Polyadenylic acid sequences in the virion RNA of poliovirus and Eastern Equine Encephalitis virus.

Poliovirus virion RNA contains a single covalently bound sequence of polyadenylic acid which is approximately 49 nucleotides long. A single, slightly longer polyadenylic acid sequence is contained in Eastern Equine Encephalitis virus RNA. Since these viruses are otherwise dissimilar these sequences may play a common role in viral replication, possibly in translation of the viral RNA.

Structure of poliovirus replicative intermediate RNA. Electron microscope analysis of RNA cross-linked in vivo with psoralen derivative.

The structure of the poliovirus replicative intermediate RNA was examined by electron microscopy after cross-linking in vivo with 4'-aminomethyl-4,5',8-trimethylpsoralen. After purification from infected cells, undenatured RI appeared as a double-stranded backbone of genome length, with an average of three (and occasionally up to eight) nascent, single-stranded tails. After denaturation, however, only single strands of heterogeneous length were visualized, indicating that the RI in the cell contains little or no duplex structure, and thus nascent chains are only transiently hydrogen-bonded to their template over short regions. The double-stranded backbone of undenatured RI, observed previously by others and in these experiments, is due to collapse of complementary chains during the deproteinization and purification procedures. The effectiveness of the in vivo cross-linking procedure was demonstrated by the complete inhibition of viral RNA synthesis in treated cells and by direct binding of [3H]AMT to RI molecules in vivo. Mature polio virions are impermeable to AMT; however, growth of virus in cells incubated with AMT in the dark resulted in normal yields of virus particles containing RNA genomes, whose infectivity could be subsequently photo-inactivated. The frequency of AMT-induced cross-linking was determined by analyses of double-stranded poliovirus RNA (RF). Cross-linking in vitro followed by spreading for electron microscopy under denaturing conditions yielded bubbled duplex structures with a minimum of one interstrand cross-link per 80 base-pairs. RF cross-linked in vivo also showed extensive cross-linking, decreased about fivefold from the in vitro cross-linked value. Thus, the failure to detect cross-linked RI under these conditions indicates that extensive base-pairing does not exist in vivo.

In situ enzyme immunodetection of surface or intracellular bacterial antigens using nitrocellulose sheets.

We describe an immunological method which allows the in situ colorimetric detection of translated DNA fragments in bacteria. In the absence of lysis only cell surface proteins are detected. For cytoplasmic proteins, lysis is required. The procedure comprises the following steps: bacteria are lysed, the proteins are transferred onto a disc of nitrocellulose sheet, the remaining protein sites are blocked, the disc is successively soaked in a solution of antibodies specific for the protein to be detected and in a solution of peroxidase-labelled anti-IgG antibody solution. Finally, the immune complexes are made visible by enzyme substrate incubation. We describe the application of this method to the detection of the LamB protein, the LacZ protein, and a LamB-polio VP1 chimera translated from cloned DNA fragment in E. coli.

Rapid analytical and preparative separation of structural polypeptides of poliovirus by reverse-phase high-performance liquid chromatography.

A reverse-phase high-performance liquid-chromatography (RP-HPLC) technique was developed for rapid (20 min) and effective separation of the structural polypeptides of poliovirus of strains of all three serological types. The method is useful for quantitative analysis as well as for isolation of polypeptides on a micropreparative scale for chemical, biochemical and immunological studies. All four virus polypeptides (VP1 to VP4) were obtained quantitatively in high purity by this one-step procedure. The solvents used were volatile and easily removed by evaporation, giving dry, amorphous polypeptides free of any additives. The separation mechanism of this RP-HPLC is based on the hydrophobicity of the protein surface. It is distinct from other separation methods such as molecular sieving and isoelectric focusing. Therefore, differences in primary and secondary structure of a polypeptide can be detected by RP-HPLC.

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.

Agarose isoelectrofocusing of intact virions.

A convenient and accurate method for determining the isoelectric points of intact virions is described. Tritium-labeled poliovirus 1 (strains Brunhilde and LSc-2) and echovirus 1 (isolates V239, V248, V212, R115 and 4CH-1) were successfully focused into sharp bands at their respective isoelectric points using a thin-layer agarose isoelectric focusing system. In situ detection of labeled virus bands in the agarose was by fluorography. Freezing and thawing of virus samples prior to isoelectric focusing did not alter their respective isoelectric points.

Protections by non-copper metal ions against copper-mediated inactivation of poliovirion RNA occurring at extraction of virions with phenol.

Al3+, Ca2+, Co2+, Cu1+, Fe2+, Fe3+, Mg2+, Mn2+, Ni2+, Pb2+, Sn4+, and Zn2+ were incubated individually with redistilled reagent-grade phenol containing impurities known from previous work to interact with Cu2+ to produce a potent inactivator(s) of the transfectivity of naked poliovirion RNA. Only the mixture with Cu1+ inactivated the RNA. Tests of each of the 11 non-copper test metal ions mixed with Cu2+ before adding the phenol showed that Ca2+ and Mg2+ do not protect, Co2+, Mn2+, Ni2+, Pb2+, and Zn2+ provide moderate protection, and Al3+, Fe2+, Fe3+, and Sn4+ give strong protection against the Cu2+-mediated inactivation. Other points of addition of protective metal ion were tested using Fe3+. Strong protection was afforded even when Fe3+ was added after synthesis of the inactivator(s) from Cu2+ and the active impurities. The relation between Cu2+ and the Fe3+ was shown to be competitive. The hypothesis that ions compete for semi-quinone anion is proposed.

[Primary structure of the crossover region in the genome of two intertype poliovirus recombinant]. / Pervichnaia struktura oblasti perekresta v genome dvukh mezhtipovykh rekombinantov poliovirusa.

The nucleotide sequence of the crossover region on genomes of two intertypic (type 3/type 1) poliovirus recombinant, has been determined by the primer extension method. No deletions, insertions or rearrangements have been observed. Identical contiguous sequences, 7 or 11 nucleotides in length, respectively, have been found in two regions of the parental genomes, involved in the recombination.