1 Institute of Virology, Philipps University Marburg, Robert-Koch-Str. 17, 35037 Marburg, Germany
2 Institute of Virology and Immunology, University of Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany
Correspondence
Andrea Maisner
maisner{at}staff.uni-marburg.de
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ABSTRACT |
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Present address: Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210, USA.
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INTRODUCTION |
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However, recent data on the release of measles virus (MV), a member of the family Paramyxoviridae, have indicated that budding polarity does not necessarily play a key role in virus spreading. As for all Paramyxoviridae, MV entry and initial replication occur in the respiratory mucosa and the virus is released apically from respiratory epithelial cells (Blau & Compans, 1995). However, in contrast to most other paramyxoviruses, which cause only local respiratory infections, MV disseminates to draining lymph nodes via infected macrophages and replicates in local lymphatic tissues followed by systemic spread of infection (Griffin & Bellini, 1996
; Moench et al., 1988
; Roscic-Mrkic et al., 2001
). This clearly indicates that MV has developed a strategy allowing systemic dissemination without direct virus release from infected respiratory epithelia to underlying tissues. Furthermore, in contrast to the model described above, apical MV release is not the result of an apical expression of the MV envelope proteins but is due to a restricted expression of the viral matrix protein (Naim et al., 2000
; Riedl et al., 2002
). We have reported previously that, despite apical virus release, the two MV surface glycoproteins, the fusion (F) protein and haemagglutinin (H), are abundantly expressed on the basolateral surface of polarized epithelia. In addition, we have demonstrated that both glycoproteins contain a tyrosine-based sorting signal in their cytoplasmic domains, which is required for basolateral protein expression and for the fusogenic activity of F/H complexes in polarized cell cultures (Maisner et al., 1998
; Moll et al., 2001
). This led us to suggest a functional role for the glycoproteins in mediating virus spread from epithelia by direct cell-to-cell fusion rather than in virus assembly.
The aim of this study was to evaluate the influence of basolateral glycoprotein targeting for the spread of MV infection from epithelial cells. We generated infectious MV from cloned cDNA without basolateral targeting signals in one or both glycoproteins (tyrosine mutants) and characterized their mode of propagation in polarized cell cultures and in vivo. For in vivo studies, we utilized cotton rats (Sigmodon hispidus) as an animal model system because they are susceptible to respiratory infection with different MV wild-type and vaccine strains (Niewiesk et al., 2000; Pfeuffer et al., 2003
; Wyde et al., 1992
). In infected cotton rats, replication of all cloned MV with replacements of tyrosine residues in the cytoplasmic tails of the glycoproteins appeared to be restricted compared with parental MV. By in situ hybridization analysis, we were able to demonstrate that only parental MV encoding basolaterally expressed F and H proteins could spread laterally within the respiratory epithelium and efficiently to cells in the subepithelial tissue. These data support our concept that, in the absence of basolateral virus budding, the basolateral targeting of both MV glycoproteins allows a more efficient spread of infection from polarized epithelial cells, most likely facilitating the systemic dissemination in vivo.
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METHODS |
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Primary tracheal cells of cotton rats were isolated by trypsination from tracheas removed from CO2-asphyxiated cotton rats and were grown in DMEM supplemented with 10 % fetal calf serum and antibiotics.
Plasmids and virus rescue.
For the generation of recombinant MV (rMV), the plasmid p(+)MVNSe (Singh et al., 1999) carrying the full-length MV Edmonston B-based genome was used. To construct the plasmid p(+)MVF549Y/A, the F gene was mutagenized in the shuttle vector peF1 (Radecke et al., 1995
). A NarIPacI fragment containing the altered F gene was subcloned into p(+)MVNSe. Plasmids p(+)MVH12Y/A and p(+)MVFHY/A were generated by ligation of a PacISpeI fragment containing the mutated H gene from the plasmid pCG-H12Y/A (Moll et al., 2001
) into p(+)MVNSe and p(+)MVF549Y/A, respectively. The sequences of all constructs were confirmed by dideoxy sequencing.
Recombinant MV Edmonston B (rMVEdm) and all mutants were rescued as described previously (Moll et al., 2002). Briefly, 293-3-46 cells mediating both artificial T7 transcription and NP and P functions were transfected with 5 µg of either p(+)MVNSe, p(+)MVF549Y/A, p(+)MVH12Y/A or p(+)MVFHY/A and 10 ng of plasmid encoding the MV polymerase (pEMC-La). The cultures were monitored daily for appearance of syncytia. Virus stocks were produced following plaque purification. RT-PCR and subsequent dideoxy sequencing confirmed the identity of the rescued viruses.
Syncytia formation and growth analysis.
To analyse fusion activity and growth of rMV in polarized cells, MDCK cells were infected as described above and seeded either on coverslips at high density or on 1 µm pore size tissue culture inserts (Falcon). At 48 h post-infection (p.i.), cells on coverslips were immunostained to visualize syncytium formation. After fixation and permeabilization, cells were incubated with monoclonal antibody (mAb) 8905 (directed against MV H protein; Chemicon) and FITC-labelled goat anti-rabbit IgG (Dako). Coverslips were mounted with Mowiol and analysed using an Axiomat fluorescence microscope (Zeiss). Virus release from filter-grown MDCK cells was determined by taking samples of apical supernatants at different times p.i., with the first sample collected immediately after washing of the filter membranes at 24 h p.i. Virus titres were determined by plaque assay.
Domain-selective surface biotinylation and immunoprecipitation.
MDCK cells were infected with rMV and seeded on 0·4 µm pore size Transwell polycarbonate filters (Costar). At 48 h p.i., cells were washed three times with PBS and either the apical or the basolateral side of the filter membranes was incubated twice for 20 min at 4 °C with PBS containing 2 mg S-NHS-Biotin ml-1 (Calbiochem). Glycine (0·1 M) was added to the opposite membranes. After washing the cells once with 0·1 M glycine and three times with PBS, filter membranes were cut out and lysed in 0·5 ml radioimmunoprecipitation assay buffer (1 % Triton X-100, 1 % sodium deoxycholate, 0·1 % SDS, 0·15 M NaCl, 10 mM EDTA, 10 mM iodoacetamide, 1 mM PMSF, 50 units aprotinin ml-1 and 20 mM Tris/HCl, pH 8·5). Cell lysates were clarified by centrifugation for 20 min at 100 000 g. Supernatants were immunoprecipitated using F- (mAb A540, kindly provided by J. Schneider-Schaulies) or H-specific (mAb 8905; Chemicon) mAbs and protein ASepharose beads (Sigma). Following SDS-PAGE and blotting on to nitrocellulose, biotinylated proteins were detected with streptavidinbiotinylated horseradish peroxidase complex (Amersham Pharmacia Biotech) and enhanced chemiluminescence (Amersham).
Cotton rats.
Cotton rats (inbred strain COTTON/NIco) were obtained from Iffa Credo (Lyon, France). Animals at 6 weeks of age of both sexes were used. The animals were pathogen free according to the breeder's specifications and were maintained in a barrier system. They were kept under controlled environmental conditions of 22±1 °C, 5560 % humidity and a 12 h light cycle.
Animal infections and virus titration.
For intranasal infection, MV was given in PBS in a volume of not more than 100 µl to ether-anaesthetized cotton rats. At different times p.i., animals were killed using CO2 and lungs were removed. Lung tissue was minced with scissors and dounced with a glass homogenizer. Serial tenfold dilutions of virus containing supernatant were assessed for the presence of infectious virus. Infectivity was quantified by the TCID50 method using B95a cells (Weidmann et al., 2000). The TCID50 was calculated by the method of Reed & Muench (1938)
. The threshold of detection was 102 TDID50 g-1 lung tissue; <102 signified no virus. Lung lavage was performed using a three-way stopcock and 10 ml PBS/1 % EDTA. Lung lavage cells were incubated in tenfold dilutions starting with 106 cells per well of a 96-well plate with 105 B95a cells. Infectivity was calculated per 107 cells using the method of Reed & Muench (1938)
; thus, the threshold of detection was <101.
In situ hybridization assay.
The assay was basically performed as described by Mrkic et al. (1998). Briefly, infected cotton rats were killed with CO2 and skulls and lungs were removed. Skulls were fixed in 4 % phosphate-buffered formaldehyde and subsequently decalcified for 710 days with Morse's solution (10 % sodium citrate, 22·5 % formic acid). Blocks of the nasal passages and sinuses were cut and embedded in paraffin and 4 µm sections were prepared. Lungs were snap frozen in liquid nitrogen and frozen 20 µm tissue sections were prepared in a cryostat. Detection of MV N mRNA in situ was performed with a digoxigenin (DIG)-labelled nucleoprotein (N) mRNA-specific probe under appropriate conditions. The hybridization probe was detected with an alkaline phosphatase-conjugated anti-DIG antibody. Sections were counterstained with haematoxylin solution.
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RESULTS |
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Apical MV glycoprotein expression prevents cell-to-cell fusion in infected polarized epithelial cells
Disruption of the basolateral targeting signals has significant negative consequences for cell-to-cell fusion in polarized epithelial cells co-expressing plasmid-encoded F and H proteins (Moll et al., 2001). To assess the ability of rMV with apically expressed glycoproteins to induce syncytia formation in polarized epithelial cells, MDCK cells were infected with rMVEdm, rMVF549Y/A, rMVH12Y/A or rMVFHY/A. Cell-to-cell fusion was monitored by immunostaining. Whereas rMVEdm-infected cells demonstrated large syncytia, cell-to-cell fusion could not be detected in any of the cultures infected with the tyrosine mutants (Fig. 3
A). These results confirmed that only cells expressing fusogenic F/H complexes on the basolateral membrane were able to fuse with neighbouring cells. Thus, apical sorting of only one of the glycoproteins completely prevents fusion in virus-infected polarized epithelial cells.
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In vitro infection of cotton rat primary tracheal cells
To study whether the abundant basolateral expression of the glycoproteins is essential for the spread of MV infection in the respiratory mucosa in vivo, we used cotton rats (S. hispidus) as a rodent model. Since it was unknown which cells of the cotton rat other than lymphocytes and macrophages can be productively infected with MV, epithelial cells from cotton rat tracheas were isolated and cultivated at low or high densities for several days. Subsequently, non-polarized and polarized cell monolayers were infected with a green fluorescent protein (GFP)-expressing rMV (Ehrengruber et al., 2002). As shown in Fig. 4
, non-polarized and polarized primary epithelial cells could be successfully infected with MV. In non-polarized tracheal cells, GFP expression in single cells was detected by 19 h p.i. From then on, infection spread rapidly throughout the monolayer resulting in the formation of large syncytia. Infection of polarized tracheal cells was delayed and GFP-positive syncytia were only found at 88 h p.i. Although syncytia formation was less pronounced than in non-polarized cells, this clearly demonstrated that polarized tracheal cells are potential MV target cells in the cotton rat respiratory tract and that parental rMVEdm can spread from these cells by direct cell-to-cell fusion.
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DISCUSSION |
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We have reported previously that MV envelope proteins F and H are abundantly expressed on the basolateral surface of polarized epithelial cells. Targeting as well as fusion activity in polarized cells has been shown to depend critically on a tyrosine residue in the cytoplasmic tails (Maisner et al., 1998; Moll et al., 2001
). Continuing this work, we have described here the successful generation of rMV with mutated basolateral targeting signals in either one or both glycoproteins. While these virus mutants did not show any obvious differences in their fusogenic activity and their growth characteristics in non-polarized cells, their cytopathic properties in polarized cell cultures and, importantly, their pathogenesis in vivo in the cotton rat model were clearly different from that of the parental rMVEdm. The exchange of cytoplasmic tyrosine residues responsible for basolateral targeting resulted in the predominant apical expression of the respective glycoprotein in infected polarized cells. Thus, both glycoproteins contain cryptic apical targeting information that becomes activated after destruction of the original basolateral signal. The finding that the mutant viruses were no longer able to induce cell-to-cell fusion in polarized epithelial cells clearly demonstrates that interaction with neighbouring cells and subsequent initiation of the fusion process strictly depend on the expression of F/H complexes at the basolateral membrane of polarized cells. The fact that tyrosine mutants promoted fusion of non-polarized cells as efficiently as parental MV showed that indeed the retargeting of the glycoproteins, and not a general defect, is responsible for the altered fusion activity in epithelia. By using cotton rats as a small-animal model, we were able to verify that not only fusion but also the spread of infection in vivo is restricted. Although it is not clear yet which cellular receptors are responsible for the infection of cotton rat cells by MV, we clearly demonstrated in vitro that non-polarized and, more importantly, polarized primary tracheal cells of cotton rats are susceptible to MV and allow virus spread by cell-to-cell fusion, thus, very likely contributing to the spread of MV infection in vivo. In agreement with this, rMV expressing apically targeted glycoproteins showed a clearly attenuated phenotype in cotton rats following intranasal infection. These viruses were less virulent and replication was significantly reduced in the respiratory tract of infected animals compared with parental rMVEdm. Even if the total number of virus-positive cells was low, the histological studies led us to suggest that differences in the infection of the respiratory epithelia are responsible for the attenuated phenotype of the virus mutants. Whereas rMVEdm was able to spread from cell to cell within the epithelial layer and from there to cells in the underlying tissue, the virus mutant rMVFHY/A showed virtually no spread of infection. Due to the experimental conditions it was not possible to determine the type of cells infected in the subepithelial tissue, but it seems likely that these cells are macrophages. Macrophages are numerous in the connective tissue of the mucosa and submucosa of the respiratory tract and, furthermore, belong to the main target cells of MV in the organism (Esolen et al., 1993
; Roscic-Mrkic et al., 2001
).
A striking observation was that, in contrast to cultured epithelial cells, spread of rMVEdm in the respiratory epithelium of cotton rats occurred without apparent formation of syncytia and epithelial damage. This is in full agreement with the observation recently published by Sinn et al. (2002) that the cell layer integrity of MV-infected primary cultures of airway epithelia is maintained. Therefore, it can be assumed that virus dissemination in vivo resulted from the transient formation of microfusion pores induced by the basolaterally expressed F and H proteins rather than from cell-to-cell fusion. This mechanism of virus spread has been already proposed for MV transmission from monocyticpromyelocytic cells (U937) to HeLa cells and between neuronal cells (Ehrengruber et al., 2002
; Firsching et al., 1999
). Moreover, this mode of propagation is in line with the observation that MV is strongly cell associated in vivo and dissemination is almost always dependent on direct cell-to-cell contact (Mrkic et al., 2000
).
Viruses have evolved a variety of mechanisms to accomplish spread from and within the respiratory epithelium. Maybe the most simple mechanism is the production of cell-free virions by budding from the surface of an infected cell and subsequent infection of neighbouring cells as shown for respiratory syncytial virus infections of well-differentiated human airway epithelial cells (Zhang et al., 2002). But this mechanism does not apply to all respiratory viruses. For example, adenoviruses have developed sophisticated mechanisms to disrupt the junctional integrity of epithelial barriers, thereby facilitating escape from the epithelium (Walters et al., 2002
). For MV, it has recently been proposed that spread of infection through the respiratory epithelium might involve pathways other than direct binding and entry through the apical surface of airway epithelia (Sinn et al., 2002
). In agreement with this, our results indicate that not only the mode of virus release from polarized cells but also the targeted glycoprotein expression has an important impact on the spread of MV within and from airway epithelia. Based on the data presented here, we propose a simplified model for the early steps of MV infection. Following aerogen transmission, MV initially replicates in polarized epithelial cells of the upper respiratory tract. Abundant basolateral expression of both MV F and H proteins in these cells promotes the transient formation of microfusion pores between epithelial cells, as well as between epithelial cells and macrophages in the subepithelial tissue. Subsequently, infectious ribonucleoproteins can translocate via microfusion pores, resulting in the infection of neighbouring epithelial cells and subepithelial macrophages without release of infectious particles or syncytia formation. Finally, infected macrophages disseminate MV infection systemically via the lymphatic and vascular system. According to this model, the basolateral expression of MV glycoproteins helps to overcome the epithelial barrier, thereby facilitating the systemic spread of the MV infection in vivo. The fact that in all MV wild-type isolates and vaccine strains known so far the tyrosine-depending sorting motifs are conserved clearly supports this idea of a functional importance for MV replication in vivo.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bomsel, M. & Alfsen, A. (2003). Entry of viruses through the epithelial barrier: pathogenic trickery. Nat Rev Mol Cell Biol 4, 5768.[CrossRef][Medline]
Ehrengruber, M. U., Ehler, E., Billeter, M. A. & Naim, H. Y. (2002). Measles virus spreads in rat hippocampal neurons by cell-to-cell contact and in a polarized fashion. J Virol 76, 57205728.
Esolen, L. M., Ward, B. J., Moench, T. R. & Griffin, D. E. (1993). Infection of monocytes during measles. J Infect Dis 168, 4752.[Medline]
Firsching, R., Buchholz, C. J., Schneider, U., Cattaneo, R., ter Meulen, V. & Schneider-Schaulies, J. (1999). Measles virus spread by cell-cell contacts: uncoupling of contact-mediated receptor (CD46) downregulation from virus uptake. J Virol 73, 52655273.
Griffin, D. E. & Bellini, W. J. (1996). Measles virus. In Fields Virology, pp. 12671312. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. New York: Raven Press.
McChesney, M. B., Miller, C. J., Rota, P. A., Zhu, Y. D., Antipa, L., Lerche, N. W., Ahmed, R. & Bellini, W. J. (1997). Experimental measles. I. Pathogenesis in the normal and the immunized host. Virology 233, 7484.[CrossRef][Medline]
Maisner, A., Klenk, H. & Herrler, G. (1998). Polarized budding of measles virus is not determined by viral surface glycoproteins. J Virol 72, 52765278.
Moench, T. R., Griffin, D. E., Obriecht, C. R., Vaisberg, A. J. & Johnson, R. T. (1988). Acute measles in patients with and without neurological involvement: distribution of measles virus antigen and RNA. J Infect Dis 158, 433442.[Medline]
Moll, M., Klenk, H. D., Herrler, G. & Maisner, A. (2001). A single amino acid change in the cytoplasmic domains of measles virus glycoproteins H and F alters targeting, endocytosis, and cell fusion in polarized MadinDarby canine kidney cells. J Biol Chem 276, 1788717894.
Moll, M., Klenk, H. D. & Maisner, A. (2002). Importance of the cytoplasmic tails of the measles virus glycoproteins for fusogenic activity and the generation of recombinant measles viruses. J Virol 76, 71747186.
Mrkic, B., Pavlovic, J., Rulicke, T., Volpe, P., Buchholz, C. J., Hourcade, D., Atkinson, J. P., Aguzzi, A. & Cattaneo, R. (1998). Measles virus spread and pathogenesis in genetically modified mice. J Virol 72, 74207427.
Mrkic, B., Odermatt, B., Klein, M. A., Billeter, M. A., Pavlovic, J. & Cattaneo, R. (2000). Lymphatic dissemination and comparative pathology of recombinant measles viruses in genetically modified mice. J Virol 74, 13641372.
Naim, H. Y., Ehler, E. & Billeter, M. A. (2000). Measles virus matrix protein specifies apical virus release and glycoprotein sorting in epithelial cells. EMBO J 19, 35763585.
Niewiesk, S., Gotzelmann, M. & ter Meulen, V. (2000). Selective in vivo suppression of T lymphocyte responses in experimental measles virus infection. Proc Natl Acad Sci U S A 97, 42514255.
Norrby, E. & Oxman, M. N. (1990). Measles virus. In Fields Virology, pp. 10131044. Edited by B. N. Fields & D. M. Knipe. New York: Raven Press.
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, 150158.[CrossRef][Medline]
Radecke, F., Spielhofer, P., Schneider, H., Kaelin, K., Huber, M., Dotsch, C., Christiansen, G. & Billeter, M. A. (1995). Rescue of measles viruses from cloned DNA. EMBO J 14, 57735784.[Abstract]
Reed, L. J. & Muench, H. (1938). A simple method of estimating fifty per cent endpoints. J Hyg 27, 493497.
Riedl, P., Moll, M., Klenk, H. D. & Maisner, A. (2002). Measles virus matrix protein is not cotransported with the viral glycoproteins but requires virus infection for efficient surface targeting. Virus Res 83, 112.[CrossRef][Medline]
Roscic-Mrkic, B., Schwendener, R. A., Odermatt, B., Zuniga, A., Pavlovic, J., Billeter, M. A. & Cattaneo, R. (2001). Roles of macrophages in measles virus infection of genetically modified mice. J Virol 75, 33433351.
Singh, M., Cattaneo, R. & Billeter, M. A. (1999). A recombinant measles virus expressing hepatitis B virus surface antigen induces humoral immune responses in genetically modified mice. J Virol 73, 48234828.
Sinn, P. L., Williams, G., Vongpunsawad, S., Cattaneo, R. & McCray, P. B. (2002). Measles virus preferentially transduces the basolateral surface of well-differentiated human airway epithelia. J Virol 76, 24032409.
Tashiro, M., James, I., Karri, S., Wahn, K., Tobita, K., Klenk, H. D., Rott, R. & Seto, J. T. (1991). Pneumotropic revertants derived from a pantropic mutant, F1-R, of Sendai virus. Virology 184, 227234.[Medline]
Tucker, S. P. & Compans, R. W. (1993). Virus infection of polarized epithelial cells. Adv Virus Res 42, 187247.[Medline]
van Binnendijk, R. S., van der Heijden, R. W. & Osterhaus, A. D. (1995). Monkeys in measles research. Curr Top Microbiol Immunol 191, 135148.[Medline]
Walters, R. W., Freimuth, P., Moninger, T. O., Ganske, I., Zabner, J. & Welsh, M. J. (2002). Adenovirus fiber disrupts CAR-mediated intercellular adhesion allowing virus escape. Cell 110, 789799.[Medline]
Weidmann, A., Maisner, A., Garten, W., Seufert, M., ter Meulen, V. & Schneider-Schaulies, S. (2000). Proteolytic cleavage of the fusion protein but not membrane fusion is required for measles virus-induced immunosuppression in vitro. J Virol 74, 19851993.
Wyde, P. R., Ambrose, M. W., Voss, T. G., Meyer, H. L. & Gilbert, B. E. (1992). Measles virus replication in lungs of hispid cotton rats after intranasal inoculation. Proc Soc Exp Biol Med 201, 8087.[Abstract]
Zhang, L., Peeples, M. E., Boucher, R. C., Collins, P. L. & Pickles, R. J. (2002). Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology. J Virol 76, 56545666.
Received 24 September 2003;
accepted 8 December 2003.