In vitro assembly of poliovirus. II. Evidence for the self-assembly of 14 S particles into empty capsids

A small (14S) virus-specific particle, isolated from poliovirus-infected HeLa cells, is able to self-assemble into a 73S particle. This 73S particle, when negatively stained and examined in the electron microscope, resembles the empty capsids found in infected cells. The kinetics of the self-assembly reaction was determined and found to be similar, but not identical, to the extract-mediated assembly of 14S particles into 73S particles. The self-assembly reaction, unlike the extract-mediated reaction, exexhibits a marked dependence on the initial concentration of 14S particles. The polypeptide content of 14S particles has been determined.     

In vitro encapsidation of plasmid DNA into human adenovirus empty capsids

Plasmid DNA, added to extracts of human adenovirus type 3-infected HeLa cells, binds to empty viral capsids and can be purified using cesium density gradient centrifugation. The fraction of DNA bound depends on the amount of DNA added to the extract, and the capsid partially protects the bound DNA from digestion by DNase I. This capsid binding of plasmid DNA does not require the presence of the adenovirus DNA packaging sequence. However, the presence of the adenovirus packaging sequence in the plasmid results in better protection of the bound plasmid molecule from cellular nucleases.     

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

Evidence is presented that the body temperature of chickens at 41° selects virus recombinants with a genome constellation necessary for pathogenicity in chickens. It could be shown that recombinants of influenza A viruses pathogenic for chicken grow equally well at 37 and 41° whereas the nonpathogenic recombinants have a significantly lower growth rate at 41°. Hybridization studies with recombinants derived from fowl plague virus and A/turkey/England/63 revealed that at 41° such isolates are selected which possess all the genes coding for the polymerase complex from one wild type parent virus. Furthermore, only pathogenic recombinants showed an increase in plaque size when the incubation temperature was shifted from 37 to 41°. The behavior of virus recombinants at elevated temperature therefore is a useful marker for rapid in vitro selection of pathogenic or nonpathogenic recombinants.     

Composition of artificially produced and naturally occurring empty capsids of poliovirus type 1

The polypeptide composition of the empty capsids of poliovirus type 1 represented either by the 73 S component isolated from sucrose gradients or by the top component from CsCl gradients is different from that ot the whole infectious virion. One protein (VP4) is absent from the empty capsids, another (VP2) is lower in relative amount, while an additional protein (NCVP6), not present in purified virions, is found in large amounts. Artificial production of 73 S particles by borate buffer, pH 10.5, treatment of purified virions results in removal of one protein component (VP4) and the RNA of the virus. The observed differences in composition of these virus-specific particles are discussed with regard to their possible role in the architecture and the assembly of the virion.     

In vitro assembly of polioviruses. IV. Evidence for the existence of two assembly steps in the formation of empty capsids from 14 S particles.

Evidence obtained from kinetic studies indicated that the assaembly reaction mediated by cytoplasmic extract prepared from poliovirus-infected HeLa cells was conposed of at least two distinct activities. One activity (assembly activity) converted mature 14 S particles into 73 S empty capsids. The other activity (activation) seemed to act on “immature” 14 S particles, enabling them to be assembled. Unlike the assembly activity, the activation reaction was depleted rapidly during the reaction in vitro. The activation factor(s) was not associated with isolated 14 S particles themselves but, like the assembly activity, appeared to be associated with the rough membrane fraction obtained from infected cells.     

In vitro assembly of polioviruses. 3. Assembly of 14 S particles into empty capsids by poliovirus-infected HeLa cell membranes

Rough membranes obtained from poliovirus-infected HeLa cells have the capacity to assemble 14 S particles into 73 S empty capsids in vitro. A corresponding fraction from uninfected cells did not possess this activity. When labeled 14 S particles were incubated with smooth membranes obtained from infected cells, a significant amount of radioactivity sedimented as heterogeneous material in the 30–70 S region of the gradient.Sucrose gradient analysis of ¹⁴C-labeled rough and smooth membrane fractions lysed with deoxycholate (DOC) demonstrated the presence of 14 S particles, 73 S empty capsids, and a 110 S structure in the rough membrane fraction. These structures were found only in trace amounts in the smooth membrane fraction. In the absence of DOC treatment, no 14 S particles were found in any of the membrane fractions. The 73 S empty capsids, on the other hand, were detected in the presence or absence of DOC treatment in the rough-membrane fraction. Therefore, it appears that 14 S particles are associated with the rough membranes where they are assembled into 73 S empty capsids and/or complete virions.     

In vitro assembly of poliovirus 14 S subunits: identification of the assembly promoting activity of infected cell extracts.

Purified poliovirus 14 S subunits are assembled into empty capsids in vitro only if their concentration exceeds a 1.6 nM threshold. This also holds true for the 14 S subunits in unpurified extracts of infected cells. Such an extract may promote the assembly of extraneous 14 S subunits, but only if it contributes enough 14 S subunits to raise their total concentration over the threshold. As a result of assembly, the concentration of 14 S subunits in an infected cell extract decreases exponentially, with a half life of 15 min at 37 degrees. When purified 14 S subunits of serotype 1 are mixed with extracts of cells infected with type 2 or 3, chimeric empty capsids are formed, thus showing the pooling of endogenous and extraneous 14 S subunits. In conclusion, the assembly promoting activity of infected cell extracts amounts to nothing more than the supply of endogenous 14 S subunits.     

Isoelectric points of polypeptides of standard poliovirus particles of different serological types and of empty capsids and dense particles of poliovirus type 1.

The isoelectric points of polypeptides of standard and dense poliovirus particles and of empty capsids have been determined by isoelectric focusing in urea and by two-dimensional analysis. Comparing virus strains belonging to the three serological types of poliovirus, differences in the pI of some, but not all of the structural polypeptides are found. The pI of polypeptides of dense particles and of empty capsids are identical with those of standard particles. Polypeptide VPo present in empty capsids has a pI between those of VP4 and VP2.     

In vitro assembly of poliovirus: V. Evidence that the self-assembly activity of 14 S particles is independent of extract assembly factor(s) and host proteins

Experiments were performed to determine whether or not the self-assembly of 14 S precursor particles into empty capsids was caused by the contamination of certain 14 S isolates with assembly factor(s) present in poliovirus-infected cell extracts. The self-assembly capacity of 14 S particle preparations was found to be directly related to the amount of viral-specific protein present. Dilution of 14 S particles markedly inhibited their self-assembly activity, whereas using ultrafiltration methods to concentrate 14 S particles increased their self-assembly activity. No consistent relationship was found for the presence or absence of particular viral or host proteins and the ability of 14 S particle preparations to self-assemble. The self-assembly capacity of 14 S particles was more sensitive to uv-inactivation than their ability to assemble into empty capsids in the presence of infec cted cell extracts. On the basis of these data and a detailed reanalysis of the effect of relative initial 14 S particle concentration on the rate of formation of empty capsids in extracts, we propose that the assembly of 14 S particles into empty capsids occurs in two steps, an initiation event and subsequent polymerization, and that extracts act by promoting the initiation event.     

Binding of neutralizing monoclonal antibodies to empty capsids of poliovirus can be blocked by monospecific antisera to structural polypeptides VP1 and VP2

Summary Binding of two neutralizing monoclonal antibodies (Nt-mAbs) to natural empty capsids (NEC) of poliovirus, type 1, was blocked to the extent of 83 per cent to 98 per cent by monospecific rabbit antisera directed against the structural polypeptides VP1 and VP2. Monospecific antisera against VP3 or VP4, however, did not show this blocking effect. It is therefore assumed that VP1 and VP2 are located close together at the antigenic sites for the two mAbs.With 1 Figure     

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.     

In vitro metabolism of phenstatin: potential pharmacological consequences

Phenstatin and its derivatives are potential anticancer drug candidates according to their inhibitory properties on tubulin polymerization, cell growth and antivascular activity. However, at the present time, neither pharmacological nor metabolic studies have been conducted in order to strengthen the relevance of phenstatine as a drug discovery candidate. In the present work, the metabolic fate of phenstatin in rat and human microsomal preparations was studied to investigate the stability of this tubulin polymerization inhibitor and any effects of the metabolites on polymerization and on PC3 cancer cell proliferation. The metabolites were separated by high-performance liquid chromatography and, after their synthesis, characterized by simultaneous LC-DAD-UV and LC-ESI-MS analyses. Thus, eight metabolites were identified. The major biotransformation pathways are carbonyl reduction, O-methylation at C-3', O-methylation after aromatic hydroxylation at the position C-2' on phenyl B ring and O-demethylation on A ring. Four of the identified metabolites were as active or more active, than phenstatin in vitro. Moreover, the better stability of phenstatin versus CA-4 and the lack of quinone formation could justify the design of new analogues which could include various substituents on phenyl rings or linker group in order to modulate the metabolism of phenstatin toward even more active metabolites and so up-regulate the pharmacological activity.     

In vitro assembly of poliovirus 14 S subunits: disoxaril stabilization as a model for the antigenicity conferring activity of infected cell extracts.

14 S subunits of poliovirus at 37 degrees undergo a rapid thermodenaturation, whereby all neutralizing epitopes are lost. This denaturation is prevented by 1 microgram/ml disoxaril, up to 42 degrees. In the absence of disoxaril, self-assembly of 14 S subunits at 37 degrees yields H-antigenic empty capsids. However, in the presence of disoxaril, and provided the molar ratio of disoxaril per 14 S subunit exceeds 7, the empty capsids generated by self-assembly are N-antigenic, and indistinguishable from native procapsids extracted from infected cells.     

In vitro selection of Junin virus antigenic variants

Summary The establishment of an experimental persistent infection with Junin virus, the aetiological agent of argentine hemorrhagic fever, involves the emergence of antigenic variants in brain and blood of the cricetidCalomys musculinus. We demonstrate that antigenic variants can also be isolated in vitro under the selective pressure of polyclonal antibodies and from a long-term infectedC. musculinus primary embryo fibroblast culture. The participation of neutralizing antibodies and host cells in the appearance of viral variants in vivo is discussed.     

In vitro cultivation of nonlymphoid thymic cells: Morphological and immunological characterization

Nonlymphoid thymic elements play an important role in T-lymphocyte development, especially in the development of recognition of transplantation antigens (H-2 in the mouse). Understanding this process will require the isolation and characterization of these cells. A simple technique for the culture of an enriched population of murine thymic epithelium is described. The epithelial nature of these cells is evidenced by their morphology, electron microscopy, and keratin content. Readily distinguishable macrophages comprise a secondary population within these cultures. Antigens encoded in the I-A region of H-2 were found on 70% of thymic epithelial cells and H-2K on 30% of thymic epithelial cells. These antigens were generally present on distinct populations but doubly positive cells were observed. Thymic macrophages were found to have conventional receptors for the Fc fragment of immunoglobulin on their surface and could ingest antibody-coated sheep erythrocytes. Thymic epithelial cells did not have such Fc receptors. A striking observation was that thymic epithelial cells could bind and internalize autologous thymocytes. This selective thymocyte ingestion by thymic epithelium may have important implications in regard to processing of T-lymphocyte precursors.     

In vitro synthesis of Trypanosoma cruzi antigenic polypeptides.

A ribosomal fraction of Trypanosoma cruzi was isolated with detergent lysis and differential ultracentrifugation. This ribosomal fraction directed in vitro protein synthesis, in a heterologous incorporation system (rat liver pH 5 fraction), leading to values up to 10 times higher than endogenous control. The ideal concentrations of Mg2+, K+, and ribosomal proteins and the time of incubation of the incorporation mixtures were 6 mM, 21 mM, 60 microliters, and 45 min, respectively. The product thus obtained was analyzed by fluorography after polyacrylamide-sodium dodecyl sulfate gel electrophoresis and showed the presence of many protein bands, but few bands were present in the immunoprecipitate obtained with serum from Chagas' disease patients. This product was able to react with anti-T. cruzi antibodies when submitted to an immunoenzymatic assay.