[An inhibitor of translation in cellular mRNA containing the 100S structure from Krebs II ascites carcinoma cells infected with encephalomyocarditis virus]. / Ingibitor transliatsii v sostave soderzhashchego kletochnuiu mRNK 100S-struktury v zarazhennykh virusom éntsefalomiokardita kletkakh astsitnoi kartsinomy Krebs II.

As shown previously, the bulk of cellular mRNA in Krebs II ascite carcinoma cells infected with encephalomyocarditis virus during active virus-specific synthesis is bound to ribosomes within the 100S structure which is inactive in protein synthesis. In order to elucidate the reasons for the translation repression of cellular mRNA within the 100S structure, a fraction of loosely bound proteins which are liberated by treatment of the 100S structure with 0.5 M KCl an which contain sum translation factors, was obtained. This fraction was shown to contain an inhibitor which non-specifically represses the translation of endogenous viral and cellular mRNA within the composition of polyribosomes and of exogenous poly(A)-containing cellular mRNAs from ascite carcinoma cells.

[4,5-S-RNAI in virus-specific polyribosomes from ascitic Krebs 2 carcinoma cells infected with encephalomyocarditis virus]. / 4,5S-RNKI v sostave virusspetsificheskikh poliribosom iz kletok astsitnoi kartsinomy Krebs 2, zarazhennykh virusom éntsefalomiokardita.

Polyribosomes of Krebs 2 ascite carcinoma cells non-infected and infected with encephalomyocarditis (EMC) virus contain a heterogeneous population of low molecular weight small RNAs. Analysis of the RNAs by polyacrylamide gel electrophoresis did not reveal any qualitative differences in the small RNA sets within the composition of polyribosomes from virus-infected and non-infected cells. However, the content of one of the small RNAs was markedly elevated in polyribosomes from virus-infected cells. As can be followed from partial determination of its primary structure, this small mRNA is identical to 4,5S-RNAI previously detected in the nuclei of Novikov hepatoma cells of the rat. The data obtained suggest that 4,5S-RNAI can be involved in the regulation of protein synthesis in virus-infected cells.

[Ability of translation factors from infected encephalomyocarditis viruses of ascites carcinoma cells to maintain the protein-synthetizing activity of cellular polyribosomes]. / Sposobnost' faktorov transliatsii iz zarazhennykh virusom éntsefalomiokardita kletok astsitnoi kartsinomy podderzhivat' beloksinteziruiushchuiu aktivnost' kletochnykh poliribosom.

A highly effective protein-synthesizing system fully dependent on translation factors was isolated from the ascite Krebs-2 carcinoma. The effect of EMC-infection on the ability of these factors to maintain the translation of polyribosomes containing cell mRNA was studied. It was shown that the activity of translation factors of infected cells does not differ from that of non-infected cells in terms of their ability to maintain the protein-synthesizing activity of cell polyribosomes in vitro.

Inhibitory effect of N-phenyl-N'-3-hydroxyphenylthiourea (PTU-23) on the reproduction of encephalomyocarditis virus in Krebs-II cells.

PTU-23, an effective in vivo wide-range enterovirus inhibitor, suppresses the reproduction of encephalomyocarditis (EMC) virus in Krebs-II ascites carcinoma cells at concentrations of 20-50 micrograms/ml, not affecting the cellular synthetic processes. The virus-specific RNA synthesis is distinctly inhibited. The studies on the kinetics of this effect point to its leading role in the inhibitory action the compound exerts on the production of infectious virions. The sedimentation profile in sucrose density gradient of the viral RNA isolated from PTU-23-treated cells by phenol extraction shows a clear inhibition of the synthesis of the single-stranded (ss) 37S RNA and to a lesser extent of the double-stranded (ds, RF) 20S RNA. The effect of the inhibitor on the RNA-dependent RNA polymerase in a cell-free RNA-synthesizing system has been studied, using an an enzyme preparation the 40000 g fraction of a nuclear-free extract from infected Krebs-II cells, containing the enzyme bound to the endogenous RNA template. It is established that the compound does, not affect the synthesis of the enzyme which could be probably explained by the incomplete (45-70%) inhibition of the viral RNA synthesis, its translatory function remaining unperturbed. The observed insignificant inhibition (20-26%) of the enzyme activity during the application of the inhibitor to the RNA-synthesizing reaction mixture cannot explain its effect on the viral RNA synthesis. An interaction of PTU-23 with another virus-specific protein component of the replication complex is suggested.

Effect of N-phenyl-N'-3-hydroxyphenylthiourea (PTU-23) on the protein synthesis in Krebs-II cells infected with encephalomyocarditis virus.

PTU-23, a selective inhibitor of the reproduction of encephalomyocarditis (EMC) virus in Krebs-II ascites carcinoma cells, counteracts the virus-induced shut-off of the host-cell protein synthesis reducing it from 32% to 19%. The compound does not inhibit the synthesis of virus-specific proteins; the electrophoretic profile in SDS-PAAG shows an increase in the peaks of some viral polypeptides, mainly A, E and epsilon. It is suggested that this effect is connected to the partial inhibition by PTU-23 of the synthesis of the viral 37S RNA, without affecting its translation activity and also to the inhibition of the synthesis of 20S (RF) RNA--an inhibitor of the virus-specific protein synthesis. PTU-23 does not affect the processing of the high-molecular-weight viral precursor polypeptide preA, which is carried out by the virus-specific protease protein p22, as shown in studies with a cell-free system.

[Reproduction of murine encephalomyocarditis virus in enucleated Krebs 2 ascitic carcinoma cells]. / Reproduktsiia virusa éntsefalomiokardita myshei v bez"iadernykh kletkakh astsitnoi kartsinomy Krebs 2.

Enucleated cells of ascites carcinoma Krebs 2 (cytoplasts) were prepared. The cells were centrifugated for this purpose in a discontinuous Ficoll density gradient containing cytochalasine B. The cytoplasts formed turbid bands in Phycol solution at the density of 1.037--1.053 g/ml. The cytoplasts failed to synthesize RNA, but proteins were synthesized for several hours. Encephalomyocarditis virus replicated in the ascites carcinoma cytoplasts, but its yield was 10 to 100-fold lower as compared to that in the intact cells. Apparently the reduction of the virus yield was not due to the low efficiency of early stages of the virus-cytoplast interaction. Viral infection resulted in inhibition of the synthesis of cellular proteins in the cytoplasts as well as in the intact cells.

Stimulation of tumor growth in mice by high doses of BCG.

The growth of Krebs-2 carcinoma in BCG-prevaccinated virgin female C57BL/6, CC57BR/M, and C3Hf mice was studied in relation to the number of living mycobacteria in the organism. When the number of mycobacteria was high, tumor growth was stimulated. After the bacteria were eliminated, tumor growth was inhibited. The effect of BCG was based, on the one hand, on the diversion of effector cells, presumably macrophages, responsible for tumor defense and, on the other hand, on the activation of the pool of these cells. The conclusions were reached that high doses of BCG may be dangerous in human cancer immunotherapy and that patients predisposed to neoplastic disease should be revaccinated with BCG.

Inhibition and stimulation of the growth of Krebs-2 carcinoma by BCG vaccine.

The effect of BCG vaccine on the growth of imtransplants of Krebs-2 carcinoma in mice was studied. The simultaneous injection of BCG and tumor cells either inhibited tumor growth (BCG given in admixture with tumor cells) or stimulated it (BCG injected contralateral to the tumor transplantation site). The BCG dose was directly related to the effect. Tumor growth was also stimulated by the ip injection of starch or liquid paraffin. In these experiments, the BCG effect was attributed to the redistribution of cells involved in nonspecific and specific tumor resistance. Shortly after BCG prevaccination, particularly when BCG doses were high and mice were susceptible to vaccine infection, BCG was either without effect or stimulated tumor growth; later, however, tumor growth was inhibited regardless of the BCG dose and the injection site of the BCG. The effect of BCG prevaccination was suggested to be due to: 1)the distraction of macrophages and T-lymphocytes to defend the host against the multiplying mycobacteria, and 2)the activation of the pool of these cells that become capable to participate in antitumor resistance after mycobacteria elimination.