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Morphogenesis of measles virus on C6 rat glioma cells
Journal of Neuroimmunology, 20 (1988) 173-176 Elsevier
173
JNI 00687
Morphogenesis of measles virus
on
C 6 rat glioma cells
Kerstin R~Sser, Wolfgang Bohn and Klaus Mannweiler Heinrich-Pette-lnstitut ffir Experimentelle Virologic und lramunologie an der Universitiit Hamburg, D-2000 Hamburg 20, F.R.G. (Received 4 July 1988) (Accepted 15 July 1988)
Key words: C 6 rat glioma cell; Measles virus, persistent infection; Viral structure, cell surface
Summary Rat glioma cells (C6) persistently infected with measles virus show a locally dissociated distribution of budding processes at the cell surface.
Introduction
Materials and methods
Systemic infection by measles virus is frequently complicated by invasion of the central nervous system (CNS) which can lead to severe neurological disease, such as subacute sclerosing panencephalitis or post-infectious encephalomyelitis (ter Meulen et al., 1983). For study of measles virus infections of the CNS, animal models have been established (Lt~ve et al., 1986) and studies have been performed in vitro using CNS-derived cell lines (Kobune et al., 1973; Miinzel and Koschel, 1982; Bellini et al., 1986). These investigations point to a restricted replication of measles virus in cells of CNS origin, the cause of which is still unknown. Using C6 rat glioma cells, we examined the structural interaction between measles virus and cellular components at the plasma membrane in the course of virus morphogenesis.
C6 cells were grown on glass coverslips and cultivated with Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum. The cells were infected with the Edmonston strain of measles virus. Viral components were labeled with monoclonal antibodies to measles virus polypeptides (Bohn et al., 1982) and with a rabbit anti-measles antiserum. Affinity-purified goat anti-mouse Igfluorescein isothiocyanate (FITC), goat anti-rabbit Ig-FITC (Sigma, Munich), and goat anti-mouse IgG-Texas Red (Dianova, Hamburg) were used as second antibodies in immunofluorescence techniques. Colloidal gold was prepared according to Frens (1973) and coated with protein A as described by Horrisberger (1985). Protein A gold labeling of virus antigens and preparation of platinum carbon (Pt/C) replicas were performed as described previously (Mannweiler et al., 1982; Hohenberg et al., 1986). Ultrathin sections were obtained from cells embedded in ERL.
Address for correspondence: Kerstin RiSser, HeinrichPette-Institut ftir Experimentelle Virologie und Immunologic an der Universit~it Hamburg, Martinistrasse 52, D-2000 Hamburg 20, F.R.G.
0165-5728/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
174
175
Results Infected C 6 cultures exhibited no cytopathic effect, despite the fact that nearly 100% of the cells contained viral antigens as d e m o n s t r a t e d b y indirect immunofluorescence with a polyclonal anti-measles antiserum. I m m u n o f l u o r e s c e n t labeling with m o n o c l o n a l antibodies indicated that the infected cells synthesized the major viral polypeptides (hemagglutinin, phosphoprotein, nucleocapsid protein, matrix protein) necessary for complete virus assembly. However, in the persistently infected cultures, only a few infectious virus partieles (1.75 T C I D s 0 / m l ) could be detected in the supernatant culture fluid. Scanning electron microscopy (Fig. 1) as well as transmission electron microscope studies of ultrathin sections and surface replicas revealed that virus b u d d i n g at the plasma m e m b r a n e was a rare event (Figs. 2-6). O n l y some of the cells exhibited viral buds which were aggregated at the basis of long cellular processes. However, plasma m e m b r a n e areas being devoid of viral buds did contain the transmembrane viral glycoprotein hemagglutinin, evenly distributed at the cell surface. I n ultrathin sections, nucleocapsids were f o u n d beneath these m e m b r a n e areas. F u r t h e r m o r e , d o u b l e immunofluorescence labeling showed a clear co-localization of virus hemagglutinin at the cell surface with actin stress filaments (Figs. 7 and 8).
limited, correlating to the low titer of infectivity in the tissue culture medium. The electron micrographs suggest that m a t u r a t i o n of virus particles only occurs at those areas of the plasma m e m brane where cell processes originate. Thus, distinct local features m a y determine the formation of b u d d i n g structures at the cell surface. The even distribution of hemagglutinin and underlying nucleocapsids at cell surface areas without viral b u d s supports a block relatively late in virus morphogenesis. T h e o b s e r v e d co-localization of hemagglutinin and actin in these cells indicates a close association of virus structures with the cytoskeleton (Bohn et al., 1986) and suggests that variations of this interaction m a y influence the formation of b u d d i n g structures in these glioma cells.
Acknowledgements This work was supported by the Gemeinniatzige Hertie-Stiftung, F r a n k f u r t / a . Main. The Heinrich-Pette-Institut is financially supported b y Freie u n d H a n s e s t a d t H a m b u r g and Bundesministerium fiir Jugend, Familie, F r a u e n und Gesundheit, Bonn. This contribution is part of a doctoral thesis by K. Rinser, Fachbereich Biologie der Universit~it Hamburg.
Discussion
References
The results indicate that infection of C 6 rat glioma cells with measles virus leads to the establishment of a persistent infection. Despite the presence of all of the structural viral c o m p o n e n t s , the quantity of replicating m a t u r e virus was
Bellini, W., Englund, G., Rammohan, K. and McFarlin, D.E. (1986) Evaluation of the structural proteins of the hamster neurotropic strain of measles virus with monoclonal antibodies. J. Neuroimmunol. 11, 149-163. Bohn, W., Rutter, G. and Mannweiler, K. (1982) Production of monoclonal antibodies to measles virus proteins by im-
Fig. 1. Scanning electron micrograph of measles virus-infected C6 cells. Bar = 5 gm; bar for insert = 2 gm. Figs. 2-6. Protein A gold labeling of hemagglutinin at the cell surface. (2) Ultrathin section of ERL-embedded cells. Bar = 1 #m. (3) Platinum carbon (Pt/C) surface replica. Bar = 1 gin. (4) Higher magnification of a section from (2); note specific labeling at the surface of mature virus particles (arrow). Bar = 0.2 #m. (5-6) Membrane areas without budding structures but containing hemagglutinin. (5) Pt/C surface replica. Bar = 0.2 #m. (6) Ultrathin section; section profiles of nucleocapsids (arrows). Bar = 0.2 gm. Figs. 7 and 8. Double immunofluorescence labeling with antibodies to actin (7) and hemagglutinin (8) on extracted cells. 950 x .
176 munization of mice with heated and detergent-treated antigens. Virology 116, 368-371. Bohn, W., Rutter, G., Hohenberg, H., Mannweiler, K. and Nobis, P. (1986) Involvement of actin filaments in budding of measles virus: studies on cytoskeletons of infected cells. Virology 149, 91-106. Frens, G. (1973) Controlled nucleation for the regulation of particle size in monodisperse gold suspensions. Nature Phys. Sci. 241, 20. Hohenberg, H., Bohn, W., Rutter, G. and Mannweiler, K. (1986) Plasma antigens detected by replica techniques. Scanning electron microscopy. In: M. Mueller, P.P. Becker, A. Boyde and J.J. Wolosewick (Eds.), Science of Biological Specimen Preparation of Microscopy and Microanalysis, SEM Inc., AMF O'Hare, Chicago, IL, pp. 235-244. Horisberger, M. and Clerc, M.F. (1985) Labeling of colloidal gold with protein A. A quantitative study. Histochemistry 82, 219-223. Kobune, K., Yamanouchi, K., Yoshikawa, Y., Hayami, M. and
Shishido, A. (1973) Growth of measles virus in continuous cell lines derived from the nervous tissue of human and rat. Arch. Virol. 61, 115-125. L6ve, A., Norrby, E. and Kristensson, K. (1986) Measles encephalitis in rodents: defective expression of viral proteins. J. Neuropathol. Exp. Neurol. 45, 258-267. Mannweiler, K., Hohenberg, H., Bohn, W. and Rutter, G. (1982) Protein A gold particles as markers in replica immunocytochemistry: high resolution electron microscope investigations of plasma membrane surfaces. J. Microsc. 126, 145-149. Miinzel, P. and Koschel, K. (1982) Alteration in phospholipid methylation and impairment of signal transmission in persistently paramyxovirus-infected C6 rat glioma cells. Proc. Natl. Acad. Sci. U.S.A. 79, 3696-3696. Ter Meulen, V., Stephenson, J.R. and Kreth, H.W. (1983) Subacute selerosing panencephalitis. In: H. Fraenkel-Conrat and R.R. Wagner (Eds.), Comprehensive Virology, Vol. 18, Plenum Press, New York, pp. 105-154.
173
JNI 00687
Morphogenesis of measles virus
on
C 6 rat glioma cells
Kerstin R~Sser, Wolfgang Bohn and Klaus Mannweiler Heinrich-Pette-lnstitut ffir Experimentelle Virologic und lramunologie an der Universitiit Hamburg, D-2000 Hamburg 20, F.R.G. (Received 4 July 1988) (Accepted 15 July 1988)
Key words: C 6 rat glioma cell; Measles virus, persistent infection; Viral structure, cell surface
Summary Rat glioma cells (C6) persistently infected with measles virus show a locally dissociated distribution of budding processes at the cell surface.
Introduction
Materials and methods
Systemic infection by measles virus is frequently complicated by invasion of the central nervous system (CNS) which can lead to severe neurological disease, such as subacute sclerosing panencephalitis or post-infectious encephalomyelitis (ter Meulen et al., 1983). For study of measles virus infections of the CNS, animal models have been established (Lt~ve et al., 1986) and studies have been performed in vitro using CNS-derived cell lines (Kobune et al., 1973; Miinzel and Koschel, 1982; Bellini et al., 1986). These investigations point to a restricted replication of measles virus in cells of CNS origin, the cause of which is still unknown. Using C6 rat glioma cells, we examined the structural interaction between measles virus and cellular components at the plasma membrane in the course of virus morphogenesis.
C6 cells were grown on glass coverslips and cultivated with Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum. The cells were infected with the Edmonston strain of measles virus. Viral components were labeled with monoclonal antibodies to measles virus polypeptides (Bohn et al., 1982) and with a rabbit anti-measles antiserum. Affinity-purified goat anti-mouse Igfluorescein isothiocyanate (FITC), goat anti-rabbit Ig-FITC (Sigma, Munich), and goat anti-mouse IgG-Texas Red (Dianova, Hamburg) were used as second antibodies in immunofluorescence techniques. Colloidal gold was prepared according to Frens (1973) and coated with protein A as described by Horrisberger (1985). Protein A gold labeling of virus antigens and preparation of platinum carbon (Pt/C) replicas were performed as described previously (Mannweiler et al., 1982; Hohenberg et al., 1986). Ultrathin sections were obtained from cells embedded in ERL.
Address for correspondence: Kerstin RiSser, HeinrichPette-Institut ftir Experimentelle Virologie und Immunologic an der Universit~it Hamburg, Martinistrasse 52, D-2000 Hamburg 20, F.R.G.
0165-5728/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
174
175
Results Infected C 6 cultures exhibited no cytopathic effect, despite the fact that nearly 100% of the cells contained viral antigens as d e m o n s t r a t e d b y indirect immunofluorescence with a polyclonal anti-measles antiserum. I m m u n o f l u o r e s c e n t labeling with m o n o c l o n a l antibodies indicated that the infected cells synthesized the major viral polypeptides (hemagglutinin, phosphoprotein, nucleocapsid protein, matrix protein) necessary for complete virus assembly. However, in the persistently infected cultures, only a few infectious virus partieles (1.75 T C I D s 0 / m l ) could be detected in the supernatant culture fluid. Scanning electron microscopy (Fig. 1) as well as transmission electron microscope studies of ultrathin sections and surface replicas revealed that virus b u d d i n g at the plasma m e m b r a n e was a rare event (Figs. 2-6). O n l y some of the cells exhibited viral buds which were aggregated at the basis of long cellular processes. However, plasma m e m b r a n e areas being devoid of viral buds did contain the transmembrane viral glycoprotein hemagglutinin, evenly distributed at the cell surface. I n ultrathin sections, nucleocapsids were f o u n d beneath these m e m b r a n e areas. F u r t h e r m o r e , d o u b l e immunofluorescence labeling showed a clear co-localization of virus hemagglutinin at the cell surface with actin stress filaments (Figs. 7 and 8).
limited, correlating to the low titer of infectivity in the tissue culture medium. The electron micrographs suggest that m a t u r a t i o n of virus particles only occurs at those areas of the plasma m e m brane where cell processes originate. Thus, distinct local features m a y determine the formation of b u d d i n g structures at the cell surface. The even distribution of hemagglutinin and underlying nucleocapsids at cell surface areas without viral b u d s supports a block relatively late in virus morphogenesis. T h e o b s e r v e d co-localization of hemagglutinin and actin in these cells indicates a close association of virus structures with the cytoskeleton (Bohn et al., 1986) and suggests that variations of this interaction m a y influence the formation of b u d d i n g structures in these glioma cells.
Acknowledgements This work was supported by the Gemeinniatzige Hertie-Stiftung, F r a n k f u r t / a . Main. The Heinrich-Pette-Institut is financially supported b y Freie u n d H a n s e s t a d t H a m b u r g and Bundesministerium fiir Jugend, Familie, F r a u e n und Gesundheit, Bonn. This contribution is part of a doctoral thesis by K. Rinser, Fachbereich Biologie der Universit~it Hamburg.
Discussion
References
The results indicate that infection of C 6 rat glioma cells with measles virus leads to the establishment of a persistent infection. Despite the presence of all of the structural viral c o m p o n e n t s , the quantity of replicating m a t u r e virus was
Bellini, W., Englund, G., Rammohan, K. and McFarlin, D.E. (1986) Evaluation of the structural proteins of the hamster neurotropic strain of measles virus with monoclonal antibodies. J. Neuroimmunol. 11, 149-163. Bohn, W., Rutter, G. and Mannweiler, K. (1982) Production of monoclonal antibodies to measles virus proteins by im-
Fig. 1. Scanning electron micrograph of measles virus-infected C6 cells. Bar = 5 gm; bar for insert = 2 gm. Figs. 2-6. Protein A gold labeling of hemagglutinin at the cell surface. (2) Ultrathin section of ERL-embedded cells. Bar = 1 #m. (3) Platinum carbon (Pt/C) surface replica. Bar = 1 gin. (4) Higher magnification of a section from (2); note specific labeling at the surface of mature virus particles (arrow). Bar = 0.2 #m. (5-6) Membrane areas without budding structures but containing hemagglutinin. (5) Pt/C surface replica. Bar = 0.2 #m. (6) Ultrathin section; section profiles of nucleocapsids (arrows). Bar = 0.2 gm. Figs. 7 and 8. Double immunofluorescence labeling with antibodies to actin (7) and hemagglutinin (8) on extracted cells. 950 x .
176 munization of mice with heated and detergent-treated antigens. Virology 116, 368-371. Bohn, W., Rutter, G., Hohenberg, H., Mannweiler, K. and Nobis, P. (1986) Involvement of actin filaments in budding of measles virus: studies on cytoskeletons of infected cells. Virology 149, 91-106. Frens, G. (1973) Controlled nucleation for the regulation of particle size in monodisperse gold suspensions. Nature Phys. Sci. 241, 20. Hohenberg, H., Bohn, W., Rutter, G. and Mannweiler, K. (1986) Plasma antigens detected by replica techniques. Scanning electron microscopy. In: M. Mueller, P.P. Becker, A. Boyde and J.J. Wolosewick (Eds.), Science of Biological Specimen Preparation of Microscopy and Microanalysis, SEM Inc., AMF O'Hare, Chicago, IL, pp. 235-244. Horisberger, M. and Clerc, M.F. (1985) Labeling of colloidal gold with protein A. A quantitative study. Histochemistry 82, 219-223. Kobune, K., Yamanouchi, K., Yoshikawa, Y., Hayami, M. and
Shishido, A. (1973) Growth of measles virus in continuous cell lines derived from the nervous tissue of human and rat. Arch. Virol. 61, 115-125. L6ve, A., Norrby, E. and Kristensson, K. (1986) Measles encephalitis in rodents: defective expression of viral proteins. J. Neuropathol. Exp. Neurol. 45, 258-267. Mannweiler, K., Hohenberg, H., Bohn, W. and Rutter, G. (1982) Protein A gold particles as markers in replica immunocytochemistry: high resolution electron microscope investigations of plasma membrane surfaces. J. Microsc. 126, 145-149. Miinzel, P. and Koschel, K. (1982) Alteration in phospholipid methylation and impairment of signal transmission in persistently paramyxovirus-infected C6 rat glioma cells. Proc. Natl. Acad. Sci. U.S.A. 79, 3696-3696. Ter Meulen, V., Stephenson, J.R. and Kreth, H.W. (1983) Subacute selerosing panencephalitis. In: H. Fraenkel-Conrat and R.R. Wagner (Eds.), Comprehensive Virology, Vol. 18, Plenum Press, New York, pp. 105-154.