Klaus Mannweiler

Involvement of actin filaments in budding of measles virus: Studies on cytoskeletons of infected cells

Cytoskeletons were prepared from measles virus infected HeLa cells to investigate the involvement of cytoskeletal filaments in virus budding at the plasma membrane. The cytoskeletons retained nearly 80% of measles virus hemagglutinin, the major viral polypeptides, including P, NP, and M, and 2 to 12% of the total cell bound infectivity. As demonstrated with platinum- and carbon-shadowed cytoskeletons, all stages of budding, i.e., virus specific strands, stub-like protrusions, and completely rounded virus particles, are associated with actin filaments composing the outer part of the cytoskeletal network. As shown with ultrathin sections of flat embedded extracted cells, actin filaments identified with heavy meromyosin almost exclusively protrude into virus particles with their barbed ends and are in close association with viral nucleocapsids. The data support previous suggestions that actin is involved in virus budding and show that budding itself is possibly the result of a vectorial growth of actin filaments.

Protein-A gold particles as markers in replica immunocytochemistry: high resolution electron microscope investigations of plasma membrane surfaces.

Due to their high atomic number contrast in transmission electron microscopy, gold particles are ideal markers in surface replicas of cultured cells. The suitability of protein‐A‐coated gold particles in replica immunocytochemistry for labelling surface antigens is demonstrated using measles virus‐infected cells as a model system. Labelled areas can easily be distinguished from unlabelled areas, and even markers positioned in the evaporation shadow of large structures can be accurately identified, which is a prerequisite for an exact quantification and mapping of antigen. In addition, the ultrastructure of labelled areas can still be visualized because of the small size of the marker.

Inhibition of measles virus budding by phenothiazines

HeLa cells infected with measles virus show an accumulation of virus-specific strands at the plasma membrane after addition of the anticalmodulin drugs trifluoperazine (TFP) and chlorpromazine (CPZ), whereas spherical virus particles are almost completely absent. At low drug concentrations (10-15 microM TFP; 30-40 microM CPZ) the inhibitory effect is dependent on the presence of extracellular calcium. The strands complete the budding process after removal of the drugs. Restoration of virus budding is not sensitive to cycloheximide and immunoprecipitation experiments give evidence that the viral protein synthesis is not qualitatively altered in the presence of TFP. The data indicate that both drugs arrest the budding process at an intermediate stage at the plasma membrane. The inability of the strands to comigrate with cytochalasin B-induced actin patches suggests that the inhibition of budding is probably the result of an impaired interaction of viral structures with the cytoskeleton.

Subclones of C6 rat glioma cells differing in intermediate filament protein expression

The C6 rat glioma cell line is shown to consist of a mixed population of cells which either contain vimentin (80% of the cells) or completely lack any cytoplasmic intermediate filament (IF) proteins. Subclones could be established with both phenotypes, indicating that these IF protein expression patterns represent stable phenotypic markers. Absence of IF proteins in C6 subclones could consistently be correlated with an altered cell morphology and a pronounced increase in the number of actin stress fibers. In vitro translation and hybridization assays suggest the absence of vimentin to result from a block at the transcriptional level. The data indicate that subcloning of the C6 cell line on the basis of IF protein expression seems to be a reasonable approach for obtaining homogeneous C6 cell populations which may represent suitable experimental models for studies on vimentin expression and glioma cell differentiation.

Production of monoclonal antibodies to measles virus proteins by immunization of mice with heated and detergent-treated antigens

Abstract By use of the mouse hybridoma technique, monoclonal antibodies were obtained with specificity for the HA(79K), P(72K), and M(36K) polypeptides of measles virus. BALB/c mice were immunized with native measles virus and measles virus treated with detergents and heat. Clones obtained after immunization of mice with native measles virus showed specificity for the HA(79K) polypeptide only. After immunization with measles virus, treated with 1% sodium sarkosyl sulfate (SSS) at 20°, a clone was obtained producing antibodies to the M(36K) polypeptide. Heating of measles virus in the presence of 1% sodium dodecyl sulfate (SDS) under reducing conditions elicited a selective immune response to the P(72K) and the NP(60K) polypeptides. Thus, clones producing antibodies to the P(72K) polypeptides were isolated.

Immunoelectron microscopy on the topographical distribution of the poliovirus receptor

The topographical distribution of the poliovirus receptor on the cell surface was demonstrated by immunoelectron microscopy using monoclonal antibodies and immunogold markers. The receptor appeared in small clusters, which were randomly distributed over the cell surface and along cellular processes. The distribution pattern of the clusters corresponded to that of absorbed and immunogold-labelled poliovirus particles and suggests a multivalent organization of poliovirus binding sites. Freeze-fracturing and ultrathin sectioning did not reveal any specific ultrastructures within the plasma membrane at labelled receptor areas. Incubation of native cells with anti-receptor antibodies did not remove the receptor molecule from the cell surface nor did it induce ultrastructural alterations within the plasma membrane. The antibody-receptor complexes exhibited lateral mobility within the plasma membrane and were able to aggregate into large immune complexes after incubation with a second ligand.

Morphogenesis of measles virus on C6 rat glioma cells

Rat glioma cells (C6) persistently infected with measles virus show a locally dissociated distribution of budding processes at the cell surface.