Uptake of poliovirus into the endosomal system of HeLa cells.

To 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 degrees 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.

Stabilisation of poliovirus with pirodavir.

Two hundred and forty pyridazinamine derivates were assayed for their ability to stabilize the Oral Polio Vaccine (OPV). Of these, pirodavir (R 77975) was selected for vaccine potency tests. Although pirodavir turned out to be an effective stabilizer of OPV, the protection induced by pirodavir was not better, nor additive to the effect by MgCl2 1 M, the usual stabilizer. Both stabilizers were effective in preventing damage to the viral proteins, but could not prevent damage of viral RNA.

Heat stabilized, infectious poliovirus.

Polioviruses type 1 (Mahoney) and type 3 (Sabin) were treated with the antiviral pyridazinamine R78206 by first binding the compound to the virus and then removing unbound compound by dialysis. As a result of this treatment, both poliovirus strains were protected against thermal inactivation at 46 degrees C. The R78206 treatment did not cause inactivation except with the Sabin 3 strain at high R78206 concentrations.

Poliovirus infection without accumulation of eclipse particles.

Poliovirus type 1 (Mahoney) was treated with the capsid-binding pyridazinamine R 78206, followed by dialysis to remove free compound. Upon infection of HeLa cells by R 78206-pretreated virus, the formation of intra- and extracellular modified particles was completely inhibited, except for a small amount of empty capsids. The synthesis of viral proteins and first cycle progeny virus was delayed by 1 h. The results suggest that poliovirus infection does not require intracellular accumulation of 135 S eclipse particles.

Thermal inactivation of oral polio vaccine: contribution of RNA and protein inactivation.

Heating the Sabin strains of poliovirus at 42 to 45 degrees C caused inactivation, loss of native antigen, and release of the viral RNA (vRNA). The loss of virion infectivity exceeded the loss of vRNA infectivity (as measured by transfection) by roughly 2 log10. Pirodavir inhibited the loss of native antigen and RNA release and reduced the loss of virion infectivity to the same level as the loss of vRNA infectivity. Thermoinactivation thus involves an RNA and a protein component, and pirodavir protected only against the latter.

Antibody neutralization of picornaviruses: can fever help?

Binding of bivalent antibody can neutralize picornaviruses by a variety of mechanisms. Some monoclonal antibodies can irreversibly neutralize the virus at temperatures that are higher than physiological by disrupting the virion, leading to ejection of the RNA. Fever may enhance this process in vivo, confirming the popular belief in the virtues of fever.

Formation of poliomyelitis subviral particles in the yeast Saccharomyces cerevisiae.

The sequence of the poliovirus genome encoding 3CD (a protease) was transferred to the yeast Saccharomyces cerevisiae on expression vectors with either a constitutive or an inducible promoter. Transformants could only be obtained with vectors carrying the inducible transcription unit. Extracts of induced cells were able to cleave cell-free synthesized P1, the precursor of the poliovirus capsid proteins, into VP0, VP3 and VP1. In yeast cells constitutively expressing P1, induction of 3CD expression resulted in only trace amounts of processed products. Processing could be improved considerably by simultaneous induction of both P1 and 3CD expression. Analysis of extracts of such induced cells revealed the presence of particles that resembled authentic subviral particles.

A competition immunoprecipitation assay of unlabeled poliovirus antigens.

An assay method is described for unlabeled, unpurified N(ative) and H(eated) antigens of poliovirus. The method is based on competition between the unlabeled antigen and a standard quantity of radiolabeled antigen, in the presence of a limiting amount of a N- or H-specific, monoclonal antibody. The immune complexes are removed by protein A-bearing, fixed staphylococci. The method is free from cross-reaction between N and H antigen, and has a detection limit of approximately 2 nM. It was applied successfully to the quantitation of poliovirus antigen synthesized by recombinant yeast expressing the viral proteins P1 and 3CD.

Discrepancy between infectivity and antigenicity stabilization of oral poliovirus vaccine by a capsid-binding compound.

Two hundred forty pyridazinamine derivatives were tested for the ability to stabilize the antigenicity and infectivity of oral poliovirus vaccine subjected to 45 degrees C for 2 h. Seven compounds stabilized the antigenicity of all three vaccine strains and neutralized the viral particles in a way that is reversible by dilution. Of these, R 77975 (pirodavir) was selected for vaccine potency tests. Sabin type 2 and type 3 strains were subjected to 4, 25, 42, and 45 degrees C for 1 week in the presence and absence of R 77975. Although R 77975 particularly stabilized the infectivity of the most thermolabile vaccine strain (Sabin type 3), the protection did not exceed that of 1 M MgCl2. When virus was inactivated in the absence of R 77975, the native or N antigenicity changed in H antigenicity. However, in the presence of the capsid-binding compound, N antigenicity was preserved in particles that had lost infectivity.

Intracellular proteolysis of poliovirus eclipse particles is abortive.

A series of weak bases and the ionophore monensin were tested for their effect on the intracellular processing of type 1 poliovirus in HeLa cells. At concentrations that did not inhibit plaque formation or viral protein synthesis, the compounds suppressed the proteolytic processing of 135S particles and the formation of 110S particles. In addition, some compounds strongly reduced transit of modified particles to the lysosomes. These results suggest that transit to lysosomes and proteolysis of subviral particles are not essential steps in the infectious pathway. The role of 135S particles as intermediates in infection is discussed.

Capsid destabilization is required for antibody-mediated disruption of poliovirus.

Three out of 36 poliovirus type 1-specific monoclonal antibodies which, at 37 degrees C in a medium of normal ionic strength (mu = 0.16), caused only aggregative neutralization (reversible by immune complex dissociation at pH 2) shifted to cause disruptive, acid-irreversible neutralization when the temperature was raised to 39 degrees C or the ionic strength was lowered to 1/100 of normal. Under both conditions, the antigenic conversion was stoichiometric, but the efficiency was lower at 39 degrees C than at low ionic strength. Antigenic conversion and irreversible neutralization under both conditions were inhibited by WIN 51711, a capsid-stabilizing compound. Complete inhibition required filling of most of the virion's binding pockets by this compound.

Monoclonal antibodies that disrupt poliovirus only at fever temperatures.

Three of thirty-six monoclonal antibodies were found to cause irreversible inactivation of type 1 poliovirus at 39 degrees C but not at 37 degrees C. Neutralization at 37 degrees C depended on aggregation and was reversible by acid-induced deaggregation; at 39 degrees C, the virions (N antigenic, 160S) were disrupted to empty capsids (H antigenic, 100S), and neutralization was irreversible. The rate of antibody-dependent conversion of N to H antigen increased steeply between 37 and 39 degrees C.

Postabsorption neutralization of poliovirus.

Nineteen neutralizing murine monoclonal antibodies against poliovirus type 1, including representatives reacting with each of the antigenic sites on the virion, were tested for their abilities to neutralize the virus either before or after attachment to susceptible cells. All antibodies neutralized unattached virus; six had reasonable titers of postabsorption neutralization (PAN). Experiments with antibodies lacking PAN activity showed that Fc-specific rabbit anti-mouse antibodies could confer PAN activity. PAN was shown to involve the prevention of the cell-mediated conversion of virus to 135S and 80S particles. Evidence that one of the PAN-positive antibodies probably bound bivalently to preabsorbed virions, whereas a PAN-negative antibody bound monovalently, is presented. Two PAN-positive antibodies were added to an excess of virus in suspension, and only one antibody caused the virus to aggregate.

Effect of a capsid-stabilizing pyridazinamine, R 78206, on the eclipse and intracellular location of poliovirus.

R 78206 (a pyridazinamine derivative) inhibits the formation of poliovirus eclipse particles. Its effect on the intracellular location of poliovirus was studied by separating subcellular fractions in iso-osmotic Nycodenz gradients. The compound did not inhibit internalization of intact virus into small lipid vesicles, but it did inhibit the release of virus from these vesicles and its entry into lysosomes.

Compartmentalization of subviral particles during poliovirus eclipse in HeLa cells.

HeLa cells were preincubated with radiolabelled poliovirus type 1 at 26 degrees C, such that the 160S virions were internalized, but not altered structurally. The temperature was then shifted to 37 degrees C to study the intracellular redistribution of the virions and the modifications they undergo at that temperature. Using subcellular fractionation in isoosmotic Nycodenz gradients, we obtained evidence for the rapid loss of virions from the plasma membrane and from a vesicular fraction, as well as for the formation of two populations of intracellular 135S particles. The first population was associated with lysosomes and was slowly converted to (RNA-containing) 110S particles. In the presence of the lysosomotropic agent chloroquine, the lysosomal 135S population was converted to 80S empty capsids. The second 135S population, which was not associated with any organelle, was converted to 80S empty capsids. Similar observations were made during unsynchronized infection at 37 degrees C. We propose a model for infection in which 135S particles cross a membrane barrier, and are uncoated in the cytosol.

Antigenic N to H conversion of poliovirus by a monoclonal antibody at low ionic strength.

Monoclonal antibody 35-1f4 at low ionic strength converted native virions (N antigen) to noninfectious H-antigenic, empty capsids. The reaction was stoichiometric, as the amount of N antigen that could be converted to H was limited to an average of 2 virions per molecule of antibody. The antibody remained associated with virus aggregates after antigenic conversion. Using antibody immobilized onto protein A-bearing staphylococci, it could be shown that the loss of antigen-converting power was concomitant with the loss of antigen-binding ability. Only a small amount of viral protein (equivalent to 0.02 empty capsid per molecule of antibody) remained attached to the antibody. Heating to 56 degrees caused most of this material to be released and restored the antibody's antigen-binding and antigen-converting abilities. Several possible explanations for the heat-reversible inactivation of the antibody are discussed.

Internalization of intact poliovirus by HeLa cells as shown by subcellular fractionation in isoosmotic Nycodenz gradients.

HeLa cells were infected with radiolabelled poliovirus at different temperatures, and the intracellular distribution of input radioactivity was studied. To this end, homogenates were fractionated by rate zonal centrifugation in linear isoosmotic (2 to 30%) Nycodenz gradients. Further purification of subcellular fractions was achieved by recentrifugation to equilibrium in 10 to 30% Nycodenz. Temperatures were kept below 30 degrees C to prevent virus capsid modification. Under these conditions, the cell-associated virions remained fully infectious. Below 18 degrees C, most of the viral label was recovered from a bottom region (BR) of the rate zonal gradients. Marker enzyme analysis and antibody accessibility showed that the BR consisted of virions bound to the plasma membrane. Between 18 degrees C and 26 degrees C, viral label also accumulated in a top region (TR) of the rate zonal gradients. According to the criterion of antibody accessibility, the virions associated with the TR were present within intracellular structures, probably lipid membranes. Electron microscopy confirmed the presence of vesicles and tubules in this region of the gradient. No correlation was found between the TR and endosomal, lysosomal or plasma membrane markers. The TR equilibrated at low density (1.10 g/ml) in Nycodenz (free virus, 1.31 g/ml). The results confirm that intact poliovirions can enter the cell and do so via lipid-bound vesicles.

Chloroquine induces empty capsid formation during poliovirus eclipse.

The poliovirus capsid (160S) is modified during eclipse in HeLa cells, which results in at least three types of particles having sedimentation coefficients of 135, 110, and 80S. The lysosomotropic agent chloroquine redirected the production of eclipse products from 135 and 110S particles (containing RNA) to 80S particles (without RNA). The effect started at 5 microM and was fully developed with 20 microM chloroquine. Viral protein synthesis and virion production remained unaffected. The results show that chloroquine can redirect the processing of input virions without interfering with productive uncoating.

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.