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Institute of Virology, Philipps University Marburg, Hans-Meerwein-Str. 2, D-35043 Marburg, Germany
Correspondence
Andrea Maisner
maisner{at}staff.uni-marburg.de
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ABSTRACT |
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Present address: Center for Infectious Medicine, Karolinska Institute, Stockholm, Sweden. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). During the course of an acute MV infection, many cell types, including polarized cells, are infected. As MV is transmitted via aerosols or droplets and replicates initially in the respiratory mucosa, polarized nasal or bronchial epithelial cells are among the first MV target cells. MV then enters local lymphatic tissues and spreads systemically through the lymphatic and blood systems. In the systemic phase of infection, monocytes and lymphocytes are the main target cells. Infected blood mononuclear cells are responsible for MV-induced transient immunosuppression (Schneider-Schaulies et al., 2001) and carry MV to various organs, such as skin, intestine, liver, lung and kidney, where different types of polarized cell are infected (Esolen et al., 1993; Osunkoya et al., 1990; Yanagi et al., 2006).
Polarized cells differ from non-polarized cells in their ability to segregate proteins and lipids into distinct surface subdomains accompanied by morphological and functional asymmetry, as occurs with the apical and basolateral surfaces in polarized epithelia and the axonal and dendritic processes in neurons (Rodriguez-Boulan & Powell, 1992). Lymphocytes can also develop a polarized phenotype if they carry out certain functions, such as cell–cell interactions or migration (Bretscher, 1996; Sanchez-Madrid & del Pozo, 1999). In migrating T cells, polarization involves the formation of a leading edge, which is enriched in receptors involved in recognition of chemokines, antigens and substrate-adhesion molecules (Negulescu et al., 1996; Nieto et al., 1997), and a trailing edge, termed the uropod (Campanero et al., 1994). The uropod selectively concentrates molecules involved in intercellular adhesion (del Pozo et al., 1995). Upon contact of T cells with other T cells or antigen-presenting cells, a characteristic polarized arrangement of molecules at cell–cell junctions, known as the immunological synapse, is induced (Grakoui et al., 1999). However, formation of synapse-like structures not only is required to respond effectively to antigenic challenge, but might also be important for the dissemination of lymphotropic retroviruses. For human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus (HTLV), cell-to-cell spread is believed to occur via a stable adhesive junction, the so-called virological synapse (Jolly & Sattentau, 2004). Thus, transient polarization of lymphocytes, similar to the permanent polarized nature of epithelia or neurons, is not only central to their physiological function, but also influences virus replication. Selective transport of viral surface and matrix proteins to a specific domain can critically determine cell-to-cell spread and targeted virus release from polarized cells (Danis et al., 2004; Deschambeault et al., 1999; Fuller et al., 1984; Lodge et al., 1997; Mora et al., 2002; Sanger et al., 2001; Tashiro et al., 1990; Zimmer et al., 2002).
MV, as a member of the family Paramyxoviridae, encodes two surface glycoproteins, the receptor-binding H protein and the fusion (F) protein. Both are required for virus entry, spread via cell-to-cell fusion and virus release. Functional virus assembly depends on the presence of the cytoplasmic domains of H and F and their interaction with the matrix (M) protein (Spielhofer et al., 1998). Furthermore, interaction of the F cytoplasmic tail was shown to be required for downregulation of MV-induced syncytium formation and cytopathogenicity (Cathomen et al., 1998a, b; Moll et al., 2002). We found previously that both MV surface glycoproteins F and H contain specific polarized sorting signals within their cytoplasmic domains, which mediate expression on the basolateral surface of epithelial cells upon both stable expression and infection with MVEdm. Targeted F and H expression is dependent on tyrosine residues in the cytoplasmic tails (Y549 in the F protein; Y12 in the H protein) and is of crucial importance for fusion of polarized epithelial cells. Mutations in the basolateral sorting signals prevent direct cell-to-cell spread in epithelial monolayers and thus compromise the ability of MV to overcome epithelial barriers, restricting virus spread in vitro and in vivo (Maisner et al., 1998; Moll et al., 2001, 2004).
As it has been shown for HIV that tyrosine-based targeting signals can also be of functional importance for the infection of lymphocytes (Deschambeault et al., 1999), we wanted to determine the impact of the basolateral sorting signals in the MV glycoproteins for propagation in lymphocytes, the main target cells during the systemic phase of infection. To study MV replication and glycoprotein targeting, lymphocytes were infected with recombinant MVs (rMVs) carrying mutations in the cytoplasmic tyrosines Y549 in the F protein and/or Y12 in the H protein (tyrosine mutants). Immunolocalization analysis in rMV-infected lymphocytes revealed that transport of F and H to the uropod of polarized lymphocytes, as well as clustering of the glycoproteins on the surface of non-polarized lymphocytes, are dependent on the cytoplasmic tyrosines. Interestingly, all tyrosine mutants had an enhanced fusion activity. rMVs carrying mutations in both glycoproteins displayed the most pronounced fusogenic phenotype and were barely released into the supernatant of infected lymphocytes. The finding that mutated glycoproteins have lost their marked colocalization with M on the surface of infected cells indicates that M–glycoprotein binding is disturbed by the tyrosine mutation in either the F or the H protein. As a consequence, M-mediated downregulation of fusion is reduced. In summary, our data indicate clearly that the cytoplasmic tyrosines in the MV glycoproteins, which are responsible for basolateral expression in polarized epithelia, also act as uropod-targeting signals in lymphocytes. Furthermore, they are involved in M–glycoprotein interaction, thereby regulating cell-to-cell fusion and virus propagation in lymphocytes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. PBLs and Jurkat cells (a human T-leukaemic cell line) were grown in RPMI 1640 medium supplemented with 10 % fetal calf serum (FCS), 100 U penicillin ml–1 and 100 µg streptomycin ml–1 (all from Gibco). B95a cells (an adherent marmoset B-cell line) and Vero cells (African green monkey kidney cells) were maintained in Dulbecco's modified minimal essential medium (DMEM; Gibco) containing 10 % FCS, penicillin and streptomycin. Madin–Darby canine kidney (MDCK) cells were grown in Eagle's minimal essential medium (MEM; Gibco) supplemented with 10 % FCS and antibiotics.
Recombinant MV Edmonston B (rMVEdm) and all rMV tyrosine mutants (rMVF549Y/A, rMVH12Y/A and rMVFHY/A) were rescued from cDNA, grown and titrated on Vero cells as described previously (Moll et al., 2004).
Immunostaining.
PBLs stimulated with 2.5 µg phytohaemagglutinin (PHA) ml–1 for 48 h and Jurkat cells, both grown in suspension, as well as adherent B95a and MDCK cells, were infected with the different rMVs at an m.o.i. of 0.5 for 2 h at 37 °C. After washing with PBS, cells were cultured in medium containing 10 % FCS together with a fusion-inhibitory peptide to prevent disruption of the cells by syncytium formation (Weidmann et al., 2000). As MV infection in Jurkat cells proceeds very fast, infected Jurkat cells were analysed at 1 day post-infection (p.i.), whereas PBLs and B95a and MDCK cells were processed at 2 days p.i. Before immunostaining, PBLs (48 h p.i.) and Jurkat cells (21 h p.i.) were seeded onto fibronectin-coated coverslips (BD BioCoat) and incubated for 1.5 h at 37 °C to allow uropod formation. Then, the cells were fixed with 4 % paraformaldehyde for 10 min at room temperature and subsequently blocked with DMEM containing 10 % FCS for 1 h at 4 °C. To visualize MV glycoproteins on the cell surfaces, cells were incubated with F- or H-specific mAbs (A504 and K83, kindly provided by S. and J. Schneider-Schaulies, Institut für Virologie und Immunbiologie, Universität Würzburg, Germany) for 1 h at 4 °C and rhodamine-conjugated goat anti-mouse IgG (Dianova) for 45 min at 4 °C. For M–H costaining, cells were incubated with an H-specific rabbit anti-MV serum (anti-Hc, kindly provided by R. Cattaneo, Mayo Clinic College of Medicine, Rochester, MN, USA) for 1 h at 4 °C. The primary antibody was detected by incubation with rhodamine-conjugated anti-rabbit IgG (Dako) for 45 min at 4 °C. After treating the cells with methanol : acetone (1 : 1) for 5 min at room temperature, the M protein was labelled with the M-specific mAb 8910 (Chemicon) for 1 h at 4 °C and fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Dako) for 45 min at 4 °C. For F–M costaining, cells were incubated with anti-F antibody (A504) and rhodamine-conjugated secondary antibody, followed by permeabilization, blocking with 5 % normal mouse serum and incubation with an FITC-labelled anti-M antibody as described previously (Moll et al., 2002). After immunostaining, cells were mounted in Mowiol (Merck) and 10 % 1.4 diazabicyclo(2.2.2)octane (Sigma), and fluorescence images were recorded by using a Zeiss ApoTome/Axiovert 200M microscope.
Fusion assay.
To analyse fusion activity of the different rMVs, 5x105 Jurkat cells were infected in suspension with rMVEdm, rMVF549Y/A, rMVH12Y/A or rMVFHY/A at an m.o.i. of 0.01. After incubation for 2 h, cells were washed and incubated with RPMI 1640 medium containing 10 % FCS at 37 °C. To study cell-to-cell fusion in adherent B95a cells, the cells were grown to confluence in 24-well plates (2.5x105 cells), then infected with the different rMVs at an m.o.i. of 0.05 and maintained in DMEM containing 10 % FCS at 37 °C. rMV-infected cells were monitored regularly for syncytium formation by phase-contrast microscopy.
Growth analysis.
Virus growth was analysed by infecting 2.5x105 Jurkat cells with rMVEdm, rMVF549Y/A, rMVH12Y/A or rMVFHY/A at an m.o.i. of 0.01 for each time point of analysis. After 2 h, cells were washed to remove unbound viruses and were cultured in 1 ml RPMI 1640 medium containing 10 % FCS at 37 °C. Every 12 h, cells were pelleted by low-speed centrifugation, and cell-free rMVs in the supernatant were titrated by plaque assay. Dilutions of the cell supernatant were adsorbed to Vero cells for 2 h, then the inoculum was removed and cells were overlaid with MEM containing 2 % FCS and 0.9 % Bacto Agar (BD). After 4 days, the plaques were stained with 0.0125 % neutral red (Merck) and counted.
In vitro proliferation assay.
Human PBLs were stimulated with PHA (2.5 µg ml–1) for 48 h and subsequently infected for 2 h with rMVEdm, rMVF549Y/A, rMVH12Y/A or rMVFHY/A at an m.o.i. of 0.5, or were left uninfected (mock). After several washings, 105 cells were seeded into a 96-well plate in a volume of 200 µl per well and incubated in the presence of PHA for 72 h. Cells were then labelled for 16 h with [3H]thymidine [18.5 kBq (0.5 µCi) ml–1]. Incorporation rates of 3H were determined by using a β-plate reader. The assay was performed in triplicate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). For immunodetection of the MV glycoproteins on the surface of rMV-infected cells, Jurkat cells and PBLs were adsorbed to fibronectin-coated coverslips at 21 and 48 h p.i., respectively. As a polarized phenotype can be induced in vitro upon adhesion to extracellular matrix components (Johansson et al., 1997), about 5–10 % of the lymphocytes form uropods under these conditions. After 1.5 h, Jurkat cells and PBLs adsorbed to coverslips were fixed with paraformaldehyde. Infected B95a cells were fixed directly at 48 h p.i. To visualize MV glycoproteins on the plasma membrane, the infected lymphocytes were incubated with anti-F protein- or anti-H-specific mAbs and rhodamine-conjugated secondary antibodies. Fig. 1 depicts the surface distribution of F and H in polarized (Fig. 1a, b) and non-polarized (Fig. 1c, d) lymphocytes. In rMVEdm-infected polarized Jurkat cells (Fig. 1a) and PBLs (Fig. 1b), standard MV glycoproteins were clearly localized at the uropod, whereas the F protein in rMVF549Y/A-infected cells, as well as the H protein in rMVH12Y/A-infected cells, were no longer concentrated at this trailing edge. In rMVFHY/A-infected cells, both MV glycoproteins were distributed more or less homogeneously all over the cell surface. This demonstrates clearly that the tyrosine-based signals in the MV glycoproteins are not only required for polarized transport in epithelia, but also act as uropod-targeting signals in polarized lymphocytes. Interestingly, the cytoplasmic tyrosines not only affect protein localization in polarized cells, but also influence H and F distribution in non-polarized lymphocytes. In both non-polarized, spherical Jurkat cells (Fig. 1c) and adherent B95a cells (Fig. 1d) infected with standard rMVEdm, F and H accumulated in large aggregates on the cell surface. In cells infected with rMV tyrosine mutants, mutated glycoproteins displayed a punctate distribution (F in rMVF549Y/A- and H in rMVH12Y/A-infected cells), whereas non-mutated glycoproteins were still found in large aggregates. In rMVFHY/A-infected cells, neither H nor F accumulated in larger clusters at the cell surface. The finding that glycoprotein distribution in Jurkat and B95a cells was changed similarly indicates that the effect of the tyrosine mutations on glycoprotein clustering at surface membranes of lymphocytes is not restricted to a certain subset of lymphocytes.
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). In both cell lines, the fusion capacity of the mutants rMVF549Y/A and rMVH12Y/A was enhanced in comparison with that of standard rMVEdm, and syncytium formation was most pronounced in rMVFHY/A-infected cells. To determine the mean size of syncytia induced by the different rMVs in B95a cells, the number of nuclei in 12 randomly chosen syncytia of each sample was counted and averaged. Syncytia induced by rMVEdm, rMVF549Y/A, rMVH12Y/A and rMVFHY/A contained on average 88, 219, 227 and 390 nuclei, respectively. Thus, the single tyrosine mutants were 2.5-fold more fusogenic than standard rMVEdm, demonstrating that mutation in only one glycoprotein already increases viral fusogenicity. rMVFHY/A was 4.4-fold more fusogenic than rMVEdm, showing that the combination of two tyrosine-mutated MV glycoproteins has an additive effect on fusion enhancement, generating a highly cytopathic virus.
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). rMVF549Y/A and rMVH12Y/A did not show a prominent difference in virus release compared with standard rMVEdm. Maximal virus titres were reached between 36 and 48 h p.i., and infected Jurkat cells constantly produced high amounts of infectious virus over at least 3 days. In contrast, growth of rMVFHY/A was clearly impaired. Maximal virus release was also observed at 36 h p.i., but the titre was 10-fold lower (8x104 p.f.u. ml–1) and dropped rapidly below 104 p.f.u. ml–1. The defective virus propagation in rMVFHY/A-infected cells is probably due to the extensive syncytium formation and the concomitant, pronounced cytopathic effect in these cells, which presumably interferes with virus protein synthesis and functional virus assembly and budding at late time points of infection. Surprisingly, the enhanced fusion capacity of rMVs with only one mutated glycoprotein (rMVF549Y/A, rMVH12Y/A) had no measurable effect on multi-step virus propagation in lymphocytes. In PBLs, the growth properties of the different rMVs were essentially the same as in Jurkat cells (Fig. 3b). This indicates that the tyrosine-dependent transport signals are of similar importance for virus propagation in lymphocytic cell lines and in primary lymphocytes.
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). To assess whether rMV tyrosine mutants affect lymphocyte proliferation differently, infected primary human PBLs were subjected to a [3H]thymidine incorporation proliferation assay in vitro (Fig. 3c
). In agreement with data published by Schlender et al. (1996), rMVEdm showed a dramatic proliferation inhibition of 76.7 % (Fig. 3c). The single tyrosine mutants (rMVH12Y/A and rMVF549Y/A) induced a similar inhibitory effect (inhibition of 67.7 and 81.5 %, respectively), whereas the double mutant rMVFHY/A caused a much less pronounced proliferation inhibition (38.6 %). This indicates that the less efficient growth of the hyperfusogenic rMVFHY/A is accompanied by a reduced inhibitory effect on contact-mediated proliferation.
Colocalization of M and the MV glycoproteins is affected by the tyrosine mutations
By using rMVs with tail-truncated glycoproteins, it has been shown that MV-induced cell-to-cell fusion is critically dependent on the interaction of F and/or H with the M protein. Binding to the glycoprotein cytoplasmic tails is obviously required for M-mediated downregulation of the glycoprotein-dependent fusion process (Cathomen et al., 1998b; Moll et al., 2002). Thus, increased syncytium formation of our rMV tyrosine mutants might be a result of deficient glycoprotein–M interaction. In order to evaluate this idea, colocalization of M and the mutated glycoproteins on the surfaces of infected cells was analysed. As the protocol for double immunostaining has been established previously for infected MDCK cells (Moll et al., 2002), colocalization studies were performed in MDCK, B95a and Jurkat cells infected with standard rMVEdm or mutants rMVH12Y/A, rMVF549Y/A or rMVFHY/A. Immunofluorescence analyses of MDCK and B95a cells were performed at 48 h p.i. and Jurkat cells were examined at 22.5 h p.i. For costaining of the F and M proteins, cell surfaces were labelled with an anti-F mAb and a rhodamine-conjugated secondary antibody. To visualize the M protein, cells were permeabilized with methanol : acetone, blocked with normal mouse serum and subsequently incubated with FITC-labelled anti-M antibodies. For H and M costaining, H proteins were labelled on the cell surfaces with a rabbit anti-H serum and M proteins were labelled after permeabilization with an anti-M mAb. Primary antibodies were then detected by rhodamine-conjugated anti-rabbit and FITC-conjugated anti-mouse sera. In Fig. 4, merged pictures of the rhodamine and FITC channels are shown. M–glycoprotein colocalization is thus indicated by a yellow colour. In Fig. 4(a), the overlay of the F+M and the H+M staining in MDCK cells is shown. As the results for the adherent B95a cells were found to be essentially the same, only H+M costaining is shown exemplarily in these cells (Fig. 4b). Fig. 4(c) depicts the double staining of F+M and H+M in Jurkat cells. In all three cell lines infected with standard rMVEdm, both MV glycoproteins accumulated in large aggregates on the cell surface and colocalized completely with the M protein. In contrast, neither F and M in rMVF549Y/A-infected cells nor H and M in rMVH12Y/A-infected cells showed a marked colocalization. In cells infected with rMVFHY/A, neither of the two MV glycoproteins colocalized markedly with the M protein. This result indicates clearly that both MV glycoproteins interact individually with the M protein and that the interaction depends on functional tyrosine residues in the cytoplasmic domains. Thus, the observed differences in the fusion activity of the rMV tyrosine mutants are probably due to defective M–glycoprotein interactions, interfering with fusion downregulation by the M protein.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Hyypia et al., 1985; Lawrence et al., 2000; Mrkic et al., 2000; Udem, 1984; van Binnendijk et al., 1994). The results presented in this paper reveal a critical role of the cytoplasmic tyrosines in the MV F and H glycoproteins for virus transmission from infected to uninfected lymphocytes, not only because they are required for localization in uropods, the cell pole involved in cell–cell interactions, but also because they mediate binding to the M protein, which downregulates H- and F-mediated cell-to-cell fusion, thereby preventing rapid syncytium formation and cell damage.
In contrast to epithelial cells, lymphocytes polarize only transiently, for example upon direct cell–cell contact with other lymphocytes or antigen-presenting cells, or in response to soluble factors such as chemokines (Krummel & Macara, 2006; Vicente-Manzanares & Sanchez-Madrid, 2004). If lymphocytes have acquired a polarized or migrating phenotype, proteins and lipids are delivered specifically to one cell domain (del Pozo et al., 1997; Gomez-Mouton et al., 2001; Sanchez-Madrid & del Pozo, 1999). However, whilst protein sorting to the apical or basolateral domain in epithelial cells is well-characterized, not much is known about polarized transport to the leading edge or the uropod of lymphocytes. As lymphocyte migration is accompanied by extensive rearrangements and polarization of microtubules and the actin cytoskeleton (Krummel & Macara, 2006), one determinant for localization at one cell pole is binding to actin-associated proteins, such as proteins of the ezrin–radixin–moesin family, which are located specifically at the uropod (del Pozo et al., 1997; Serrador et al., 1998). In addition, interaction with proteins of the polarity network, e.g. Scrib, Lgl, Dlg and PAR, or specific recruitment into detergent-insoluble, glycolipid-enriched membrane domains (rafts) may account for polarized protein localization. It has been shown that rafts are essential for the generation, maintenance and functionality of T-cell anteroposterior polarity and that acquisition of a migrating phenotype in T lymphocytes results in the asymmetrical redistribution of ganglioside GM3- and GM1-enriched raft domains to the leading edge and to the uropod, respectively (Gomez-Mouton et al., 2001; Krummel & Macara, 2006; Millan et al., 2002). Also, viral proteins can be transported selectively to one subdomain in lymphocytes (Danis et al., 2004; Millan et al., 2002). For example, localization of the influenza virus haemagglutinin (HA) at the uropod has been linked to its raft association (Millan et al., 2002). In contrast to HA, the MV F protein has only a weak intrinsic ability to associate with rafts, and MV H is supposed to be recruited into these membrane domains exclusively via its interaction with the F protein. Only 15–40 % of F or H proteins are located within rafts (Manie et al., 2000; Vincent et al., 2000). Thus, raft association is probably not the cause of the uropod localization of MV F and H observed in this study. The fact that mutations in the cytoplasmic tyrosines responsible for basolateral transport in polarized epithelial cells prevented concentration in uropods rather indicates that these residues also serve as transport signals in polarized lymphocytes. A similar mechanism might account for the localization of the Env protein of HIV-1, a basolateral protein that also localizes in uropods (Lodge et al., 1997; Nguyen & Hildreth, 2000). It can therefore be assumed that lymphocytes possess a pathway of transport to the uropod reminiscent of that used for its specific targeting to the basolateral surface of epithelial cells, probably involving cellular adaptor proteins recognizing tyrosine-containing motifs in the cytoplasmic domain of membrane proteins (Bonifacino & Dell'Angelica, 1999; Rodriguez-Boulan et al., 2005).
As lymphocytes recruit bystander cells through their uropod (del Pozo et al., 1997), the localization of F and H at one cell pole might assure rapid and efficient binding to the receptor on the uninfected neighbouring cell, thereby facilitating directed virus transmission. Similar to MV, spread of HTLV-1 is also dependent on direct cell contacts, because naturally infected lymphocytes produce very few cell-free virions. It has been shown that HTLV-1 transmission among CD4+ T cells occurs via a virological synapse, defined as a cytoskeleton-dependent, stable adhesive junction across which virus is transmitted by directed transfer (Igakura et al., 2003). The same method of propagation has also been described for HIV-1 spread between T cells or between dendritic cells and T cells (Jolly et al., 2004; McDonald et al., 2003; Turville et al., 2004). Like the Env proteins of HIV-1 and HTLV-1, the MV envelope proteins F, H and M concentrate at the contact sides between infected and uninfected Jurkat cells (N. Runkler, unpublished data). Furthermore, we recently observed a relocalization of the microtubule-organizing centre to these cell-contact sides, similar to what has been reported for the virological synapse in HTLV-1-infected cells (Igakura et al., 2003). This suggests strongly that MV is also transmitted via a virological synapse. Directed budding of virus into the synaptic cleft would not only facilitate virus transfer into uninfected target cells, but would also protect against neutralization by antibodies or the complement system.
Lymphopenia, cytokine imbalance and the inability of PBLs to expand in response to polyclonal or antigen-specific stimulation ex vivo are hallmarks of generalized immunosuppression caused by MV (Borrow & Oldstone, 1995). Several studies have shown that T cells can no longer proliferate in response to antigenic stimulation after contact with MV particles or MV-infected cells carrying F and H glycoproteins on the surface. Interaction of the MV glycoprotein complexes with uninfected T cells interferes with activation of the PI3/Akt kinase pathway and rearrangements of the cortical actin cytoskeleton, thus perturbing the ability of T cells to adhere, spread and cluster receptors essential for sustained T-cell activation (Avota et al., 2001; Muller et al., 2006; Schlender et al., 1996). As localization of both MV glycoproteins at cell-contact sides is required for T-cell silencing, changes in the H and F surface distribution might influence not only virus dissemination from infected lymphocytes, but also MV-induced immunosuppression. This idea is clearly supported by the finding that rMVFHY/A had a reduced inhibitory effect on PBL proliferation (Fig. 3c).
For efficient MV assembly, all virus components must interact specifically with each other at the plasma membrane. The M protein is known to play the key role during the assembly and budding process (Cathomen et al., 1998a; Peebles, 1991), because it mediates the contact between the outer surface glycoproteins and the inner nucleocapsids. We have shown very recently that M is required for nucleocapsid transport from intracellular inclusions to the plasma membrane (Runkler et al., 2007). At the inner side of surface membranes, M is able to form large aggregates by self-aggregation, and budding is induced after recruiting the glycoproteins via M binding to the F and H cytoplasmic tails. For functional assembly, the glycoproteins must thus colocalize in M clusters at the plasma membrane. Here, we demonstrated that this colocalization depends critically on one amino acid, the cytoplasmic tyrosine in the H and F proteins. The importance of a tyrosine motif in the glycoprotein cytoplasmic tail has also been proposed for Sendai virus. Here, binding of the HN glycoprotein to M depends on an SYWST motif (Takimoto et al., 1998). In agreement with the independent binding of each MV glycoprotein to M, cell-to-cell fusion of infected and uninfected lymphocytes is enhanced if only one MV glycoprotein fails to colocalize with M clusters at the cell surface. As it is generally assumed that interaction of the glycoproteins with large M aggregates lowers the lateral mobility of F and H in the plasma membrane of the infected cell, thereby downregulating the formation of active fusion complexes, as well as accumulation of these complexes at the sites of cell-to-cell fusion (Henis et al., 1989), defective interaction of one MV glycoprotein with M probably increases lateral mobility. This effect is synergistic if both glycoproteins are mutated, as demonstrated by dramatically enhanced syncytium formation in rMVFHY/A-infected cells. As virus release, in contrast to fusogenic properties, is not changed in rMVF549Y/A-infected and rMVH12Y/A-infected cells, the virus-assembly process is obviously not disturbed markedly if only one glycoprotein has lost its ability to bind stably to M clusters. A negative effect on virus release was only found in rMVFHY/A-infected cells, which is probably due to the extensive cytopathic properties of this virus, destroying the infected cell before new virions can be assembled. Supporting this view, we found that the amount of cell-associated virus in rMVFHY/A-infected cells was reduced similarly to the virus titres in the supernatant (N. Runkler, unpublished data). Thus, reduced virus release is accompanied by lower intracellular virus production. However, the possibility that the simultaneous lack of M interaction with both glycoproteins has a direct negative effect on virus assembly by affecting the amount of surface glycoproteins incorporated into budding virions, thereby decreasing the infectivity of the cell-free viruses, cannot be excluded completely.
Our data emphasize the critical role of the tyrosine residues in the cytoplasmic tails of both MV glycoproteins for efficient MV propagation. These residues not only account for basolateral glycoprotein expression in polarized epithelia, thus allowing fusion of infected epithelial cells with neighbouring or underlying cells, helping the virus to overcome epithelial barriers, but they are also responsible for F and H transport to one pole of polarized lymphocytes, the main MV target cells in acute MV infection. Accumulation at cell-contact sides probably allows direct virus transfer to uninfected cells, perhaps via a virological synapse. As the cytoplasmic tyrosine residues also mediate interaction with the M protein, cell-to-cell fusion is limited, and virus assembly and propagation are not prevented by an overshooting cytopathic effect.
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ACKNOWLEDGEMENTS |
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Received 29 August 2007;
accepted 28 November 2007.
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, rMVEdm; 
, rMVF549Y/A; 
, rMVH12Y/A; 
, rMVFHY/A. The values plotted represent mean results from two experiments. (c) PHA-stimulated PBLs were infected with rMVs at an m.o.i. of 0.5 and activated with PHA (2.5 µg ml–1). Proliferative activity was determined after 72 h by labelling with [3H]thymidine for 16 h and is indicated in relation to uninfected, PHA-stimulated PBLs (mock).