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A fractionated cell-free system from Krebs-2 cells was prepared which contained ribosomes and a high-speed supernatant. When this system was programmed with encephalomyocarditis virus RNA, the synthesis of a precursor of capsid proteins, polypeptide preA, proceeded at a rate not very different from that observed in unfractionated extracts, whereas the synthesis of more distally encoded proteins, in particular polypeptide F, was greatly retarded, if not abolished. A protein was purified from the cytoplasmic extracts of Krebs-2 cells which greatly enhanced production of polypeptide F as well as other noncapsid proteins in the fractionated system. By several criteria, this protein was identified as eukaryotic elongation factor 2 (eEF-2). By using the ADP-ribosylation assay, it was found that the fractionated system contained about 15% of the amount of eEF-2 present in the unfractionated extracts. The results suggest that changes in the eEF-2 content may affect the elongation rate differently at different regions of the RNA template.

A low-molecular-mass RNA from the cytoplasm of mouse Krebs II cells was found to be covalently linked to a protein with an apparent molecular mass of 13 kDa. The protein appears to be attached to the 5'-terminus of the RNA molecule, which is approx. 20 nucleotides long.

A series of intertypic (type 3/type 1) poliovirus recombinants was obtained whose crossover sites were expected to be located in the middle of the viral genome, between the loci encoding type-specific antigenic properties, on the 5' side, and an altered sensitivity to guanidine, on the 3' side. The primary structures of the crossover regions in the genomes of these recombinants were determined by the primer extension method. The length of the crossover sites (the uninterrupted sequences shared by the recombinant and both parental genomes that are flanked, in the recombinant RNAs, by two heterotypic segments) varied between 2 and 32 nucleotides, but the majority of the sites were 5 nucleotides long or shorter. The crossover sites were nonrandomly distributed over the presumably available genome region: only a single such site was found within the gene for polypeptide 2A, whereas an apparent clustering of the crossover sites was encountered in other genomic segments. When the crossover sites were superimposed on a model of the secondary structure of the relevant region of the viral RNA molecule, a pattern consistent with the previously proposed mechanism of poliovirus recombination (L.I. Romanova, V.M. Blinov, E.A. Tolskaya, E.G. Viktorova, M.S. Kolesnikova, E.I. Guseva, and V.I. Agol (1986) Virology 155, 202-213) was observed. It is suggested that the nonrandom distribution of the crossover sites in the genomes of intertypic poliovirus recombinants was due to two factors: the existence of preferred sites for recombination, and selection against recombinants with a lowered level of viability.

Antibodies were raised by immunization of Macaca fascicularis monkeys with extracts of M. fascicularis kidney cells separately infected with one of the three poliovirus serotypes. Preparations of antibodies were shown to be strictly type-specific in the neutralization test but formed immune complexes adsorbable on Staphylococcus aureus, Cowan I strain cells, with heterotypic as well as homotypic virus-specific polypeptides present in extracts from the virus-infected cells. The presence of intertypic antigenic determinants was demonstrated by this technique on both the non-capsid and capsid poliovirus polypeptides. Structural variations of poliovirus polypeptides were studied by analysing products of their partial proteolysis. Non-capsid polypeptides encoded in the central portion of the virus genome (polypeptides 5b and X) as well as in the 3'-terminal region (NCVP2 and NCVP4) were found to be highly conserved, whereas capsid polypeptides VP1, VP2, and VP3, which are encoded in the 5'-terminal region of the virus RNA, displayed a much greater variability.

Two cell lines, M10-45-2 and L-41, were studied, each of which possessed specific resistance either to poliovirus or to coxsackievirus. Infection of M10-45-2 cells with poliovirus ribonucleic acid (RNA) and L-41 cells with infectious coxsackievirus RNA was accompanied by production of complete viruses in each of the resistant cell lines. During incubation of the cells with the virus to which they were resistant, the amount of infectious virus did not decrease. Treatment with glycine-HCl buffer solution (pH 2.5) of resistant M10-45-2 cells after incubation with poliovirus at 0 C did not result in recovery of infectious virus, although such release did take place after treatment of sensitive M10 cells. Infection of resistant cells with virus containing poliovirus RNA and coxsackievirus proteins resulted in production of poliovirus in M10-45-2 cells but not in L-41 cells. The resistant cells are apparently unable to adsorb the virus to which they are resistant.

When the osmotic pressure of the maintenance medium is decreased, poliovirus reproduction is inhibited. Poliovirus strains may vary in their sensitivity to the effect of hypotonic solutions. Mutants have been selected (designated as osm mutants) the reproduction of which is characterized by particularly high resistance to media with reduced osmotic pressure. Initial stages of the virus-cell interaction proceed at similar rates in physiologic and hypotonic solutions. Reactions sensitive to hypotonic solutions take place in the second half of the latent period and during the stage of virus maturation. The multiplication of viruses in moderately hypotonic solutions exhibits a marked dependence upon the temperature and the presence of cystine in the medium, even though these factors have relatively small influence upon the reproduction of the viruses in isotonic solutions. Despite complete cessation of the production of infectious virus in certain hypotonic media, there is a considerable synthesis of virus-induced RNA. At least some of the RNA produced under these conditions is infectious. With a further decrease of the osmotic pressure of the medium the synthesis of the viral RNA is inhibited. Some possible explanations of the observed facts are briefly discussed.