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B Rombaut

Publications and source records attributed to B Rombaut.

At least 37 records · Page 2Linked to original sources
PubMed1991

Formation of subviral particles by in vitro translation of subgenomic poliovirus RNAs.

Rabbit reticulocyte lysates were programmed with either RNA extracted from purified poliovirus or a mixture of mRNAs encoding the capsid precursor, P1, and proteinase 3CD. In both cases, 14S subunits were formed at 30 degrees C and empty capsids at 37 degrees C. Both the 14S subunits and empty capsids had the expected polypeptide composition and neutralization epitopes. It is concluded that the proteinase 3CD gene is the only viral genetic information needed for the correct processing of P1 and the formation of 14S subunits, and their assembly into antigenically correct empty capsids.

3C Viral Proteases↗
PubMed1991

A comparison of WIN 51711 and R 78206 as stabilizers of poliovirus virions and procapsids.

The thermal denaturation of poliovirus virions and procapsids in the 42 to 48 degrees C range was studied using N- and H-specific monoclonal antibodies. The half-life of the N antigen of Mahoney and Sabin 1 virions was extended 50- to 250-fold by either 10 microM WIN 51711 or R 78206. The minimum concentrations required for full stabilization at 46 degrees C (1.0 microM for WIN 51711, 0.2 microM for R 78206) were independent of the strain or serotype of the virus; 30 to 60 molecules of stabilizer per virion were required for full protection. R 78206 was the most efficient stabilizer of Mahoney procapsids; the half-life of the N-specific epitopes of these particles at 44 degrees C was extended from less than 1 min to 1 day.

Antiviral Agents↗
PubMed1990

Use of high-performance size-exclusion chromatography for the separation of poliovirus and subviral particles.

The size-dependent separation of viral and subviral particles in the range 10(5)-10(7) daltons was undertaken by high-performance liquid chromatography. A combination of Ultrahydrogel 2000 and 1000 size-exclusion columns, equilibrated and developed with Tris buffer (pH 7.4), was used to fractionate extracts of cells infected with radiolabelled poliovirus. Poliovirions (30 nm) and subviral particles (20 nm) were separated according to size with full retention to their biological activities. Procapsids (same size as virions, but devoid of RNA) could not be separated from virions. Sample recoveries as determined with radiolabelled material constantly exceeded 70%. The method was successfully applied to the separation of viral and subviral particles from complex mixtures.

Chromatography, Gel↗
PubMed1990

Creation of an antigenic site in poliovirus type 1 by assembly of 14 S subunits.

Using a set of neutralizing monoclonal antibodies targeted against the four known antigenic sites of poliovirus type 1, it was shown that three out of four antigenic sites are already present on 14 S subunits (pentamers of the structural unit). Site 3B, in contrast, is formed upon assembly of 14 S subunits into capsids. The data support the hypothesis that site 3B spans the boundary between pentamers in the virion.

Antibodies, Monoclonal↗
PubMed1990

New evidence for the precursor role of 14 S subunits in poliovirus morphogenesis.

In poliovirus-infected HeLa cells incubated as 30 degrees, 14 S subunits are selectively labeled and no virions are assembled. When the temperature is subsequently shifted to 37 degrees, the radioactivity of the 14 S material is transferred mainly to virions, showing that 14 S subunits are virion precursors. When 14 S subunits synthesized at 30 degrees are incubated in vitro at 37 degrees, they assemble to empty capsids.

Electrophoresis, Polyacrylamide Gel↗
PubMed1990

Purification of poliovirus 14 S subunits by sucrose gradient ultracentrifugation and high-performance size-exclusion chromatography.

Purification of 14 S subunits from extracts of poliovirus-infected HeLa cells was achieved by a combination of sucrose gradient ultracentrifugation and high-performance size-exclusion chromatography. The particles were free of admixtures of other subviral particles, of nonstructural viral proteins, and of host cell proteins. The purified material retained the physical and antigenic properties of native 14 S subunits fully, as well as their ability to assemble to empty capsids in vitro.

Antibodies, Monoclonal↗
PubMed1990

Disoxaril stabilization and immunogenicity of poliovirus procapsids.

Disoxaril (5-[7-[4(4,5-dihydro-2-oxazolyl)phenoxy]-heptyl]-3- methylisoxazole) protects poliovirus procapsids against alkaline dissociation and thermal denaturation up to 42 degrees C. When added during the purification of procapsids, it enhances their yield and antigenic quality. Disoxaril increases the immunogenicity of both purified virions and procapsids in mice. Whereas untreated procapsids mainly elicit H-specific antibodies, disoxaril-treated procapsids yield high titres of neutralizing antibodies. The prospect of using disoxaril-treated procapsids in vaccines is discussed.

Animals↗
PubMed1989

Denaturation of poliovirus procapsids.

The denaturation of poliovirus procapsids at pH 6.5 was studied, both in the frozen and liquid condition. Denaturation involved alteration of antigenic and physical features (isoelectric pH, alkali dissociability, sedimentation coefficient). Magnesium was a stabilizing factor, and the sedimentation coefficient was the most denaturation-sensitive feature.

Capsid↗
PubMed1988

Response of cultured rat Kupffer cells to lipopolysaccharide.

The effects of Salmonella abortus equi lipopolysaccharides (LPS) on pure cultures of rat Kupffer cells (Kc) were studied. In vitro, LPS is ingested by Kc and located in vacuoles and secondary lysosomes. Culture of Kc in the presence of 1-50 micrograms LPS/ml during 24 h did not affect the viability of the Kc as measured by trypan blue exclusion, neutral red uptake, lactate dehydrogenase leakage and cell survival and spreading. LPS treatment did not influence the ultrastructure of Kc. The exposure of Kc to LPS in vitro did not change the phagocytic activity. Several biochemical processes were stimulated: glucose consumption, MTT-tetrazolium salt reduction, total protein synthesis and secretion of proteins. LPS activated Kc to tumoricidal activity against L929 mouse fibrosarcoma cells. In the light of the above observations, it is concluded that purified LPS is not cytotoxic for pure Kc in culture. On the contrary, LPS stimulates several biochemical and functional processes.

Animals↗
PubMed1987

A pH-dependent dissociation of poliovirus procapsids.

At pH 7.6-8.2, poliovirus procapsids are dissociated in the cold to 14 S subunits which exhibit the same antigenicity and ability to be reassembled to empty capsids as naturally occurring 14 S subunits. The dissociation of the procapsids at pH 8.2 follows exponential kinetics. At pH 8.4 or 8.5, the procapsids are converted to 80 S H-antigenic, empty capsids.

Capsid↗
PubMed1986

Assembly factors in poliovirus morphogenesis.

Extracts of poliovirus-infected HeLa cells promoted the in vitro assembly of 14 S subunits into empty capsids antigenically indistinguishable from procapsids. When infected cells were treated with iodoacetamide, the extract lacked the assembly promoting activity. This activity was restored by the addition of heat-disrupted virions, but the empty capsids formed in this system were antigenically different from procapsids. This and other observations introduce a distinction between the "assembly promoting" and "antigenicity conferring" activities of infected-cell extracts.

Animals↗
PubMed1985

Characterization and assembly of poliovirus-related 45 S particles.

Using detergents, 45 S particles could be extracted from poliovirus-infected Vero and human embryonic kidney monolayer cells, but not from HeLa suspension cells. They were composed of the capsid proteins VP0, VP1, and VP3, devoid of RNA, and extremely sensitive to heat or to a slightly alkaline pH. The 45 S particles possessed neutralization epitopes of the N1 and N2 classes, as well as a VP3-linked epitope. The N2 epitopes were lost upon denaturation. In the presence of cell extracts 45 S particles were, like 14 S subunits, assembled to 71 S empty capsids expressing the N1 and N2 epitopes.

Animals↗
PubMed1985

Quantitation of bovine immunoglobulin G2 by a competitive fluorescence immunoassay.

Bovine immunoglobulin G2 (IgG2) was quantitated by fluoroimmunoassay (FIA) based on competition with fluorescein-conjugated IgG2 for Sepharose-bound anti-IgG2 antibodies. The optimal range was 0.1 to 1 microgram IgG2. The FIA was compared with the single radial immunodiffusion (SRD) test. Both methods gave comparable means and standard errors for IgG2 in serum. The FIA method was 30 times more sensitive.

Animals↗
PubMed1985

Hit-and-run neutralization of poliovirus.

At physiological ionic strength, monoclonal antibody 35-1f4 has previously been shown to neutralize poliovirus type 1 by antibody-mediated polymerization. At low ionic strength, this antibody neutralized the virus by a hit-and-run mechanism: the virions were converted to non-infectious, empty capsids devoid of antibodies. These empty capsids resembled those formed by thermal denaturation of native polio virions in their sedimentation coefficient (80S), antigenicity (H) and isoelectric pH (6.3).

Antibodies, Monoclonal↗
PubMed1985

Stabilization by host cell components and Mg2+ of the neutralization epitopes of poliovirus.

When type 1 poliovirions were extracted from infected HeLa cells and heated at 56 degrees C in the presence of 1 mM-Mg2+, RNA-free, pluri-antigenic particles were formed. These particles possessed neutralization epitopes as well as epitopes associated with H antigen and with the isolated capsid protein VP3. The heating caused the formation of exclusively H antigenic particles when the Mg2+ concentration was 10(-4) M or lower, or when the virus was further purified in order to remove remaining cellular components. The ability of purified virus to form pluri-antigenic particles upon heating could be restored by incubation at 37 degrees C with infected cell extract.

Antibodies, Monoclonal↗
PubMed1984

In vitro assembly of poliovirus empty capsids: antigenic consequences and immunological assay of the morphopoietic factor.

The assembly of poliovirus 14 S particles into empty capsids was studied without cell extract (self-assembly) and in extracts of infected or uninfected HeLa cells. The products were analyzed using monoclonal antibodies specific for N1, N2, or H epitopes. The empty capsids formed in infected cell extract, and only those, possessed N2 epitopes like the procapsids formed in vivo, thus showing that a virally encoded or induced factor determines the antigenicity of the assembly product. Based on this observation, a simple immunological assay for the activity of the morphopoietic factor is presented. This factor is shown to lack serotype specificity.

Antibodies, Monoclonal↗
PubMed1984

Intertypic cross-reactions of nonneutralizing, monoclonal poliovirus antibodies.

Quantitative data are presented on (i) the intertypic cross-reactions of polyclonal, guinea pig antibodies directed against the N or H antigen of type 1 poliovirus and (ii) a set of five nonneutralizing, mouse hybridoma antibodies raised against N antigen or a mixture of capsid polypeptides VP1, VP2, and VP3. Three of these antibodies recognize H antigen and VP1, the fourth H antigen only, and the fifth VP3 only. The antibodies recognize either only homotypic antigens or the antigens of the three serotypes.

Animals↗