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Factors involved in measles virus budding
252
Biology of the Cell (1998) 90, 247290
Factors involved in measles virus budding Danikle Spehner *, Toni Cathomerfl Skverine Vincentf Denis Gerlierg and Robert Drillien*
Roberto Cattaneo”,
*CJF 94-03, Etablissement de Transfusion Sanguine de Strasbourg, IO rue Spielmann, 6 7065, Strasbourg, France; “Institut fiir Molekularbiologie, Abteilung I, UniversiW Ziirich, Htinggerberg, 8093 Ziirich, Suisse; #Immune-VirologieMolkculaire et Cellulaire, UMR 30, CNRS, Facult6 de Mkdecine, RTH Laennec, rue GuiliaumeParadin,69008 Lyon, France Measles virus, a member of the Paramyxoviridae family, is an enveloped virus that encapsidates a 16kb single stranded RNA genome of negative polarity. The viral genome encodes six structural proteins involved in the formation of virions. Three of these proteins (the nucleoprotein, the phosphoprotein and the large protein) are required for the encapsidation of the genome and its subsequent transcription and replication. The nucleoprotein enwraps the viral RNA into tightly coiled helical structures designated nucleocapsids. The phosphoprotein and the large protein associate into a polymerase complex that attaches to nucleocapsids and transcribes the viral genome upon infection. Three other structural proteins (the hemagglutinin, the fusion protein and the matrix protein) participate in the migration of viral nucleocapsids to the plasma membrane and their budding at the cell surface into enveloped particles. The hemagglutinin and the fusion proteins are transmembrane proteins that form spikes on the viral envelope. The hemagglutinin behaves as an attachment protein by binding to receptors on the cell surface whereas the fusion protein promotes fusion of the viral envelope with the cell membrane thus enabling penetration of nucleocapsids into the cytoplasm. The matrix protein lies beneath the viral envelope and presumably interacts with the cytoplasmic domains of the viral transmembrane proteins on one side and the nucleocapsids on the other, thereby behaving as a major organiser in the assembly process. The assembly of measles virus particles requires not only viral encoded proteins but also host cell factors. This study focuses on the role of the matrix protein and as yet uncharacterised host cell factors in the phenomenon of virus budding at the infected cell surface.
Role of the matrix protein A strategy developed several years ago enables one to obtain infectious measles virus from full length genomic cDNA (Radeckc ef al. (1995)
EMBO J 14, 5773-5784). This has allowed the precise engineering of mutant genomes and the study of the phenotypes they display. To investigate the role of the matrix protein, the region of the genome encoding it, from the Edmonston strain, was deleted (Cathomen ef al. 1998 EMBOf in press). Transfection of the corresponding cDNA allowed the recovery of infectious virus indicating that the matrix protein is not absolutely essential for infectivity. Nevertheless, both cell associated and extracellular virus titers of the matrix-less virus were about 100 to 1000 fold lower than those of the parental virus. Furthermore, the matrix-less virus induced the formation of larger syncytia than the wild type suggesting that this process was controlled directly or indirectly by the matrix protein. To study the effect of deleting the matrix gene on virus assembly, Vero cells (a permiGve monkey cell line) were infected and incubated, after permeabilisation, with anti-bodies recognising several of the measles virus proteins. After washing to remove unattached antibodies the cells were labelled with immunogold conjugates directed against the primary antibodies. Immunogold labelling was performed using either 1Onm gold congugates for recognition of antigens at the cell surface or ultra-small gold conjugates (=lnm) for recognition of intracellular antigens. Cells were then embedded in epon, cut into ultrathin sections and observed by transmission electron microscopy. Silver enhancement was carried out directly on samples deposited on the electron microscope grids when ultra-small gold particles had been employed. Large cytoplasmic inclusions containing the nucleocapsid structures could be readily detected both after infection with the wild type and the matrix-less virus. This indicated that the mutant virus infects cells and its genome is transcribed, replicated and encapsidated similarly to the wild type virus. When the localisation of nucleocapsids was examined more closely it was noticed that many of them could be found close to the plasma
Abstracts Trinoculaire ‘98 des Microscopies, Strasbourg-lllkirch, France, l-3 July 1998 membrane in wild type infected cells but not in mutant infected cells. Thus the matrix protein was essential for the accumulation of nucleocapsids at the cell surface. Examination of the localisation of the two viral glycoproteins showed that both the hemagglutinin and the fusion protein could be immunolabelled at the cell surface of wild type or mutant infected cells. In wild type infected cells these proteins were concentrated over limited areas of the cell surface where cell protrusions, characteristic of viral budding, appeared. Nucleocapsids were also noticed in the immediate vicinity of the surface glycoproteins. In contrast, the viral glycoproteins were more spread out over the surface of cells infected with the matrix-less virus and no areas of budding or underlying nucleocapsids were visible. Thus, the matrix protein clearly plays an important role in 1” the migration of nucleocapsids to the plasma membrane 2” the concentration of viral glycoproteins at the cell surface and 3” the budding of new virions. Despite the defect in these steps, the matrix-less virus gave rise to a small amount of infectious extracellular virus. In fact, some extracellular viral particles were apparent in the samples infected with the matrix-less virus although many of them appeared abberant. Such virus could result from the release of vesicles, coated with viral glycoproteins, at the cell surface. These vesicles could contain, in a haphazard fashion, viral nucleocapsids. The observation that the matrix-less virus induced more efficient cellto-cell fusion than the wild type virus could be accounted for by a more diffuse distribution of the glycoproteins at the cell surface and possibly a different conformation of the fusion protein due to lack of interaction with the matrix protein. Finally, it is interesting to note that measles virus isolates from patients with subacute sclerosing panencephalitis display defects in the matrix protein that could explain the behaviour of this virus in the brain by analogy with the matrixless virus described here : propagation of infection by cell-to-cell fusion and lack of normal levels of extracellular virus.
253
Control of virus budding by the host cell A major receptor of measles virus is the complement control protein CD46 (Naniche et al. (1993) J Viral 67, 602560321, found at the surface of most human cell types. Mouse cell lines are not permissive for measles virus but may become permissive if they are transfected with human CD46. Nevertheless, some mouse lines, such as the L cell line, still remain non permissive despite expression of human CD46. This suggests that one or several cell factors, not present in L cells, are required for measles infection. To determine the step in the infectious cycle that is controlled by such cell factors we examined measles virus infection of L cells permanently transfected with CD46 (L-CD46). Infection of these cells with the Halle strain of measles virus led to the formation of characteristic syncytia although very little infectious virus was produced. The expression of measles virus proteins measured by flow cytometry and immunofluorescence microscopy was found to be similar in non permissive L-CD46 cells and HeLa cells (a permissive human cell line). Notably, the amount of the nucleoprotein, the phosphoprotein, the matrix protein and the hemagglutinin were even higher in L-CD46 cells than in HeLa cells and the nucleoprotein appeared to co-localize at the plasma membrane with the viral glycoproteins. Furthermore, nucleocapsids were assembled to a high level in LCD46 cells as judged by gradient analysis. Examination of the assembly events by immunogold labelling and transmission electron microscopy showed that no enveloped virus particles were apparent at the surface of L-CD46 cells in contrast to the large amount of particles at the surface of HeLa cells. However, the surface of L-CD46 cells could be indirectly immunolabelled with collo’idal-gold coupled antibodies against viral glycoproteins and numerous nucleocapsids were apparent near the plasma membrane. These results indicate that measles virus assembly in L-CD46 cells is defective in the formation of budding particles despite the presence of the viral proteins required for this process and their normal localisation. Further studies are needed in order to understand why measles virus particles fail to bud at the surface of L-CD46 cells and which cellular factors control this event.
Biology of the Cell (1998) 90, 247290
Factors involved in measles virus budding Danikle Spehner *, Toni Cathomerfl Skverine Vincentf Denis Gerlierg and Robert Drillien*
Roberto Cattaneo”,
*CJF 94-03, Etablissement de Transfusion Sanguine de Strasbourg, IO rue Spielmann, 6 7065, Strasbourg, France; “Institut fiir Molekularbiologie, Abteilung I, UniversiW Ziirich, Htinggerberg, 8093 Ziirich, Suisse; #Immune-VirologieMolkculaire et Cellulaire, UMR 30, CNRS, Facult6 de Mkdecine, RTH Laennec, rue GuiliaumeParadin,69008 Lyon, France Measles virus, a member of the Paramyxoviridae family, is an enveloped virus that encapsidates a 16kb single stranded RNA genome of negative polarity. The viral genome encodes six structural proteins involved in the formation of virions. Three of these proteins (the nucleoprotein, the phosphoprotein and the large protein) are required for the encapsidation of the genome and its subsequent transcription and replication. The nucleoprotein enwraps the viral RNA into tightly coiled helical structures designated nucleocapsids. The phosphoprotein and the large protein associate into a polymerase complex that attaches to nucleocapsids and transcribes the viral genome upon infection. Three other structural proteins (the hemagglutinin, the fusion protein and the matrix protein) participate in the migration of viral nucleocapsids to the plasma membrane and their budding at the cell surface into enveloped particles. The hemagglutinin and the fusion proteins are transmembrane proteins that form spikes on the viral envelope. The hemagglutinin behaves as an attachment protein by binding to receptors on the cell surface whereas the fusion protein promotes fusion of the viral envelope with the cell membrane thus enabling penetration of nucleocapsids into the cytoplasm. The matrix protein lies beneath the viral envelope and presumably interacts with the cytoplasmic domains of the viral transmembrane proteins on one side and the nucleocapsids on the other, thereby behaving as a major organiser in the assembly process. The assembly of measles virus particles requires not only viral encoded proteins but also host cell factors. This study focuses on the role of the matrix protein and as yet uncharacterised host cell factors in the phenomenon of virus budding at the infected cell surface.
Role of the matrix protein A strategy developed several years ago enables one to obtain infectious measles virus from full length genomic cDNA (Radeckc ef al. (1995)
EMBO J 14, 5773-5784). This has allowed the precise engineering of mutant genomes and the study of the phenotypes they display. To investigate the role of the matrix protein, the region of the genome encoding it, from the Edmonston strain, was deleted (Cathomen ef al. 1998 EMBOf in press). Transfection of the corresponding cDNA allowed the recovery of infectious virus indicating that the matrix protein is not absolutely essential for infectivity. Nevertheless, both cell associated and extracellular virus titers of the matrix-less virus were about 100 to 1000 fold lower than those of the parental virus. Furthermore, the matrix-less virus induced the formation of larger syncytia than the wild type suggesting that this process was controlled directly or indirectly by the matrix protein. To study the effect of deleting the matrix gene on virus assembly, Vero cells (a permiGve monkey cell line) were infected and incubated, after permeabilisation, with anti-bodies recognising several of the measles virus proteins. After washing to remove unattached antibodies the cells were labelled with immunogold conjugates directed against the primary antibodies. Immunogold labelling was performed using either 1Onm gold congugates for recognition of antigens at the cell surface or ultra-small gold conjugates (=lnm) for recognition of intracellular antigens. Cells were then embedded in epon, cut into ultrathin sections and observed by transmission electron microscopy. Silver enhancement was carried out directly on samples deposited on the electron microscope grids when ultra-small gold particles had been employed. Large cytoplasmic inclusions containing the nucleocapsid structures could be readily detected both after infection with the wild type and the matrix-less virus. This indicated that the mutant virus infects cells and its genome is transcribed, replicated and encapsidated similarly to the wild type virus. When the localisation of nucleocapsids was examined more closely it was noticed that many of them could be found close to the plasma
Abstracts Trinoculaire ‘98 des Microscopies, Strasbourg-lllkirch, France, l-3 July 1998 membrane in wild type infected cells but not in mutant infected cells. Thus the matrix protein was essential for the accumulation of nucleocapsids at the cell surface. Examination of the localisation of the two viral glycoproteins showed that both the hemagglutinin and the fusion protein could be immunolabelled at the cell surface of wild type or mutant infected cells. In wild type infected cells these proteins were concentrated over limited areas of the cell surface where cell protrusions, characteristic of viral budding, appeared. Nucleocapsids were also noticed in the immediate vicinity of the surface glycoproteins. In contrast, the viral glycoproteins were more spread out over the surface of cells infected with the matrix-less virus and no areas of budding or underlying nucleocapsids were visible. Thus, the matrix protein clearly plays an important role in 1” the migration of nucleocapsids to the plasma membrane 2” the concentration of viral glycoproteins at the cell surface and 3” the budding of new virions. Despite the defect in these steps, the matrix-less virus gave rise to a small amount of infectious extracellular virus. In fact, some extracellular viral particles were apparent in the samples infected with the matrix-less virus although many of them appeared abberant. Such virus could result from the release of vesicles, coated with viral glycoproteins, at the cell surface. These vesicles could contain, in a haphazard fashion, viral nucleocapsids. The observation that the matrix-less virus induced more efficient cellto-cell fusion than the wild type virus could be accounted for by a more diffuse distribution of the glycoproteins at the cell surface and possibly a different conformation of the fusion protein due to lack of interaction with the matrix protein. Finally, it is interesting to note that measles virus isolates from patients with subacute sclerosing panencephalitis display defects in the matrix protein that could explain the behaviour of this virus in the brain by analogy with the matrixless virus described here : propagation of infection by cell-to-cell fusion and lack of normal levels of extracellular virus.
253
Control of virus budding by the host cell A major receptor of measles virus is the complement control protein CD46 (Naniche et al. (1993) J Viral 67, 602560321, found at the surface of most human cell types. Mouse cell lines are not permissive for measles virus but may become permissive if they are transfected with human CD46. Nevertheless, some mouse lines, such as the L cell line, still remain non permissive despite expression of human CD46. This suggests that one or several cell factors, not present in L cells, are required for measles infection. To determine the step in the infectious cycle that is controlled by such cell factors we examined measles virus infection of L cells permanently transfected with CD46 (L-CD46). Infection of these cells with the Halle strain of measles virus led to the formation of characteristic syncytia although very little infectious virus was produced. The expression of measles virus proteins measured by flow cytometry and immunofluorescence microscopy was found to be similar in non permissive L-CD46 cells and HeLa cells (a permissive human cell line). Notably, the amount of the nucleoprotein, the phosphoprotein, the matrix protein and the hemagglutinin were even higher in L-CD46 cells than in HeLa cells and the nucleoprotein appeared to co-localize at the plasma membrane with the viral glycoproteins. Furthermore, nucleocapsids were assembled to a high level in LCD46 cells as judged by gradient analysis. Examination of the assembly events by immunogold labelling and transmission electron microscopy showed that no enveloped virus particles were apparent at the surface of L-CD46 cells in contrast to the large amount of particles at the surface of HeLa cells. However, the surface of L-CD46 cells could be indirectly immunolabelled with collo’idal-gold coupled antibodies against viral glycoproteins and numerous nucleocapsids were apparent near the plasma membrane. These results indicate that measles virus assembly in L-CD46 cells is defective in the formation of budding particles despite the presence of the viral proteins required for this process and their normal localisation. Further studies are needed in order to understand why measles virus particles fail to bud at the surface of L-CD46 cells and which cellular factors control this event.