A COMPARISON OF THE CHANGES IN FINE STRUCTURE OF L CELLS DURING SINGLE CYCLES OF VIRAL MULTIPLICATION, FOLLOWING THEIR INFECTION WITH THE VIRUSES OF MENGO AND ENCEPHALOMYOCARDITIS

The virus of encephalomyocarditis (EMC), examined by the negative-contrast method, is indistinguishable from the serologically related Mengovirus. The particles are 270 to 280 A in diameter. The surface of EMC is composed of an undetermined number of subunits. Frequent sampling of infected cells was carried out throughout one-step cycles of viral multiplication to observe cytopathic changes occurring in L cells infected by these two related RNA viruses. EMC and Mengovirus, which multiply at equal rates, in most respects elicit similar alterations in cell fine structure. Rearrangement and changes in nuclear material accompanied by formation of small vesicles in the centrosphere region commence at 4 to 6 hours after infection. Thereafter a progressive degeneration of the nucleus and vesiculation of the cytoplasm are observed up to 18 to 20 hours. Increased numbers of small dense granules, indistinguishable from ribonucleoprotein particles, appear in the cytoplasm between 8 and 14 hours after infection. L cells infected with Mengovirus become permeable to Erythrocin more slowly than those infected with EMC. Only in the case of Mengovirus infection are large aggregates of dense material first observed in the cytoplasm at 8 hours, followed by the appearance of crystals probably composed of Mengovirus particles, at 12 hours. Differences in the rates of cell permeability after infection with EMC and Mengovirus are discussed in relation to formation of virus crystals and plaque-type mutants.     

Polyadenylated RNA of Volvox: isolation and partial characterization

Polyadenylated RNA from Volvox carteri has been isolated and partially characterized. Electrophoretic profiles of total cellular poly(A)-associated RNA of Volvox spheroids indicate a hetero-disperse distribution of size classes with the range extending from an apparent sedimentation value of approximately 10S to greater than 38S. The radioactive labelling kinetics of this material are typical for rapidly-turning-over RNA. The profiles of poly(A) RNA from different cell types show marked differences in average migration rate. Terminally-differentiated somatic cells contain a greater proportion of material of higher molecular weight than either gonidia (germ cells) or cleaving embryos. The poly(A) segments associated with cellular RNA, obtained by selective RNase digestion are heterogeneous in size as determined by gel electrophoresis with the largest tracts estimated to be 75-80 nucleotides long. Gonidia and embryos display the greatest degree of size heterogeneity, while somatic cells show predominantly the largest classes of poly(A) tract. It is apparent that gross changes in poly(A) RNA metabolism accompany development and cellular differentiation in Volvox.     

Translational control by messenger RNA competition for eukaryotic initiation factor 2

Translation of globin mRNA in a micrococcal nuclease-treated reticulocyte lysate was studied in the presence of increasing amounts of Mengovirus RNA, under conditions in which the number of translation initiation events remains constant as judged by the transfer of label from N-formyl[35S]methionyl-tRNAf into protein. The translation of globin mRNA is progressively inhibited by low concentrations of Mengovirus RNA, free of detectable traces of double-stranded RNA, concomitant with the increasing synthesis of Mengovirus RNA-directed products. On a molar basis, Mengovirus RNA apparently competes about 35 times more effectively than globin mRNA for a critical component in translation. The competition is relieved by the addition of highly purified eukaryotic initiation factor 2 (eIF-2). Addition of eIF-2 does not stimulate overall protein synthesis, but shifts it in favor of globin synthesis. No stimulation of globin mRNA translation by eIF-2 is seen when Mengovirus RNA is absent. These experiments show that Mengovirus RNA competes, directly or indirectly, with globin mRNA for eIF-2. In direct binding experiments using isolated mRNA and eIF-2, Mengovirus RNA is shown to compete with globin mRNA for eIF-2 and to exhibit a 30-fold higher affinity for this factor. The binding of Mengovirus RNA to eIF-2 is much more resistant to increasing salt concentrations than is the binding of globin mRNA, again reflecting its high affinity. These results reveal a direct correlation between the ability of these mRNA species to compete in translation and their ability to bind to initiation factor eIF-2. They suggest that the affinity of a given mRNA species for eIF-2 is essential in determining its translation, relative to that of other mRNA species. Messenger RNA competition for eIF-2 may contribute significantly to the selective translation of viral RNA in infected cells.     

Expression of Madin-Darby canine kidney cell Na(+)-and Cl(-)-dependent taurine transporter in Xenopus laevis oocytes.

Expression of a Madin-Darby canine kidney (MDCK) cell taurine transporter was examined in Xenopus oocytes that had been injected with poly(A)+ RNA extracted from MDCK cells. Compared with water-injected oocytes, injection of total poly(A)+ RNA resulted in an increase in Na(+)-dependent taurine uptake which was directly related to the amount of RNA injected. The magnitude of expression in poly(A)+ RNA-injected oocytes was 5-10-fold higher than that of water-injected oocytes. Since the Vmax of taurine uptake in MDCK cells is increased by culture in hypertonic medium, we compared oocyte taurine uptake after injection with poly(A)+ RNA from MDCK cells cultured in hypertonic medium with uptake in oocytes injected with poly(A)+ RNA from hypertonic cells elicited twice the taurine uptake elicited by poly(A)+ RNA from isotonic cells. The transporter expressed in oocytes was like that in MDCK cells: it was completely dependent on external sodium and was also anion dependent (Cl- greater than or equal to Br- greater than SCN- much greater than gluconate-). Other beta-amino acids, beta-alanine and hypotaurine, inhibited taurine uptake, but L-alanine and 2-(methylamino) isobutyric acid did not. The apparent Km of the transporter was 7.0 microM. After size fractionation on a sucrose density gradient, poly(A)+ RNA encoding for the MDCK taurine transporter was found in the fraction whose average size was 4.4 kilobases.     

Polyadenylation of RNA in immature oocytes and early cleavage of Urechis caupo

The poly(A) content of RNA extracted from four stages of immature oocytes, mature oocytes, and cleavage embryos through the eight-cell stage was determined by hybridization with [³H]-poly(U). During oogenesis the poly(A) content per cell gradually increases from 0.007 pg of poly(A)/cell in the 10- to 39-μm oocytes to 0.20 pg of poly(A)/cell in the 125-μm mature oocytes. After fertilization there is an additional increase to approximately 1.1 pg of poly(A)/embryo at the two-cell stage which is followed by a slight decline between the two- and eight-cell stages. Most of the increase in poly(A) after fertilization occurs in a 45-min interval coincident with the appearance of the polar bodies. The size distribution of the poly(A) in RNA from the different stages of development was determined based on the length of RNase-resistant poly(U) obtained from poly(U)-poly(A) hybrids. The size distribution of the poly(A) sequences is constant through each stage of development which indicates that the increase in the poly(A) content of the cells is the result of polyadenylation of new RNA sequences.     

Polyadenylic acid on poliovirus RNA. III. In vitro addition of polyadenylic acid to poliovirus RNAs.

A crude RNA polymerase preparation was made from HeLa cells infected for 3 h with poliovirus. All virus-specific RNA species labeled in vitro (35S RNA, replicative intermediate RNA [RI], and double-stranded RNA [dsRNA]) would bind to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. After incubation for 45 min with [3-H]ATP in the presence of the other three nucleoside triphosphates, the labeled poly(A) on the RI and dsRNA migrated on gels as relatively homogenous peaks approximately 200 nucleotides in length. In contrast, the poly(A) from the 35S RNA had a heterogeneous size distribution ranging from 50 to 250 nucleotides. In the absence of UTP, CTP, and GTP, the size of the newly labeled poly(A) on the dsRNA and RI RNA was the same as it was in the presence of all four nucleoside triphosphates. However the poly(A) on the 35S RNA lacked the larger sequences seen when the other three nucleoside triphosphates were present. When [3-H]ATP was used as the label in infected and uninfected extracts, heterogeneous single-stranded RNA sedimenting at less than 28S was also labeled. This heterogeneous RNA probably represents HeLa cytoplasmic RNA to which small lengths of poly(A) (approximately 15 nucleotides) had been added. These results indicate that in the in vitro system poly(A) can be added to both newly synthesized and preexisting RNA molecules. Furthermore, an enzyme capable of terminal addition of poly(A) exists in both infected and uninfected extracts.     

Polyadenylic acid on poliovirus RNA. II. poly(A) on intracellular RNAs.

The content, size, and mechanism of synthesis of 3'-terminal poly(A) on the various intracellular species of poliovirus RNA have been examined. All viral RNA species bound to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. At 3 h after infection, the poly(A) on virion RNA, relicative intermediate RNA, polyribosomal RNA, and total cytoplasmic 35S RNA was heterogeneous in size with an average length of 75 nucleotides. By 6 h after infection many of the intracellular RNA's had poly(A) of over 150 nucleotides in length, but the poly(A) in virion RNA did not increase in size suggesting that the amount of poly(A) which can be encapsidated is limited. At all times, the double-stranded poliovirus RNA molecules had poly(A) of 150 to 200 nucleotides. Investigation of the kinetics of poly(A) appearance in the replicative intermediate and in finished 35S molecules indicated that poly(A) is the last portion of the 35S RNA to be synthesized; no nascent poly(A) could be detected in the replicative intermediate. Although this result indicates that poliovirus RNA is synthesized 5' leads to 3' like other RNA's, it also suggests that much of the poly(A) found in the replicative intermediate is an artifact possibly arising from the binding of finished 35S RNA molecules to the replicative intermediate during extraction. The addition of poly(A) to 35S RNA molecules was not sensitive to guanidene.     

Dimethyl-10,12-benz(a)acridine; evidence for differential effects on the synthesis of RNA of mammalian or avian fibroblasts and some RNA viruses.

We have studied the differential effect of dimethyl-10,12-benz(a)acridine (DBMAcr) on the synthesis of RNA of chicken or mouse fibroblasts in culture and that of some RNA-containing viruses such as Rous sarcoma virus and Mengovirus. DMBAcr at low concentrations blocks the cell multiplication of both normal and Rous sarcoma virus-transformed chicken fibroblasts in culture; it affects transformed cells more than normal ones. The cell growth inhibiting effect of DMBAcr is reversible after short periods of incubation. DMBAcr depresses the synthesis of cellular DNA and RNA in parallel. Concurrently the synthesis of protein proceedes at a relatively high rate in DMBAcr-treated cultures. Its inhibitory effect on cellular RNA synthesis is mostly due to a block in the formation of 28 S and 18 S ribosomal RNA species; in contrast, the synthesis of 45 S ribosomal RNA precursor is proceeding at almost control rate. Also, the synthesis of heterogeneous nuclear RNA is not blocked by DMBAcr. The production of Rous sarcoma virus in transformed fibroblasts is not affected by DMBAcr. Since this is correlated with persisting high rates of protein and heterogenous nuclear RNA synthesis, the effects of DMBAcr suggest that the synthesis of Rous sarcoma virus-RNA shares the specificity of messenger and heterogeneous nuclear RNA. DMBAcr inhibits the synthesis of viral RNA of Mengovirus under conditions where the synthesis of total cellular RNA is not appreciably depressed, suggesting its differential effect on the DNA-directed and the RNA-directed RNA synthesis.     

An investigation of the stability of messenger RNAs in cell-free,translational systems from uninfected and mengovirus-infected Ehrlich ascites tumor cells

The stabilities and translation of Ehrlich ascites tumor cell poly(A)-containing mRNA and mengovirus RNA in fractionated cell-free protein synthesizing systems from uninfected and mengovirus-infected Ehrlich ascites tumor cells were studied. During incubation of the systems about 20% of the input RNA is reduced in size and associated with ribosomes engaged in polypeptide synthesis; the remainder is rapidly degraded by RNases. At the end of active translation, both mRNA and nascent proteins are bound to polysomes which are of the same size as those formed during active protein synthesis. The kinetics of protein synthesis closely follow those of RNA hydrolysis. The stabilities of mengovirus RNA and poly(A)-containing mRNA from Ehrlich ascites tumor cells are the same in both systems.     

The synthesis and properties of polysomal RNA in potato tuber slices during the early stage of aging

The synthesis, molecular size, and coding properties of polysome-associatedpolyadenylated RNA[poly(A)(+)RNA]and non-polyadenylated RNA[poly(A)(–)RNA] were investigated in potato tuber discsduring the early stage of aging. Tissue discs were labeled for6 hr with ³H-uridine in the presence of 5-fluorouracil to suppressrRNA synthesis, and polysomal RNA was isolated from the discs.Poly(A)(+)RNA accounted for 70% of the radioactivity in polysomalRNA and had a molecular size ranging from 6S to 30S with a peakat about 15S, when measured by formamide-polyacrylamide gelelectrophoresis. The rest of the radioactivity was in poly(A)(–)RNAwhich had nearly the same range in molecular size, but had noconspicuous peaks on the gel. The polysomal RNA could programthe synthesis of a wide variety of polypeptides in a cell-freetranslation system of wheat germ. Seventy percent of the translationalcapacity of polysomal RNA was attributed to poly(A)(+)RNA. Theelectrophoretic behaviour of the majority of the products frompoly(A)(+)RNA was similar to that of products from poly(A)(–)RNA,but the former could program the synthesis of five polypeptidesin addition to those translated from the latter. There was atendency for poly(A)(–)RNA to be a more efficient messengerfor large polypeptides. ¹Present address: Department of Agricultural Chemistry, Facultyof Horticulture, Chiba University, Matsudo 271, Japan. (Received November 16, 1979; )     

Length of the polyadenylated sequence in cytoplasmic ribonucleic acid isolated from fetal calf myoblasts differentiating in vitro

Poly(adenylic acid) [poly(A)] containing cytoplasmic ribonucleic acid (RNA) in differentiating fetal calf myoblasts cultivated in vitro was examined by hybridization with radioactive poly(uridylic acid). The size distribution of the poly(A)-containing RNA after sucrose-gradient centrifugation was similar in cells before and after differentiation. There was no apparent correlation between the length of the poly(A) segment and the change in stability of messenger RNA which occurs on differentiation, nor with the polysomal or nonpolysomal localization of the RNA in the cytoplasm. The average length of the poly(A) segments in cytoplasmic RNA in the steady state was found to be dependent on the size of the RNA: the longer the RNA, the longer the average length of the poly(A) sequence. In contrast, in pulse-labeled RNa, the length of poly(A) is similar in all size classes of RNa.     

RNA metabolism and membrane-bound polysomes in relation to globulin biosynthesis in cotyledons of developing field beans (Vicia faba L.).

In cotyledon cells of developing field beans the RNA content per cell does not change in the second half of developmental period 2, whereas globulin biosynthesis continues. The constant RNA content per cell results from an equilibrium between RNA synthesis and degradation. All types of RNA are synthesized until the end of globulin biosynthesis, but poly(A)-containing RNA was preferentially labelled during maximum globulin formation. During stage 2 of seed development of poly(A)-containing RNA fraction represents a discrete peak in the 12--18-S region on agarose gels and corresponds to the peak of poly(A)-containing RNA isolated from polysomes. alpha-Amanitin inhibits selectively the labelling of poly(A)-containing RNA and concomitantly globulin formation. Translation of total poly(A)-containing RNA, free and membrane-bound polysomes in a cell-free wheat germs demonstrates that the globulins are preferentially produced on membrane-bound polysomes and that poly(A)-containing RNA includes the mRNA for both vicilin and legumin.     

A CYTOCHEMICAL DESCRIPTION OF THE MULTIPLICATION OF MENGOVIRUS IN L-929 CELLS

A correlation of cytochemical changes with virus production has been studied in L cells infected with Mengovirus. After a latent period of about 2 hours, virus was produced rapidly, reaching maximum titers of up to 12,000 particles per cell in 6 to 8 hours. The earliest cytological change was in the nucleus and consisted of a slight condensation of chromatin. There is no evidence, however, for the multiplication of either the viral RNA or protein in the nucleus. RNA, of high molecular weight, accumulated in the perinuclear area of the cytoplasm and was later found in inclusions. The perinuclear RNA was digestible with RNase and may be located in or on ribosomes. The inclusion RNA was resistant to RNase but could be removed by pepsin or potassium permanganate; it is probably in completed virus particles. Viral antigen was first observed in a perinuclear location and later in the above-mentioned inclusions. Although the viral protein contains appreciable amounts of arginine and lysine, it is not a basic protein of the histone type. Phase-contrast microscopy of living cells clearly demonstrated the role of the inclusions in release of virus from infected cells. A comparison is made between these cytological changes in Mengo-infected cells and those which have been found by other workers in polio-infected cells. There are many very similar changes.     

In vitro synthesis of full-length influenza virus complementary RNA.

Influenza virus-specific RNA has been synthesized in vitro, using cytoplasmic or microsomal fractions of influenza virus-infected MDCK cells. The RNA polymerase activity was stimulated 5-30 times by priming with ApG. About 20-30% of the product was polyadenylated. Most of the in vitro product was of positive polarity, as shown by hybridization to strand specific probes and by T1 fingerprinting of the poly(A)+ and poly(A)- RNA segments encoding haemagglutinin and nucleoprotein. The size of poly(A)- RNA segments, determined on sequencing gels, was indistinguishable from that of virion RNA, whereas poly(A)+ RNA segments contain poly(A) tails approximately 50 nucleotides long. The size of in vitro synthesized RNA segments was also determined by gel electrophoresis of S1-treated double-stranded RNAs, obtained by hybridization of poly(A)+ or poly(A)- RNA fractions with excess of unlabelled virion RNA. The results of these experiments indicate that poly(A)- RNA contains full-length complementary RNA. This conclusion is further substantiated by the presence of additional oligonucleotides in the T1 fingerprints of in vitro synthesized poly(A)- haemagglutinin or nucleoprotein RNA, selected by hybridization to cloned DNA probes corresponding to the 3' termini of the genes.     

Polyadenylate Sequences on Newcastle Disease Virus mRNA Synthesized In Vivo and In Vitro

Polyadenylate [poly(A)] sequences are associated with the 35 and 50S Newcastle disease virus (NDV)-specific RNAs as well as all six to seven of the 18-22S NDV-specific messenger RNAs extracted from infected chicken embryo cells. The poly(A) associated with the 18-22S RNA has an average size of 120 to 130 nucleotides. The 18-22S RNA synthesized in vitro by NDV's virion-bound polymerase contains six to seven species of the same size and relative proportions as its intracellular counterpart. This in vitro synthesized 18-22S RNA also contains covalently linked poly(A) sequences which, although variable in size, are usually larger and more heterogeneous than those from the infected cell. In vitro RNA synthesis is supported not only by magnesium (at an optimal concentration of mM) but by manganese (at an optimal concentration of 0.5 to 1.0 mM) as well. However, the major product made in the presence of manganese, although sedimenting at 18 to 22S, differs somewhat from the product made in the presence of magnesium.