Effect of isonicotinic acid hydrazide-copper complex on Rous sarcoma virus and its genome RNA.

The copper complex of the antituberculous drug, insonicotinic acid hydrazide (INH), inhibits the RNA-dependent DNA polymerase of Rous sarcoma virus and inactivates its ability to malignantly transform chick embryo cells. The INH-copper complex binds to the 70S genome RNA of Rous sarcoma virus (RSV), which may account for its ability to inhibit the RNA-dependent DNA polymerase. The complex binds RNA more effectively than DNA in contrast to M-IBT-copper complexes, which bind both types of nucleic acids equally. The homopolymers, poly rA and poly rU, are bound by the INH-copper complex to a greater extent than poly rC. Isonicotinic acid hydrazide alone and CuSO4 alone bind neither DNA, RNA, poly (rA), poly (rU), nor poly (rC). However, CuSO4 alone binds poly (rI); INH alone does not. In addition to viral DNA synthesis, chick-embryo cell DNA synthesis is inhibited by the INH-copper complex. The extent of inhibition of cellular DNA synthesis is greater than that of cellular RNA and protein synthesis. No selective inhibition of transformation in cells previously infected with Rous sarcoma virus is observed.     

Nature of Ribonuclease-resistant Nucleic Acid of Chick Embryo Cells transformed by Schmidt–Ruppin Strain of Rous Sarcoma Virus

THE mode of replication of RNA or RNA-containing tumour viruses is not understood. The recent studies on Rous sarcoma and other RNA-containing oncogenic viruses suggest that the replicative cycle of the RNA of these viruses might not be associated with ribonuclease-resistant structures (double stranded RNAs), but might involve the synthesis of a DNA intermediate specific to viral RNA^1–3. Two groups of workers, however, presented evidence for the presence of a double stranded RNA in 78 Al cell line of rat embryo fibroblasts which had been transformed and chronically infected with the murine sarcoma-leukaemia virus complex (MSV-MLV)^4,5 and it was suggested that the mode of replication of oncogenic viral RNAs was the same as that of non-oncogenic viral RNAs⁴. This apparent discrepancy prompted me to look for ribonuclease-resistant RNA structures in the chick embryo cells transformed by Schmidt-Ruppin Rous sarcoma virus (SR-RSV).     

New procedure for isolation of Rous sarcoma virus-specific RNA from infected cells.

The use of mercurated "strong stop" complementary DNA (complementary to the 5'-terminal 101 nucleotides of Rous sarcoma virus RNA) in the isolation of virus-specific RNA from infected chicken embryo fibroblasts is described. Strong stop Rous sarcoma virus complementary DNA was mercurated chemically, and, as a result of the low complexity of this DNA, short hybridization times (up to 15 min) and heating in the absence of formamide were found to be adequate conditions for the isolation of virus-specific RNA. The purity of the isolated RNA was demonstrated by analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. The isolated RNA could be translated in the in vitro protein synthesis system derived from rabbit reticulocytes, and an analysis of polypeptides programmed by isolated RNA before and after immunoprecipitation further demonstrated both the purity of the isolated mRNA and the quantitative nature of the isolation procedure.     

The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro. Transformation by rous sarcoma virus induces a switch in the collagen type synthesis and enhances fibronectin expression

Epithelial-like chondrocytes obtained from chick embryo were transformed with Rous sarcoma virus. Cellular transformation was monitored looking at the morphology change, the cell growth, and the expression of plasminogen activator. Analysis on polyacrylamide gel of intracellular and secreted proteins showed: 1) a disappearance of the specific products of differentiated chondrocytes; 2) a switch in the collagen synthesis from the type II, the chondrocyte-specific type, to the type I, characteristic of fibroblasts and other cells of mesenchymal origin; 3) an enhancement of fibronectin synthesis. Analysis of the proteins from chondrocytes infected with Rous-associated virus 1, a virus unable to induce cell transformation in vitro, indicated that the altered expression of the differentiated proteins in Rous sarcoma virus-infected chondrocytes depended upon the action of src gene product.     

Reduced levels of adenosine deaminase in chick embryo fibroblasts transformed by Rous sarcoma virus.

Adenosine deaminase activities in chick embryo fibroblasts were substantially reduced after infection and transformation by Rous sarcoma virus. Concomitant with the reduction in adenosine deaminase activities, the incorporation of exogenous adenosine into RNA species of the virus transformed cells was moderately increased. The significance between reduction in adenosine deaminase activity and malignant transformation by Rous sarcoma virus remains to be eleucidated.     

Changes in the synthesis and phosphorylation of cellular proteins in chick fibroblasts transformed by two avian sarcoma viruses

35S- and 32P-labeled proteins from control chick embryo fibroblasts and from fibroblasts transformed by UR2 sarcoma virus, or by a temperature-sensitive mutant (tsLA29) of Rous sarcoma virus, were separated by two-dimensional electrophoresis on giant gels to detect transformation-specific changes in protein synthesis and total phosphorylation. A nontransforming avian retrovirus, UR2-associated virus (UR2AV), was also studied. Virus-coded proteins appear in whole cell lysates of all infected cells. The structural proteins can be identified by comparison with proteins immunoprecipitated with antivirus serum. The transforming proteins pp60src and p68ros, present in cells transformed with Rous sarcoma virus and UR2, respectively, are phosphorylated in vivo. Eighteen increases and eight decreases in cellular phosphoproteins are associated with transformation, and revert toward normal levels when cells infected with tsLA29 are incubated at 42 degrees C. These changes are more extensive than previously reported, but none represent new phosphorylations, since all phosphoproteins seen in transformed cells also appear to be phosphorylated to a certain extent in control cells. Fifteen cellular proteins show increased relative rates of synthesis apparently related either to transformation or to growth at 42 degrees C. Four other proteins are increased exclusively in cells incubated at 42 degrees C, but not at 37 degrees C, whether transformed or not. Eleven additional increases in the synthesis of cellular proteins, many quite large, and one seemingly a de novo induction, appear to be specific for transformation. These changes occur in cells transformed by either UR2 or Rous sarcoma virus at 37 degrees C, do not occur with UR2AV infection, and tend to revert in cells infected with tsLA29 incubated at 42 degrees C. These 11 changes may represent increases in cellular gene expression that are related specifically to the maintenance of the transformed state.     

A mutation in the short 5''-proximal open reading frame on Rous sarcoma virus RNA alters virus production.

The 5'-proximal open reading frame on Rous sarcoma virus RNA encodes a seven-amino-acid peptide and is conserved in all avian sarcoma-leukosis retroviruses. Ribosome-binding site analysis in intact chick cells showed that the 5'-proximal AUG codon is a strong site for initiation of translation in vivo. Removal of the 5'-proximal AUG codon by site-specific mutagenesis resulted in a virus with a reduced ability either to replicate or to transform a population of chicken embryo fibroblasts. These results establish a procedure for determining sites of ribosome binding and initiation of translation on mRNAs in intact eucaryotic cells and strongly suggest that the 5'-proximal open reading frame (or its AUG codon) on Rous sarcoma virus RNA has an important role in regulating viral activity.     

Effect of microinjected amine and diamine oxidases on the ultrastructure of eukaryotic cultured cells

Diamine oxide and serum amine oxidase, which catalyse the oxidation of diamines and polyamines, respectively, were trapped within reconstituted Sendai virus envelopes. These loaded envelopes were incubated with cultured normal chick fibroblasts or with fibroblasts transformed by Rous sarcoma viruses. The binding of the reconstituted envelopes to the cultured cells was confirmed by scanning electron microscopy. It has been shown that the reconstituted envelopes (1-3 microns diameter) were attached to the eukaryotic cells. No significant changes in the morphology of the normal chick embryo fibroblasts were noted upon treatment with enzyme-loaded envelopes. On the other hand, chick embryo fibroblasts transformed by Rous sarcoma virus were affected by the microinjected amine oxidases. Scanning electron microscopy demonstrated the formation of holes in the microinjected cells. Similar morphological changes were also observed when diamine oxidase was microinjected into cultured glioma cells. These holes may be the result of the ejection of the nucleus. These findings are in line with the observed effect of the injected amine oxidases on macromolecular synthesis in normal and transformed chick embryo fibroblasts.     

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.     

Use of recombinant plasmids to characterize collagen RNAs in normal and transformed chick embryo fibroblasts.

Two recombinant plasmids containing chick collagen DNA sequences have been used to characterize messenger RNAs for pro-alpha1 (type I) and pro-alpha2 collagen. Poly(A)-containing RNA from chick embryo calvaria and long bones, tissues which are very active in collagen synthesis, were electrophoresed on agarose gels containing methylmercuric hydroxide and transferred to diazobenzyloxymethyl paper; these covalently bound RNAs were hybridized to 32P-labeled pro-alpha1 or pro-alpha2 collagen DNA sequences derived from the recombinant plasmids. The pro-alpha1 collagen probe identified two RNAs, a major species of 5000 bases and a minor species of 7100 bases; the pro-alpha2 collagen probe hybridized to a major species very similar in size to the pro-alpha1 mRNA, about 5200 bases, and a minor species of 5700 bases. It is possible that the 7100 and 5700 base RNAs represent precursors of pro-alpha1 and pro-alpha2 collagen mRNA, respectively. When similar hybridization experiments were performed with RNA from chick embryo fibroblasts, both the pro-alpha1 and pro-alpha2 collagen mRNAs were observed, as well as their corresponding larger species. With RNAs from fibroblasts transformed by Rous sarcoma virus, however, the levels of all RNA species which hybridized with the pro-alpha1 and pro-alpha2 collagen DNA probes were significantly reduced.     

Effects of inhibitors of lipid synthesis on the replication of rous sarcoma virus. A specific effect of cerulenin on the processing of major non-glycosylated viral structural proteins

The effects of two inhibitors of lipid biosynthesis on the replication of Rous sarcoma virus Prague C strain in chick embryo fibroblasts have been examined in media containing delipidated serum. 25-Hydroxycholesterol, which markedly inhibits the incorporation of [1-¹⁴C]acetate into sterols, had no effect on the formation of infectious virions or on the synthesis and processing of intracellular virion proteins. Cerulenin strongly inhibited [1-¹⁴C]acetate incorporation into fatty acids and partially inhibited its incorporation into sterols in chick embryo cells. Rous sarcoma virus production as measured by focus formation and by the production of [³⁵S]methionine-labeled virions was strongly inhibited within 5 h after cerulenin addition to infected cultures. Examination of extracts of these cells revealed the accumulation of the 76 000 dalton precursor (Pr76) of the major non-glycosylated virion structural proteins, p27, p19, p15 and p12. The failure to process the 76 000 dalton precursor was coincident in time with the decrease in viron production. Neither whole serum nor mixtures of fatty acids plus cholesterol were able to reverse the effects of cerulenin.     

Modifications of drug metabolism by disulfiram and diethyldithiocarbamate. I. Mixed-function oxygenase.

Disulfiram and diethyldithiocarbamate were administered to rats for 4 days alone (300 mg/kg, daily, per os) or in combination with phenobarbital (80 mg/kg, daily, i.p.), in order to observe the effects of these compounds on the microsomal membrane components and on the mixed-function oxygenase system. Both disulfiram and diethyldithiocarbamate increased the liver to body weight ratio, and the total hepatic protein content. Disulfiram significantly increased also the microsomal protein and phospholipid contents. Diethyldithiocarbamate and disulfiram partially prevented the increase of microsomal protein and phospholipid contents caused by phenobarbital. Disulfiram and diethyldithiocarbamate decreased the amount of cytochrome P-450 and P-420, and the activity of p-nitroanisole O-demethylase. These changes were more pronounced after diethyldithiocarbamate than after disulfiram treatment. On the contrary, the activity of NADPH-cytochrome c reductase was enhanced only by disulfiram. The induction by phenobarbital of cytochrome P-450 and p-nitrosanisole O-demethylase was partially prevented on concomitant treatment with disulfiram and diethyldithiocarbamate. These compounds. however, had an additive effect with phenobarbital in enhancing the microsomal NADPH-cytochrome c reductase activity.     

Plasminogen activator in chick embryo muscle cells: induction of enzyme by RSV, PMA and retinoic acid.

To explore the generality of the effects of sarcoma viruses, tumor-promoting phorbol esters and retinoic acid, we have studied plasminogen activator production in differentiating chick myogenic cultures. Although slightly higher than in chick fibroblast cultures, the level of spontaneously synthesized enzyme is low; it reaches a peak shortly after maximum cell fusion has been completed and then declines. Rous sarcoma virus (RSV) transformation of differentiating myotubes was accomplished by infecting myoblasts with a temperature-sensitive mutant, maintaining cultures at the nonpermissive temperature until completion of fusion and shifting to permissive temperatures at selected times thereafter. RSV transformation, phorbol myristate acetate (PMA) and retinoic acid all induced high levels of plasminogen activator production by differentiating myotubes in the absence of DNA synthesis. In comparison with fibroblasts, virus-induced enzyme synthesis by myogenic cultures proceeded more slowly but ultimately reached comparably high levels. Whereas cAMP strongly repressed RSV- and PMA-induced plasminogen activator production by chick fibroblasts, it weakly stimulated enzyme synthesis by myotubes. This suggests that enzyme induction by RSV and PMA is not mediated primarily through effects on cAMP metabolism.     

Effects of inhibitors of lipid synthesis on the replication of Rous sarcoma virus. A specific effect of cerulenin on the processing of major non-glycosylated viral structural proteins.

The effects of two inhibitors of lipid biosynthesis on the replication of Rous sarcoma virus Prague C strain in chick embryo fibroblasts have been examined in media containing delipidated serum. 25-Hydroxycholestetate into sterols, had no effect on the formation of infectious virions or on the synthesis and processing of intracellular virion proteins. Cerulenin strongly inhibited [1(-14C)]acetate incorporation into fatty acids and partially inhibited its incorporation into sterols in chick embryo cells. Rous sarcoma virus production as measured by focus formation and by the production of [35S]methionine-labeled virions was strongly inhibited within 5 h after cerulenin addition to infected cultures. Examinatin of extracts of these cells revealed the accumulation of the 76 000 dalton precursor (Pr76) of the major non-glycosylated virion structural proteins, p27, p19, p15 and p12. The failure to process the 76 000 dalton precursor was coincident in time with the decrease in viron production. Neither whole serum nor mixtures of fatty acids plus cholesterol were able to reverse the effects of cerulenin.     

RNA-directed DNA polymerase of Rous sarcoma virus: initiation of synthesis with 70 S viral RNA as template

DNA synthesis by the RNA-directed DNA polymerase of Rous sarcoma virus with 70 S viral RNA as template initiates by the covalent attachment of dAMP to the 3′ terminal adenosine of an RNA molecule. Initiation continues throughout the course of a 90-minute enzymatic reaction, and chain propagation occurs on most if not all of the dAMP residues attached to primer RNA. The nature of the primer molecules was established in two ways. First, the RNA was tagged by attachment of radioactive mono- and oligodeoxynucleotides. Second, primers were isolated directly from their covalent complexes with nascent DNA. The results of both procedures indicate that DNA synthesis initiates on the 3′ termini of 4 S RNA molecules hydrogen-bonded to 70 S RNA. Purified primer RNA has a nucleotide composition (G + C = 64%) different from that (G + C = 60%) of other 4 S RNAs found hydrogen-bonded to the 70 S RNA of Rous sarcoma virus.     

Cell Cycle-Dependent Activation of Rous Sarcoma Virus-Infected Stationary Chicken Cells: Avian Leukosis Virus Group-Specific Antigens and Ribonucleic Acid

Stationary chicken embryo fibroblasts exposed to Rous sarcoma virus (RSV) remained stably infected for at least 5 days, but they did not release infectious virus or become transformed until after cell division. These infected stationary cells did not contain avian leukosis virus group-specific antigens or ribonucleic acid (RNA) hybridizable to deoxyribonucleic acid (DNA) made by the RSV endogenous RNA-directed DNA polymerase activity.