Poliovirus-induced inhibition of polypeptide initiation in vitro on native polyribosomes.

The inhibition of HeLa cell protein synthesis by poliovirus was studied by examining initiation in vitro on endogenous host polyribosomes. At an early stage, before major viral RNA replication and protein synthesis begins, the initiation of translation on cellular mRNA is strongly inhibited. Fractionation of extracts from infected cells shows that the lesion is associated mainly with the crude polyribosome fraction. The cellular mRNA appears unchanged and is as active as mRNA from control cells in stimulating incorporation. The native ribosomal subunits and KCl-washed polyribosomes from the infected cells are also active. Only the ribosomal wash fraction prepared from the inhibited polyribosomes had reduced activity. However, the reduction in the ribosomal wash activity measured in a reconstructed system is not as large as the inhibition seen with "native" polyribosomes. The results indicate that a viral induced inhibition is probably associated with the ribosomal wash fraction, but the reconstructed system is not equivalent to the "native" inhibited system.     

Regulation of Protein Synthesis Initiation in HeLa Cells Deprived of Single Essential Amino Acids

In HeLa cells deprived of valine, histidine, or methionine initiation of protein synthesis decreases rapidly and disaggregation of polyribosomes also occurs. The mechanism of inhibition does not seem to involve the supply of RNA in the cell, and thus it differs from the initiation of inhibition at elevated temperatures. Polyribosomes rapidly form again if the missing amino acid is restored, even in the presence of actinomycin D.     

Protein synthesis studies in skeletal muscle of aging rats. II. In vitro studies with 0.5M potassium chloride washed polyribosomes

To investigate further the age-related reduction in muscle protein synthesis activity found previously using a crude polyribosome/pH 5 system (Pluskal et al., 1984), a 0.5M KCl washing procedure was utilized to remove the nonribosomal factors from polyribosomes isolated from male Sprague-Dawley rats in the following age groups: young (1 to 2 months), mature (12 months), and aged (22 to 24 months). Using a common source of enriched elongation factor fraction from young animals, it was not possible to demonstrate any significant difference (p greater than .05) in protein synthesis between the 0.5M KCl-washed polyribosomes isolated from the various age groups. Using a cell-free system containing young salt washed polyribosomes stimulated by the addition of 0.5M KCl-wash fractions, however, it was shown that the mature and aged salt-wash fractions were less (p less than .05) active than material from young animals. Thus, the observed decline in protein synthesis efficiency during aging may be attributed to a reduced capacity to promote initiation/elongation by the nonribosomal salt wash fractions of muscle polyribosomes.     

Virus-Specific Messenger RNA and Nascent Polypeptides in Polyribosomes of Cells Replicating Murine Sarcoma-Leukemia Viruses

We present evidence that virus-specific RNA is present in polyribosomes of transformed cells replicating the murine sarcoma-leukemia virus complex and that it serves as messenger RNA for the synthesis of viral-coded proteins. Both virus-specific RNA (detected by hybridization with the [(3)H]DNA product of the viral RNA-directed DNA polymerase) and nascent viral polypeptides (measured by precipitation with antiserum to purified virus) were found in membrane-bound and free polyribosomes. Membrane-bound polyribosomes contained a higher content of both virus-specific RNA and nascent viral polypeptides. From 60 to 70% of viral RNA sequences were released from polyribosomes with EDTA, consistent with a function as messenger RNA. Maximum amounts of both virus-specific RNA and nascent viral polypeptides were found in the polyribosome region sedimenting at about 350 S.     

Stimulation of Polypeptide Initiation In Vitro After Protein Synthesis Inhibition In Vivo in HeLa Cells

Crude cytoplasmic extracts prepared from HeLa cells actively incorporate amino acids but show little initiation of new peptides (as seen by labeling of N-terminal amino acids). In contrast, extracts prepared from cells subjected to prior inhibition of protein synthesis show a significant amount of polypeptide initiation indicated by formation of peptides with radioactive N-terminal methionine. The same result was obtained whether prior inhibition occurred with cycloheximide or by starvation for an essential amino acid. Cellular response to suppression of protein synthesis appears to be mediated through production of RNA, since it is inhibited by actinomycin but appears in the presence of cycloheximide. The crude extracts continue initiating new polypeptides for at least 10 min in vitro. It is postulated that enhancement of in vitro initiation described here is related to the apparent stimulation of initiation of translation seen in vivo.     

Synthesis of Rauscher murine leukemia virus-specific polypeptides in vitro.

The biosynthesis of specific polypeptides directed by purified viral messenger RNA from JLS-V9 cells infected with Rauscher leukemia virus has been studied in a rabbit reticulocyte lysate. The 35S viral mRNA gives rise to two major products of 65,000 and 72,000 molecular weight. The synthesis of specific polypeptides was also investigated in lysates derived from infected cells. The main products were polypeptides with molecular weights of 65,000, 76,000, and 82,000, and were preferentially made in association with membranes. The relative content of the virus-specific polypeptide of 65,000 molecular weight, synthesized in a cell-free system supplemented with purified polyribosomes, is considerably higher for membrane-bound polyribosomes.     

In vitro inhibition of protein synthesis by purified cores from vaccinia virus.

The mechanism of the shutoff of cellular protein synthesis in vaccinia virus-infected cells has been investigated by using in vitro systems. Purified vaccinia cores cause inhibition of endogenous mRNA translation in nonpreincubated reticulocyte lysates and Ehrlich ascites tumor cell-free systems. Translation of viral mRNA from turnip yellow mosaic virus is also impaired in wheat germ cell-free extracts. The block induced by vaccinia cores in protein synthesis is not due to a decrease in the availability of mRNA but rather to an alteration of the cellular translational machinery. No nucleolytic activity able of digesting mRNA could be detected in purified vaccinia cores with three sensitive tests. There is a lack of inhibition in the poly(Phe)-poly(U) system, which bypasses the normal initiation process. An almost complete disaggregation of polyribosomes in the reticulocyte lysate appears when vaccinia cores are present. These results indicate that mRNA translation in a cell-free system is affected predominantly at the level of polypeptide chain initiation.     

Separation of Specific Initiation Factors Involved in the Translation of Myosin and Myoglobin Messenger RNAs and the Isolation of a New RNA Involved in Translation

Two messenger specific factors, phosphocellulose fractions 3 and 4, have been isolated from the initiation factor 3 fraction of red muscle initiation factors by chromatography on phosphocellulose. When added to a reticulocyte cell-free system containing both myoglobin and myosin mRNAs, phosphocellulose fraction 3 is found to specifically stimulate the synthesis of myoglobin while phosphocellulose fraction 4 is found to specifically stimulate the synthesis of myosin. In addition, a new RNA, isolated from the initiation factor 3 fraction, is shown to specifically inhibit the translation of heterologous mRNAs.     

Endogenous RNA-Directed DNA Polymerase Activity in Uninfected Chicken Embryos

Early chicken embryos that are either positive or negative for group-specific antigens of avian leukosis viruses contained endogenous RNA-directed DNA polymerase activity. This endogenous DNA polymerase activity was not increased after mixture of soluble DNA polymerases isolated from chicken embryos with disrupted chicken embryo cells. The endogenous activity was resistant to treatment with deoxyribonuclease, and the initial rate of DNA synthesis was partially resistant to actinomycin D. In contrast, over 90% of the endogenous polymerase activity was destroyed by ribonuclease in medium with high salt concentration. The DNA product of the endogenous DNA polymerase activity from chicken embryos did not hybridize with RNA of Rous sarcoma virus or reticuloendotheliosis virus, whereas about 40% of this DNA product hybridized with the RNA from the same chicken-cell fraction. Antibody against DNA polymerase of avian myeloblastosis virus did not neutralize the chicken endogenous DNA polymerase activity. These results demonstrate that uninfected chicken embryo cells contain endogenous RNA-directed DNA polymerase activity that is not derived from avian leukosis or reticuloendotheliosis viruses.     

Phosphorylation of eukaryotic protein synthesis initiation factors.

Phosphorylation of eukaryotic initiation factors was examined both in intact cells and in vitro with purified components. Intact rabbit reticulocytes were incubated in a medium containing[32P]phosphate, and eight initiation factors were isolated and partially purified. The purified factors were analyzed on dodecyl sulfate/polyacrylamide gels and compared with highly purified nonradioactive factors. Significant amounts of radioactivity were found associated with initiation factors eIF-2, polypeptide 2 (molecular weight 53,000); eIF-3, polypeptides 2 and 4 (molecular weights 110,000 and 67,000); and eIF-4B. Purfied initiation factors from rabbit reticulocytes were also treated in vitro with [gamma-32P]ATP and a cyclic AMP-independent protein kinase isolated from rabbit erythrocytes. Only the factor polypeptides phosphorylated intracellularly were phosphorylated in vitro. The results suggest that the cyclic AMP-independent protein kinase is responsible for the phosphorylation of specific initiation factors in cells active in protein synthesis and that it may play a role in regulating translation.     

Increased RNA Synthesis in Nuclear Monolayers of WI-38 Cells Stimulated to Proliferate

Nuclear monolayers of WI-38 cells prepared by the method of Tsai and Green were used to determine RNA synthesis in isolated nuclei in situ. In nuclear monolayers, incorporation of [(3)H]UTP into RNA is dependent on the presence of the other three nucleotide triphosphate and is abolished by actinomycin D. The extent of RNA synthesis under these conditions was measured in density-inhibited WI-38 human diploid fibroblasts at various intervals after cell proliferation was stimulated by a change of medium.RNA synthesis increases 15 min after the nutritional change and reaches a peak at 18 hr, which is also the peak of DNA synthesis. Thereafter RNA synthesis declines. Essentially similar results are obtained whether the endogenous RNA polymerase or a bacterial polymerase is used. Replacement of the stimulating medium by conditioned medium stops the increase in RNA synthesis that occurs in cultures subject to continuous stimulation. Finally, RNA synthesis in nuclear monolayers, using the endogenous RNA polymerase, occurs by chain elongation only, while re-initiation occurs with the bacterial RNA polymerase.     

Messenger RNA species partially in a repressed state in mouse sarcoma ascites cells.

Four major mRNA species of mouse sarcoma ascites cells, coding for polypeptides designated P65, P40, P36, and P21, occur predominantly as untranslated messenger ribonucleoprotein particles. Cloned cDNA probes were used to study their distribution in cytoplasmic extracts of these cells. A considerable portion of the mRNA molecules sedimented as small particles, whereas the rest was present in polyribosomes. In contrast, the actin mRNA was present almost exclusively in polyribosomes. Incubation of the ascites cells in culture medium, particularly after a starvation treatment, caused an enhancement in polypeptide chain initiation relative to elongation in these cells, as evidenced by a shift of ribosomes into the polyribosome fraction and by an increase in polyribosome size. Exposure of the cells to a low concentration of cycloheximide, an inhibitor of the elongation step, had a similar effect. The actin mRNA and the active P65, P40, P36, and P21 mRNA molecules were shifted to larger polyribosomes in the treated cells, but no shift of molecules from small particles to polyribosomes was observed. The incubation in culture also led to considerable increases in the proportion of P65 and P40 mRNA molecules in the untranslated state. The results indicate that the untranslated state cannot be attributed to poor initiation efficiency. It is suggested that a portion of the mRNA molecules is maintained in a repressed state and that mRNA repression may represent an important translation control process.     

Infection with paramyxoviruses stimulates synthesis of cellular polypeptides that are also stimulated in cells transformed by Rous sarcoma virus or deprived of glucose

Infection of several types of cultured cells with the paramyxoviruses simian virus 5 and Sendai virus stimulates synthesis of four polypeptides (I-IV) with molecular weights of approximately 99,000, 97,000, 86,000, and 78,000, respectively. That these are host polypeptides encoded in cellular mRNAs has been shown by the inhibition of their synthesis by actinomycin D and by the similarity of the peptide maps of them and of polypeptides with the same electrophoretic mobility from uninfected cells. Peptide mapping as well as identical migration in polyacrylamide gels has also indicated that polypeptides I, II, and IV are the same as plasma membrane polypeptides whose synthesis is enhanced in cells transformed by Rous sarcoma virus and in normal cells by glucose deprivation or treatment with 2-deoxyglucose. Polypeptides I and II appear to be the same polypeptides, with the observed differences in migration reflecting the glycosylation of polypeptide I, a relationship previously shown to exist between polypeptides in glucose-deprived and glucose-fed cells. Infection with paramyxoviruses does not significantly increase the transport of glucose by cells, and the maintenance of a high concentration of glucose in the medium does not prevent the enhanced synthesis of these polypeptides. This is in contrast to the situation in transformed cells in which stimulation of synthesis of these polypeptides is secondary to depletion of glucose in the medium due to increased glucose uptake by the cells. Thus, although paramyxovirus infection and transformation by Rous sarcoma virus result in stimulation of the synthesis of the same membrane polypeptides, the mechanism of stimulation differs.     

Protein synthesis studies in skeletal muscle of aging rats. I. Alterations in nitrogen composition and protein synthesis using a crude polyribosome and pH 5 enzyme system

The effect of aging on rat skeletal muscle protein synthesis was studied using a cell-free system. The activity of crude polyribosomes from hind-limb skeletal muscle was reduced by 40% in aged animals (22 to 24 months) and by 20% in the mature (12 months) animal compared with young (2 month) rats. In a poly Uridylic-acid-directed incorporation system the ribosomes from aged and mature animals showed a decrease in activity. Sucrose density gradient analysis also demonstrated a loss of heavy polyribosomes in aged and mature animals. The pH 5 enzyme fraction from aged and mature animals was less efficient than that from young rats in support of protein synthesis, suggesting a decreased activity of soluble factors. In conclusion, aging leads to a progressive decline in the efficiency of protein synthesis, associated with both the ribosome and soluble fractions of the cell.     

Synthesis of a Brain-Specific Protein (S100 Protein) in a Homologous Cell-Free System Programmed with Cerebral Polysomal Messenger RNA

Polyribosomes, carrying nascent polypeptide chains, were prepared from whole brain, cortex, and hindbrain-medullary white matter of young adult rats. In a homologous cell-free system, a brain-specific protein (S100 protein) was identified in the mixture of polypeptides released from the polyribosomes during incubation for 1 hr at 37 degrees . De novo synthesis of the S100 protein was achieved in a reconstituted cerebral cell-free system containing polysome-derived mRNA and 40S + 60S subunits. The radioactively labeled S100 protein synthesized in vitro was identified by precipitation with antibody to S100 after addition of purified S100 as a carrier, and migration of the solubilized precipitate on acrylamide gels in the presence of sodium dodecyl sulfate. In vitro synthesis of the S100 protein did not occur in analogous cell-free systems derived from hepatic tissue or in a heterologous system containing liver polyribosomes and cerebral enzymes.     

In Vitro Synthesis of Ribosomal Proteins Directed by Escherichia coli DNA

In vitro synthesis of a number of E. coli 30S ribosomal proteins has been demonstrated in a cell-free system consisting of ribosomes, initiation factors, RNA polymerase, a fraction containing soluble enzymes and factors, and E. coli DNA. DNA-dependent synthesis of the following 30S proteins has been demonstrated: S4, S5, S7, S8, S9, S10, S13, S14, S16, S19, and S20.     

Inactivation of cap-binding proteins accompanies the shut-off of host protein synthesis by poliovirus.

Infection of HeLa cells with poliovirus results in a rapid shut-off of host protein synthesis. It has been suggested that inactivation of a protein that binds to the cap structure of cellular mRNAs would explain the selective inhibition of host protein synthesis because the naturally uncapped poliovirus RNA can be translated by a cap-independent mechanism. To test directly for the presence of cap-binding proteins in poliovirus-infected and mock-infected cells, we analyzed initiation factor preparations for their ability to specifically crosslink to the 5' cap structure of oxidized reovirus mRNA. The data presented here show that the crosslinking ability of the different cap-binding proteins (24-, 28-, 32-, 50-, and 80-kilodalton polypeptides) is reduced in preparations from poliovirus-infected as compared to mock-infected cells. This reduction correlates with the inability of initiation factor preparations from infected cells to restore translation of capped mRNAs in extracts of poliovirus-infected cells. In addition, initiation factor preparations from poliovirus-infected cells have the ability to rapidly inactivate cap-binding proteins and can also impair the restoring activity of initiation factors from mock-infected cells.