are representative of one of three independent experiments.

 
To confirm that NCORE interacts with Fc{gamma}RIIB1, we performed blocking experiments using anti-Fc{gamma}RII mAb KB61. Biotinylated Cl120 and Cl25 mAbs were used to detect NCORE and NTAIL binding, respectively (Fig. 1a). Therefore, we used L-CD32 cells (Fig. 1b), since biotinylated Cl120 mAb cross-reacted slightly with these cells. As illustrated in Fig. 1(d), L-CD32 cells efficiently bound MV-N, NTAIL and NCORE. As expected, the addition of KB61 mAb partially inhibited MV-N binding to L-CD32 cells (Fig. 1d). Interestingly, NCORE was responsible for binding to Fc{gamma}RII, as judged by the complete inhibition of binding to L-CD32 cells in the presence of KB61 mAb (Fig. 1d). The fact that NCORE binding to L-CD32 cells in the presence of the high-affinity Fc{gamma}RII inhibitory antibody KB61 was lower than that observed with the negative control (dashed line) may be accounted for by the cross-reactivity of Cl120 mAb with L-CD32 cells. Thus, the addition of KB61 mAb would block NCORE binding as well as non-specific binding of Cl120 mAb to L-CD32 cells. As expected, NTAIL binding to murine NR expressed on L-CD32 cells was not affected by KB61 mAb, as indicated by the superimposition of the histogram profiles obtained with and without anti-Fc{gamma}RII mAb (Fig. 1d). Similarly, KB61 mAb did not affect NTAIL binding to NR expressed on either HT144 or HT144IIB1 cells (data not shown). Moreover, L Orient cells bound MV-N and NTAIL, but not NCORE (data not shown). Taken together, these results indicated the presence of NCORE–Fc{gamma}RIIB1 and NTAIL–NR interactions.

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 Fc{gamma}RIIB1 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.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2. Effect of MV-N, NCORE and NTAIL on spontaneous human cell growth. (a, b) At 2 days post-treatment, the effects of MV-N, NCORE and NTAIL on HT144 (a) and HT144IIB1 (b) cell proliferation were determined by measuring [3H]thymidine incorporation. Cells were treated with increasing amounts of MV-N ({bullet}), NCORE ({blacksquare}) or NTAIL ({blacktriangleup}). Each value represents the mean±SD of triplicates. (c) Cell-cycle analysis of HT144 and HT144IIB1 cells by 7AAD/PY staining. HT144 (open bars) and HT144IIB1 (filled bars) cells were treated for 24 h with 34 µg N, 68 µg NCORE or 13 µg NTAIL per well in a 48-well plate. The increase in the percentage of G0/G1-arrested cells compared with untreated cells was evaluated and expressed as a percentage. Results represent the mean±SD of at least three independent experiments.

 
In contrast, NCORE, as MV-N and NTAIL, inhibited HT144IIB1 cell proliferation by up to 60 % (Fig. 2b). Thus, the apparent discrepancy in the inhibitory effect between NTAIL and MV-N was more pronounced in HT144 cells than in HT144IIB1 cells (Fig. 2a versus b). This phenomenon was due to cumulative binding effects, i.e. the fact that MV-N binds to both Fc{gamma}RIIB1 and NR in HT144IIB1, while it binds only to NR in HT144 cells. In all cases, the observed inhibition was dose dependent (Fig. 2a and b), indicating that inhibition of cell proliferation specifically relies upon either NCORE–Fc{gamma}RIIB1 or NTAIL–NR interactions.

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{gamma}RIIB1 and NR, respectively.

NCORE–Fc{gamma}RIIB1 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{gamma}RIIB1.



View larger version (122K):
[in this window]
[in a new window]
 
Fig. 3. Cellular morphology and apoptosis of HT144 and HT144IIB1 cells 24 h after treatment with MV-N, NCORE or NTAIL. HT144IIB cells were treated with 34 µg MV-N, 68 µg NCORE or 13 µg NTAIL per well in a 48-well plate. Control experiments were carried out using untreated cells. Photomicrographs were taken under a light microscope at the same original magnification (x400; Leica DM IRB). One representative experiment of three is shown. The percentage of attached and floating cells positive for activated pan-caspase was determined using FITC–VAD-FMK. Results correspond to the mean from at least three independent experiments (SD<15 %).

 
We next investigated whether MV-N and NCORE mediated apoptosis via Fc{gamma}RIIB1 by measuring caspase activation. Interestingly, HT144IIB1 cells underwent apoptosis when cultured with either MV-N (38·9 %) or NCORE (20·6 %), while NTAIL only slightly increased the level of apoptosis (5·9 %) compared with untreated cells (0·5 %; Fig. 3). Apoptosis of HT144IIB1 cells was also observed after CD32 cross-linking by Abs (AT10/CD32 plus rabbit anti-mouse, data not shown), suggesting that apoptosis resulted from clustering of Fc{gamma}RIIB1. A lower degree of apoptosis was observed in HT144 cells treated with MV-N and NTAIL (8·5 and 3·3 %; Fig. 3) compared with NCORE (1·6 %). These results strongly suggested that NCORE-induced Fc{gamma}RIIB1 clustering triggered apoptosis, while NR-mediated apoptosis appeared to provide a minor contribution. In order to demonstrate that NCORE-induced apoptosis was strictly dependent on Fc{gamma}RIIB1 clustering, we performed experiments on HT144 cells expressing Fc{gamma}RIIB1 but lacking its intra-cytoplasmic tail (HT144IIB1/IC). In these cells, no signal can be transduced via Fc{gamma}RII clustering. As expected, MV-N, NCORE and NTAIL bound to HT144IIB1/IC cells and the cells expressed Fc{gamma}RIIB1 at a level similar to that of HT144IIB1 cells (Fig. 4a, and data not shown). Interestingly, neither MV-N nor NCORE triggered significant caspase-3 activation in HT144IIB1/IC cells (Fig. 4b), even though in some cases NCORE-binding detection was higher than that currently observed (data not shown). In contrast, MV-N and NCORE induced 24·7 and 14·2 % caspase-3 activation in HT144IIB1 cells, respectively (Fig. 4b). As expected, HT144IIB1 cells underwent apoptosis following the addition of either MV-N or NCORE as shown by nuclear condensation and fragmentation, in contrast to HT144IIB1/IC cells (Fig. 5a). Whichever cell line was used, NTAIL was not toxic, even with doses as high as 68 µg per well, as judged by pan-caspase activation, caspase-3 activation and Hoechst staining (Fig. 4b, 5a, and data not shown).



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 4. Caspase-3 activation in HT144 cells expressing functional Fc{gamma}RIIB1 following incubation with MV-N, NCORE or NTAIL. (a) Binding of MV-N, NCORE and NTAIL to HT144IIB1 cells lacking the intra-cytoplasmic tail of Fc{gamma}RIIB1 (HT144IIB1/IC). Prior to flow cytometry analysis, MV-N, NCORE and NTAIL binding were detected as described in Fig. 1(c). One representative experiment out of three is shown. (b) Activated caspase-3 detection in HT144, HT144IIB1 and HT144IIB1/IC cells at 24 h post-treatment. Cells were treated with 34 µg MV-N, 68 µg NCORE or 13 µg NTAIL per well in a 48-well plate. Control experiments were carried out using untreated cells. The percentage of attached cells with activated caspase-3 was determined by flow cytometry analysis. Results are representative of one of two independent experiments.

 


View larger version (72K):
[in this window]
[in a new window]
 
Fig. 5. Effect of MV-N and NCORE on apoptosis of HT144IIB1 and HT144IIB1/IC cells. Cells were incubated in the absence (a) or in the presence (b) of the pan-caspase inhibitor zVAD-FMK (100 µM) for 1 h at 37 °C prior to incubation with 34 µg MV-N, 68 µg NCORE or 13 µg NTAIL per well in a 48-well plate. Control experiments were carried out using untreated cells. Apoptosis was measured by Hoechst staining after 24 h. Attached cells were observed using a Leica DM IRB microscope at a magnification of x400. White arrows indicate nuclear condensation and fragmentation. Data are representative of one experiment of two or three independent experiments.

 
To demonstrate further that caspase activation was involved in MV-N- and NCORE-induced apoptosis through interaction with Fc{gamma}RIIB1, cells were incubated in the presence of the pan-caspase inhibitor zVAD-FMK and nuclear morphology was analysed at day 1. In HT144IIB1 cells treated with MV-N and NCORE, zVAD-FMK totally inhibited apoptosis, and the nuclei of untreated or NTAIL-treated cells remained intact (Fig. 5b). Taken together, these data demonstrated that both MV-N and NCORE specifically trigger apoptosis via binding to Fc{gamma}RIIB1 in a caspase-3-dependent manner.

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 401–420, 489–506 and 517–525) (Diallo et al., 1994). To test this possibility, we purified three NTAIL deletion proteins carrying different combinations of such boxes, NTAIL{Delta}1, NTAIL{Delta}2,3 and NTAIL{Delta}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).



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 6. Mapping of the NR binding site within NTAIL. (a) Schematic representation of NTAIL and NTAIL deletion proteins. NTAIL{Delta}3, NTAIL{Delta}2,3 and NTAIL{Delta}1 mutants are devoid of Box3, Box2 plus Box3, and Box1, respectively. The three NTAIL deletion proteins contained a six-His tag fused to their N terminus (white boxes) and a C-terminal FLAG (black boxes). The {alpha}-helical molecular recognition element, i.e. the region involved in induced folding of NTAIL through binding to P (Bourhis et al., 2004), is indicated as an {alpha}-helix. (b) Purification profile of NTAIL deletion proteins from bacteria, analysed by 15 % SDS-PAGE and stained with Coomassie brilliant blue. TF, Bacterial lysate (total fraction); SN, clarified supernatant (soluble fraction); IMAC, eluent from IMAC. Molecular mass markers (kDa) are shown. (c) Binding of NTAIL deletion proteins to NR expressed on HT144 cells. Cells were incubated with 5 µg purified NTAIL deletion proteins per well for 1 h at 4 °C (thin lines). Binding was detected with biotinylated (Biot) Cl25 mAb and revealed with Av–PE prior to flow cytometry analysis. As a negative control, cells were incubated with biotinylated Cl25 mAb and Av–PE in the absence of NTAIL deletion proteins (dashed lines). The results are representative of one of three independent experiments. (d) Competition between MV-N and NTAIL deletion proteins. Cells were incubated with MV-N (2·5 µg per well, corresponding to 50 % binding) and increasing amounts of NTAIL deletion proteins. MV-N binding was then detected using mAb anti-N (Cl120) and Av–PE prior to flow cytometry analysis. Results are expressed as MFI of binding and are representative of one of three independent experiments. (e) Effect of NTAIL, NTAIL{Delta}3, NTAIL{Delta}2,3 and NTAIL{Delta}1 on spontaneous HT144 cell proliferation at 2 days post-treatment. Thymidine incorporation was determined as described in Methods. One representative experiment of three is presented.

 
We then compared the ability of the NTAIL{Delta}1, NTAIL{Delta}2,3 and NTAIL{Delta}3 deletion proteins to bind to NR. Biotinylated Cl25 mAb was used to detect specific binding of these deletion proteins to HT144 cells. As shown in Fig. 6(c), both NTAIL{Delta}3 and NTAIL{Delta}2,3 bound to NR, demonstrating that Box2 and Box3 are dispensable for NR binding. On the other hand, no detectable NTAIL{Delta}1 binding to NR was observed, even with amounts as high as 20 µg per well (data not shown), indicating that NTAIL binding strictly requires Box1. Similar results were obtained when binding of deletion proteins was revealed with anti-FLAG mAb (data not shown). Binding of NTAIL{Delta}3 and NTAIL{Delta}2,3, but not NTAIL{Delta}1, was also observed on both murine L and human thymic epithelial cell lines (data not shown). The intrinsic disorder of NTAIL rules out the possibility that the inability of NTAIL{Delta}1 to bind to cells may arise from structural changes induced by removal of Box1. We further performed competition experiments by co-incubating MV-N with increasing amounts of NTAIL deletion proteins. Binding of MV-N was then detected using biotinylated Cl120 mAb, specifically recognizing the NCORE domain. Both NTAIL{Delta}3 and NTAIL{Delta}2,3 significantly inhibited MV-N binding (19–76 %) in a dose-dependent manner, whereas NTAIL{Delta}1 failed to do so (Fig. 6d). Collectively, these results support the conclusion that the region responsible for specific binding to NR is located within aa 401–420 of MV-N.

Finally, we investigated whether NR engagement by the deletion proteins affected HT144 cell proliferation. As illustrated in Fig. 6(e), NTAIL{Delta}2,3 and NTAIL{Delta}3 deletion proteins inhibited human cell proliferation in a dose-dependent manner, although to a lower extent than NTAIL. As expected, NTAIL{Delta}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 401–420 region of MV-N is required for NR binding and, as a result, is responsible for cell proliferation inhibition.


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The mechanisms responsible for MV-induced immunosuppression are multifactorial and involved MV-infected immune cells, apoptosis of both MV-infected and uninfected cells, interference with cytokine synthesis, induction of soluble inhibitory factors and abnormalities in both antigen-presenting cell and lymphocyte functions (Gerlier et al., 2005; Schneider-Schaulies & ter Meulen, 2002). However, the overall frequency of infected peripheral blood lymphocytes is usually low during viraemia, supporting the notion that indirect mechanisms based on viral protein effects are mainly involved. Thus, the F–H complex binding to an as yet unknown receptor on uninfected T cells was shown to disrupt intracellular signalling pathways leading to the inhibition of lymphocyte proliferation (Avota et al., 2001). Likewise, MV-N exerts a suppressive activity after interaction with cell-surface receptors expressed on uninfected cells (Gerlier et al., 2005). In the present study, we have identified the MV-N domains involved in binding to human Fc{gamma}RIIB1 and NR, and shown that MV-N mediates either suppression of cell proliferation or apoptosis, depending on the type of receptor involved.

Our results point to an exclusive interaction of NCORE with Fc{gamma}RIIB1 and show that MV-N binds to Fc{gamma}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{gamma}RIIB1. Fc{gamma}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{gamma}RIIB1, rinderpest virus (RPV) N does not (Laine et al., 2003), thus supporting specific NCORE binding to Fc{gamma}RIIB1. The distinctive behaviour of RPV-N may be due to its unique sequence properties within the putative region of interaction with Fc{gamma}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 122–144 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{gamma}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{gamma}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 520–525) is conserved in MV NTAIL (aa 519–523) (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 401–420), 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 407–414 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 NTAIL–NR interaction in a human melanoma line, whereas apoptosis was mediated primarily by the NCORE–Fc{gamma}RIIB1 interaction in melanoma cells expressing Fc{gamma}RIIB1. Fc{gamma}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 NCORE–Fc{gamma}RIIB1 interaction on cell proliferation can be ascribed to B cells, while the NTAIL–NR 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{gamma}RIIB1 by NCORE triggers apoptosis via the intra-cytoplasmic tail of Fc{gamma}RIIB1. Previous data have already demonstrated that cross-linking of murine Fc{gamma}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{gamma}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 NTAIL–NR interaction, with MV, CDV and PPRV being also involved in NCORE–Fc{gamma}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{gamma}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{gamma}RIIB, as well as for cell-cycle manipulation of rapidly proliferating cells expressing NR.


   ACKNOWLEDGEMENTS
 
We thank Drs D. Gerlier, C. Servet-Delprat, Y. Zaffran and Y. Leverrier for their scientific advice during this work. We particularly thank Professor M. Oglesbee and Dr P. O. Vidalain for critical reading of the manuscript. The authors wish to thank C. Bella for expert technical assistance with the flow cytometry platform on IFR128. This work was supported in part by institutional grants from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche, INSERM, and by additional supports from ARC (CRC 5753) and the Programme de Recherche de la Région Rhône-Alpes (UR503-2A7-HHC02F). This study has also been carried out with financial support from the Commission of the European Communities, specific RTD programme (QLK2-CT2001-01225). D. L. was supported by a fellowship from MENRT (2000/2003) and ARC (2004).


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Avota, E., Avots, A., Niewiesk, S., Kane, L. P., Bommhardt, U., ter Meulen, V. & Schneider-Schaulies, S. (2001). Disruption of Akt kinase activation is important for immunosuppression induced by measles virus. Nat Med 7, 725–731.[CrossRef][Medline]

Bankamp, B., Horikami, S. M., Thompson, P. D., Huber, M., Billeter, M. & Moyer, S. A. (1996). Domains of the measles virus N protein required for binding to P protein and self-assembly. Virology 216, 272–277.[CrossRef][Medline]

Beckford, A. P., Kaschula, R. O. & Stephen, C. (1985). Factors associated with fatal cases of measles. A retrospective autopsy study. S Afr Med J 68, 858–863.[Medline]

Bourhis, J., Johansson, K., Receveur-Bréchot, V., Oldfield, C. J., Dunker, K. A., Canard, B. & Longhi, S. (2004). The C-terminal domain of measles virus nucleoprotein belongs to the class of intrinsically disordered proteins that fold upon binding to their physiological partner. Virus Res 99, 157–167.[CrossRef][Medline]

Buckland, R., Giraudon, P. & Wild, F. (1989). Expression of measles virus nucleoprotein in Escherichia coli: use of deletion mutants to locate the antigenic sites. J Gen Virol 70, 435–441.[Abstract]

Cassard, L., Cohen-Solal, J. F., Galinha, A. & 7 other authors (2002). Modulation of tumor growth by inhibitory Fc{gamma} receptor expressed by human melanoma cells. J Clin Invest 110, 1549–1557.[Abstract/Free Full Text]

Choi, K.-S., Nah, J.-J., Ko, Y.-J., Choi, C.-U., Kim, J.-H., Kang, S.-Y. & Joo, Y.-S. (2003). Characterization of antigenic sites on the rinderpest virus N protein using monoclonal antibodies. J Vet Sci 4, 57–65.[Medline]

Choi, K.-S., Nah, J.-J., Ko, Y.-J., Kang, S.-Y., Yoon, K.-J. & Joo, Y.-S. (2004). Characterization of immunodominant linear B-cell epitopes on the carboxy terminus of the rinderpest virus nucleocapsid protein. Clin Diagn Lab Immunol 11, 658–664.[Abstract/Free Full Text]

Cohen-Solal, J. F., Cassard, L., Fridman, W. H. & Sautes-Fridman, C. (2004). Fc {gamma} receptors. Immunol Lett 92, 199–205.[CrossRef][Medline]

Curran, J. & Kolakofsky, D. (1999). Replication of paramyxoviruses. Adv Virus Res 54, 403–422.[Medline]

Curran, J., Homann, H., Buchholz, C., Rochat, S., Neubert, W. & Kolakofsky, D. (1993). The hypervariable C-terminal tail of the Sendai paramyxovirus nucleocapsid protein is required for template function but not for RNA encapsidation. J Virol 67, 4358–4364.[Abstract]

Diallo, A., Barrett, T., Barbron, M., Meyer, G. & Lefevre, P. C. (1994). Cloning of the nucleocapsid protein gene of peste-des-petits-ruminants virus: relationship to other morbilliviruses. J Gen Virol 75, 233–237.[Abstract]

Etchart, N., Desmoulins, P. O., Chemin, K., Maliszewski, C., Dubois, B., Wild, F. & Kaiserlian, D. (2001). Dendritic cells recruitment and in vivo priming of CD8+ CTL induced by a single topical or transepithelial immunization via the buccal mucosa with measles virus nucleoprotein. J Immunol 167, 384–391.[Abstract/Free Full Text]

Fujinami, R. S., Sun, X., Howell, J. M., Jenkin, J. C. & Burns, J. B. (1998). Modulation of immune system function by measles virus infection: role of soluble factor and direct infection. J Virol 72, 9421–9427.[Abstract/Free Full Text]

Gerlier, D., Valentin, H., Laine, D., Rabourdin-Combe, C. & Servet-Delprat, C. (2005). Subversion of the immune system by measles virus: a model for the intricate interplay between a virus and the immune system. In Microbial Subversion of Host Immunity, pp. 1–81. Edited by P. Lachmman & M. B. Oldstone. Norfolk: Horizon Scientific Press.

Giraudon, P. & Wild, T. F. (1981). Monoclonal antibodies against measles virus. J Gen Virol 54, 325–332.[Abstract]

Giraudon, P., Jacquier, M. F. & Wild, T. F. (1988). Antigenic analysis of African measles virus field isolates: identification and localization of one conserved and two variable epitope sites on the NP protein. Virus Res 10, 137–152.[CrossRef][Medline]

Graves, M., Griffin, D. E., Johnson, R. T., Hirsch, R. L., de Soriano, I. L., Roedenbeck, S. & Vaisberg, A. (1984). Development of antibody to measles virus polypeptides during complicated and uncomplicated measles virus infections. J Virol 49, 409–412.[Medline]

Grazia Cappiello, M., Sutterwala, F. S., Trinchieri, G., Mosser, D. M. & Ma, X. (2001). Suppression of IL-12 transcription in macrophages following Fc{gamma} receptor ligation. J Immunol 166, 4498–4506.[Abstract/Free Full Text]

Griffin, D. E. (1995). Immune responses during measles virus infection. Curr Top Microbiol Immunol 191, 117–134.[Medline]

Hahm, B., Arbour, N., Naniche, D., Homann, D., Manchester, M. & Oldstone, M. B. (2003). Measles virus infects and suppresses proliferation of T lymphocytes from transgenic mice bearing human signaling lymphocytic activation molecule. J Virol 77, 3505–3515.[Abstract/Free Full Text]

Harty, R. N. & Palese, P. (1995). Measles virus phosphoprotein (P) requires the NH2- and COOH-terminal domains for interactions with the nucleoprotein (N) but only the COOH terminus for interactions with itself. J Gen Virol 76, 2863–2867.[Abstract]

Heaney, J., Barrett, T. & Cosby, S. L. (2002). Inhibition of in vitro leukocyte proliferation by morbilliviruses. J Virol 76, 3579–3584.[Abstract/Free Full Text]

Ilonen, J., Makela, M. J., Ziola, B. & Salmi, A. A. (1990). Cloning of human T cells specific for measles virus haemagglutinin and nucleocapsid. Clin Exp Immunol 81, 212–217.[Medline]

Jacobson, S., Sekaly, R. P., Jacobson, C. L., McFarland, H. F. & Long, E. O. (1989). HLA class II-restricted presentation of cytoplasmic measles virus antigens to cytotoxic T cells. J Virol 63, 1756–1762.[Medline]

Johansson, K., Bourhis, J. M., Campanacci, V., Cambillau, C., Canard, B. & Longhi, S. (2003). Crystal structure of the measles virus phosphoprotein domain responsible for the induced folding of the C-terminal domain of the nucleoprotein. J Biol Chem 278, 44567–44573.[Abstract/Free Full Text]

Karlin, D., Longhi, S. & Canard, B. (2002). Substitution of two residues in the measles virus nucleoprotein results in an impaired self-association. Virology 302, 420–432.[CrossRef][Medline]

Karlin, D., Ferron, F., Canard, B. & Longhi, S. (2003). Structural disorder and modular organization in Paramyxovirinae N and P. J Gen Virol 84, 3239–3252.[Abstract/Free Full Text]

Laine, D., Trescol-Biemont, M. C., Longhi, S. & 8 other authors (2003). Measles virus (MV) nucleoprotein binds to a novel cell surface receptor distinct from Fc{gamma}RII via its C-terminal domain: role in MV-induced immunosuppression. J Virol 77, 11332–11346.[Abstract/Free Full Text]

Liston, P., Batal, R., DiFlumeri, C. & Briedis, D. J. (1997). Protein interaction domains of the measles virus nucleocapsid protein (NP). Arch Virol 142, 305–321.[CrossRef][Medline]

Longhi, S., Receveur-Brechot, V., Karlin, D., Johansson, K., Darbon, H., Bhella, D., Yeo, R., Finet, S. & Canard, B. (2003). The C-terminal domain of the measles virus nucleoprotein is intrinsically disordered and folds upon binding to the C-terminal moiety of the phosphoprotein. J Biol Chem 278, 18638–18648.[Abstract/Free Full Text]

Malbec, O., Fridman, W. H. & Daeron, M. (1999). Negative regulation of hematopoietic cell activation and proliferation by Fc {gamma} RIIB. Curr Top Microbiol Immunol 244, 13–27.[Medline]

Marie, J. C., Kehren, J., Trescol-Biemont, M. C. & 8 other authors (2001). Mechanism of measles virus-induced suppression of inflammatory immune responses. Immunity 14, 69–79.[CrossRef][Medline]

McChesney, M. B., Kehrl, J. H., Valsamakis, A., Fauci, A. S. & Oldstone, M. B. (1987). Measles virus infection of B lymphocytes permits cellular activation but blocks progression through the cell cycle. J Virol 61, 3441–3447.[Medline]

McChesney, M. B., Altman, A. & Oldstone, M. B. (1988). Suppression of T lymphocyte function by measles virus is due to cell cycle arrest in G1. J Immunol 140, 1269–1273.[Abstract/Free Full Text]

Miller, D. L. (1964). Frequency of complications of measles, 1963. Report on a national inquiry by the Public Health Laboratory Service in collaboration with the Society of Medical Officers of Health. Br Med J 5401, 75–78.[Medline]

Mondal, B., Sreenivasa, B. P., Dhar, P., Singh, R. P. & Bandyopadhyay, S. K. (2001). Apoptosis induced by peste des petits ruminants virus in goat peripheral blood mononuclear cells. Virus Res 73, 113–119.[CrossRef][Medline]

Moss, W. J. & Polack, F. P. (2001). Immune responses to measles and measles vaccine: challenges for measles control. Viral Immunol 14, 297–309.[CrossRef][Medline]

Naniche, D., Reed, S. I. & Oldstone, M. B. (1999). Cell cycle arrest during measles virus infection: a G0-like block leads to suppression of retinoblastoma protein expression. J Virol 73, 1894–1901.[Abstract/Free Full Text]

Niewiesk, S., Eisenhuth, I., Fooks, A., Clegg, J. C., Schnorr, J. J., Schneider-Schaulies, S. & ter Meulen, V. (1997). Measles virus-induced immune suppression in the cotton rat (Sigmodon hispidus) model depends on viral glycoproteins. J Virol 71, 7214–7219.[Abstract]

Norrby, E. & Gollmar, Y. (1972). Appearance and persistence of antibodies against different virus components after regular measles infections. Infect Immun 6, 240–247.[Medline]

Okada, H., Kobune, F., Sato, T. A., Kohama, T., Takeuchi, Y., Abe, T., Takayama, N., Tsuchiya, T. & Tashiro, M. (2000). Extensive lymphopenia due to apoptosis of uninfected lymphocytes in acute measles patients. Arch Virol 145, 905–920.[CrossRef][Medline]

Okada, H., Sato, T. A., Katayama, A. & 8 other authors (2001). Comparative analysis of host responses related to immunosuppression between measles patients and vaccine recipients with live attenuated measles vaccines. Arch Virol 146, 859–874.[CrossRef][Medline]

Olszewska, W., Erume, J., Ripley, J., Steward, M. W. & Partidos, C. D. (2001). Immune responses and protection induced by mucosal and systemic immunization with recombinant measles nucleoprotein in a mouse model of measles virus-induced encephalitis. Arch Virol 146, 293–302.[CrossRef][Medline]

Pearse, R. N., Kawabe, T., Bolland, S., Guinamard, R., Kurosaki, T. & Ravetch, J. V. (1999). SHIP recruitment attenuates Fc{gamma}RIIB-induced B cell apoptosis. Immunity 10, 753–760.[CrossRef][Medline]

Pfeuffer, J., Puschel, K., Meulen, V., Schneider-Schaulies, J. & Niewiesk, S. (2003). Extent of measles virus spread and immune suppression differentiates between wild-type and vaccine strains in the cotton rat model (Sigmodon hispidus). J Virol 77, 150–158.[CrossRef][Medline]

Planz, O., Pleschka, S., Oesterle, K., Berberich-Siebelt, F., Ehrhardt, C., Stitz, L. & Ludwig, S. (2003). Borna disease virus nucleoprotein interacts with the CDC2–cyclin B1 complex. J Virol 77, 11186–11192.[Abstract/Free Full Text]

Pulford, K., Ralfkiaer, E., MacDonald, S. M., Erber, W. N., Falini, B., Gatter, K. C. & Mason, D. Y. (1986). A new monoclonal antibody (KB61) recognizing a novel antigen which is selectively expressed on a subpopulation of human B lymphocytes. Immunology 57, 71–76.[Medline]

Ravanel, K., Castelle, C., Defrance, T., Wild, T. F., Charron, D., Lotteau, V. & Rabourdin-Combe, C. (1997). Measles virus nucleocapsid protein binds to Fc{gamma}RII and inhibits human B cell antibody production. J Exp Med 186, 269–278.[Abstract/Free Full Text]

Ravetch, J. V. & Bolland, S. (2001). IgG Fc receptors. Annu Rev Immunol 19, 275–290.[CrossRef][Medline]

Realdon, S., Gerotto, M., Dal Pero, F., Marin, O., Granato, A., Basso, G., Muraca, M. & Alberti, A. (2004). Proapoptotic effect of hepatitis C virus CORE protein in transiently transfected cells is enhanced by nuclear localization and is dependent on PKR activation. J Hepatol 40, 77–85.[Medline]

Reth, M. (1989). Antigen receptor tail clue. Nature 338, 383–384.[Medline]

Sanchez-Lanier, M., Guerin, P., McLaren, L. C. & Bankhurst, A. D. (1988). Measles virus-induced suppression of lymphocyte proliferation. Cell Immunol 116, 367–381.[CrossRef][Medline]

Schlender, J., Schnorr, J. J., Spielhoffer, P., Cathomen, T., Cattaneo, R., Billeter, M. A., ter Meulen, V. & Schneider-Schaulies, S. (1996). Interaction of measles virus glycoproteins with the surface of uninfected peripheral blood lymphocytes induces immunosuppression in vitro. Proc Natl Acad Sci U S A 93, 13194–13199.[Abstract/Free Full Text]

Schneider-Schaulies, S. & ter Meulen, V. (2002). Modulation of immune functions by measles virus. Springer Semin Immunopathol 24, 127–148.[CrossRef][Medline]

Schobesberger, M., Summerfield, A., Doherr, M. G., Zurbriggen, A. & Griot, C. (2005). Canine distemper virus-induced depletion of uninfected lymphocytes is associated with apoptosis. Vet Immunol Immunopathol 104, 33–44.[CrossRef][Medline]

Servet-Delprat, C., Vidalain, P.-O., Valentin, H. & Rabourdin-Combe, C. (2003). Measles virus and dendritic cell functions: how specific response cohabits with immunosuppression. Curr Top Microbiol Immunol 276, 103–123.[Medline]

Stolte, M., Haas, L., Wamwayi, H. M., Barrett, T. & Wohlsein, P. (2002). Induction of apoptotic cellular death in lymphatic tissues of cattle experimentally infected with different strains of rinderpest virus. J Comp Pathol 127, 14–21.[CrossRef][Medline]

Sun, X., Burns, J. B., Howell, J. M. & Fujinami, R. S. (1998). Suppression of antigen-specific T cell proliferation by measles virus infection: role of a soluble factor in suppression. Virology 246, 24–33.[CrossRef][Medline]

tenOever, B. R., Servant, M. J., Grandvaux, N., Lin, R. & Hiscott, J. (2002). Recognition of the measles virus nucleocapsid as a mechanism of IRF-3 activation. J Virol 76, 3659–3669.[Abstract/Free Full Text]

Toba, K., Winton, E. F., Koike, T. & Shibata, A. (1995). Simultaneous three-color analysis of the surface phenotype and DNA–RNA quantitation using 7-amino-actinomycin D and pyronin Y. J Immunol Methods 182, 193–207.[CrossRef][Medline]

Tsubata, T. (1999). Co-receptors on B lymphocytes. Curr Opin Immunol 11, 249–255.[CrossRef][Medline]

Valentin, H., Azocar, O., Horvat, B., Williems, R., Garrone, R., Evlashev, A., Toribio, M. L. & Rabourdin-Combe, C. (1999). Measles virus infection induces terminal differentiation of human thymic epithelial cells. J Virol 73, 2212–2221.[Abstract/Free Full Text]

van Binnendijk, R. S., Poelen, M. C., de Vries, P., Voorma, H. O., Osterhaus, A. D. & Uytdehaag, F. G. (1989). Measles virus-specific human T cell clones. Characterization of specificity and function of CD4+ helper/cytotoxic and CD8+ cytotoxic T cell clones. J Immunol 142, 2847–2854.[Abstract/Free Full Text]

Wang, M., Libbey, J. E., Tsunoda, I. & Fujinami, R. S. (2003). Modulation of immune system function by measles virus infection. II. Infection of B cells leads to the production of a soluble factor that arrests uninfected B cells in G0/G1. Viral Immunol 16, 45–55.[CrossRef][Medline]

Yao, Z. Q., Eisen-Vandervelde, A., Ray, S. & Hahn, Y. S. (2003). HCV core/gC1qR interaction arrests T cell cycle progression through stabilization of the cell cycle inhibitor p27Kip1. Virology 314, 271–282.[CrossRef][Medline]

Zhang, X., Glendening, C., Linke, H., Parks, C. L., Brooks, C., Udem, S. A. & Oglesbee, M. (2002). Identification and characterization of a regulatory domain on the carboxyl terminus of the measles virus nucleocapsid protein. J Virol 76, 8737–8746.[Abstract/Free Full Text]

Received 26 November 2004; accepted 11 February 2005.