Raf Vrijsen

Eclipse products of poliovirus after cold-synchronized infection of HeLa cells.

The particles derived from radioactively labeled poliovirus, in cold-synchronized infection of HeLa cells, were studied. After temperatures shift-up, radioactivity was transferred rapidly from the infecting virions (160 S) to 135 S, and from there more slowly to 110 S, a previously unreported eclipse product. Both the 135 and 110 S particles contained RNA, lacked VP4, and were H-antigenic. In the 135 S particles, part of the VP1 complement was lost; in addition to this, the VP2 polypeptide of the 110 S particles was cleaved in situ. The formation of eclipse products was insensitive to cycloheximide. Arildone totally blocked the formation of 135 S particles, but not their further processing.

Hybridoma antibodies to poliovirus N and H antigen

SummaryA set of 13 hybridoma antibodies to type 1 poliovirus has been studied with regard to neutralization and binding to N antigen, H antigen and capsid proteins. Two hybridomas produced heterogeneous antibodies, even after repeated attempts to reclone them.

A simple quantitative protein A micro-immunoprecipitation method; assay of antibodies to the N and H antigens of poliovirus.

Staphylococcus aureus (Cowan strain I) was used to absorb immune complexes from antiserum to poliovirus to which labeled N or H poliovirus antigens had been added, and the radioactivity in the pelleted organisms and in the supernatant was measured. Excellent agreement was obtained between values calculated separately from the pellet and supernatant readings, validating the use of supernatant measurements from a microtitration plate method.

A pH-dependent antigenic conversion of empty capsids of poliovirus studied with the aid of monoclonal antibodies to N and H antigen

Abstract The antigenic specificity and sedimentation coefficient of poliovirus empty capsids prepared at 4° from infected HeLa cells differ according to the pH of the extraction buffer: at pH 6.0 these particles are N antigenic (65 S), but at pH 8.5 they are H (80 S). N (65 S) particles are rapidly converted to H (80 S) at pH 8.5, 4°. The time course and the influence of temperature and pH on this conversion is studied. The N antigen associated with 65 S particles is more sensitive to alkaline pH than that of 160 S particles.

The poliovirus-induced shut-off of cellular protein synthesis persists in the presence of 3-methylquercetin, a flavonoid which blocks viral protein and RNA synthesis

In poliovirus-infected cells, the viral protein and RNA synthesis were severely reduced, provided 3-methylquercetin was present between 1 and 2 h post-infection. Under these conditions, the virally induced host shut-off remained in effect. On the other hand, in uninfected HeLa cells, protein and RNA synthesis was inhibited only slightly by 3-methylquercetin. The inhibition of poliovirus cytopathogenicity in Vero cells by 3-methylquercetin exhibited a similar time dependence.

Epitope evolution in poliovirus maturation

SummaryEight monoclonal antibodies specific for native (N) poliovirus antigen all recognized empty capsids extracted from infected HeLa cells; only three of them recognized 14S particles. The facts point to the existence of two different epitopes N1 and N2, only one of which (N1) is present on 14S particles (these observations confirm and extend findings byEminiet al). Another epitope of 14S particles is recognized by antibodies to heated (H) poliovirus antigen.Infected cell extract-catalyzed polymerization of 14S particles yielded mainly empty capsids carrying the N1 and N2, but no H epitopes.

Metabolic activation of quercetin mutagenicity

The mutagenicity of quercetin was reinvestigated using the Salmonella/microsome test. The mutagenicity of quercetin was enhanced by the cytosolic fraction of liver extract (S100), or by ascorbate, and even more by the complete liver supernatant (S9) in the presence of cofactors (NADP and glucose-6-phosphate). The formation of metabolites by the S9 enzymes was demonstrated by reverse-phase HPLC.

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.

Uptake of poliovirus into the endosomal system of HeLa cells.

SummaryTo understand the topology and mechanism of poliovirus uncoating, the question of whether intact virions can be endocytosed by the host cell was studied by a combination of various techniques. In order to prevent alteration of the virus to subviral particles, Hela cells were infected at 26 °C. At this temperature the majority of cell-associated virions remained at the plasma membrane, whereas a smaller amount accumulated in vesicles having the same mobility (upon free-flow electrophoresis) and migration behaviour on Nycodenz density gradients as early and late endosomes. Co-localization of native poliovirions with endosomal markers was verified by peroxidase-induced diaminobenzidine density-shift of endosomal vesicles. Internalization of poliovirions into endosomes makes it likely, but does not prove that viral RNA can be released into the cytoplasm from the vesicular compartment.

Denaturation of poliovirus procapsids.

SummaryThe 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.