Impairment of reovirus mRNA methylation in extracts of interferon-treated Ehrilich ascites tumor cells: further characteristics of the phenomenon.

We reported earlier that the methylation of unmethylated reovirus mRNA (reo mRNAU) by the cellular methylating enzymes is impaired in extracts of uninfected, interferon-treated Ehrilich ascites tumor cells (S30INT). We find now that after the methylation of reo mRNAU has stopped in S30INT, the RNA can be reisolated and further methylated in an extract of control cells (S30C). Thus the impairment of methylation in S30INT cannot be due to cleavage or irreversible inactivation of reo mRNAU. Freshly added reo mRNAU can be methylated in S30INT in which the methylation of previously added reo mRNAU has stopped. This indicates that the impairment is due to the depletion of S-adenosylme thionine (the methyl donor), the accumulation of S-adenosylhomocysteine (an inhibitor of methylation), or the irreversible inactivation of reo mRNAU. Freshly added reo mRNAU can be methylated in S30INT in which the methylation of previously added reo mRNAU has stopped. This indicates that the impairment is not due to the depletion of S-adenosylmethionine (the methyl donor), the accumulation of S-adenoxylhomocysteine (an inhibitor of methylation), or the irreversible inactivation of the methylating enzymes. It may be due, however, to the unavailability of reo mRNAU for methylation. The extent of the impairment of reo mRNAU methylation in S30INT decreases with an increasing concentration of reo mRNAU but is not affected by added poly (U), ribosomal RNA, or encephalomyocarditis virus RNA (an mRNA that is probably not capped or methylated at its 5' end). The methylation of reo mRNAU is also impaired in an extract from cells that have not been treated with interferon but with the interferon inducer poly(I) - poly(C). The inhibitor is apparently a macromolecule that is inactivated during incubation. It decreases the methylation at the 7 position of the 5' terminal guanylate residue. In vitro, the rate of reo mRNA synthesis by reovirus cores in the presence of S30INT is the same as in the presence of S30C. However, the methylation of the de novo synthesized reo mRNA by the core-associated methylating enzyme(s) in vitro is inhibited by S30INT but not by S30C. The relevance of these phenomena to the inhibition of reovirus replication in interferon-treated cells remains to be established.     

Maintenance of protein synthesis in spite of mRNA breakdown in interferon-treated HeLa cells infected with reovirus.

Interferon induces the synthesis of an enzyme which synthesizes 2',5'-oligoadenylate [2',5'-oligo(A)] when activated by double-stranded RNA. The 2',5'-oligo(A) in turn activates an endonuclease (RNase L). Concentrations of 2',5'-oligo(A) sufficient to activate RNase L are formed in interferon-treated HeLa cells infected with reovirus, and a large fraction of cellular mRNA is degraded (T. W. Nilsen, P. A. Maroney, and C. Baglioni, J. Virol. 42:1039-1045, 1982). We report here that in spite of this mRNA degradation, protein synthesis was not significantly inhibited in these cells. When mRNA synthesis was inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, protein synthesis was markedly decreased, as shown by reduced incorporation of labeled amino acids and a decrease in polyribosomes. This suggested that the turnover of mRNA could be compensated for by increased production of mRNA. The relative concentration of specific mRNAs was measured with cloned cDNA probes. The amount of these mRNAs present in control cells was comparable to that in interferon-treated cells infected with reovirus, whereas it was decreased in the latter cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.     

Inhibition of protein synthesis in reovirus-infected HeLa cells with elevated levels of interferon-induced protein kinase activity

Protein synthesis was inhibited in one line of interferon-treated HeLa cells (line 2) upon infection with reovirus, but not in different HeLa cells (line 1) treated in the same way. The inhibition resulted in polysome runoff, suggesting that it was due to an impairment of peptide chain initiation. Interferon induces the synthesis of a protein kinase, which is activated in cell-free systems by double-stranded RNA and phosphorylates the alpha subunit of eukaryotic initiation factor 2, thus inhibiting the initiation of protein synthesis. Therefore, we measured the level of this protein kinase in extracts prepared from the two HeLa cell lines. Cells of line 2 showed about 3-4 times more protein kinase activity than cells of line 1. The inhibition of protein synthesis upon infection with reovirus was correlated with an increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 in interferon-treated cells labeled with 32P. The kinase was presumably activated in intact cells by viral double-stranded RNA, but this activation resulted in inhibition of protein synthesis only in cells with elevated levels of the kinase.     

Translation of reovirus messenger ribonucleic acids synthesized in vitro into reovirus proteins in a mouse L cell extract

Ten species of reovirus mRNAs were synthesized by incubating ATP, CTP, GTP, and UTP with reovirus particles which had been treated with chymotrypsin. The mRNAs obtained promote the synthesis of seven or more proteins in a cell-free system prepared from mouse L fibroblasts and the mobilities of these proteins during electrophoresis through polyacrylamide gels are indistinguishable from those of reo capsid proteins. Three antisera were prepared in rabbits: the first against the large size class of reo virion proteins, the second against the medium, and the third against the small. From the proteins whose synthesis was directed in the cell-free system by reo mRNAs each antiserum precipitates only those which correspond in size to the virion proteins against which the antiserum was prepared. The translation of reo mRNA occurs on large polysomal structures. Translation of peptide chains is initiated in the reo mRNA-directed cell-free system for at least 30 min. The average half-life of the various reo mRNAs during protein synthesis in our system is about 15 min. The optimal ionic conditions for reo mRNA translation are very different from those for encephalomyocarditis virus mRNA translation.     

Protein Synthesis in Cell-Free Systems: an Effect of Interferon

The activity of ribosome and cell-sap fractions from interferon-treated and control chick embryo fibroblasts was compared in mixed chick-mouse and purely chick cell-free systems capable of the synthesis of viral polypeptide(s) in response to viral ribonucleic acid (RNA). Interferon treatment of cells did not affect the intrinsic amino acid incorporation activity of these systems or their response to polyuridylic acid. With encephalomyocarditis (EMC) virus RNA as messenger, however, a fraction of the ribosomes from interferon-treated cells appeared less active than parallel controls. The results obtained with the corresponding cell-sap fractions were variable. Although competition between endogenous and added messengers cannot be excluded in these systems, a reduced level of translation of EMC RNA with interferon-treated cell ribosomes was also suggested by the results of analyses of tryptic digests of the products formed in response to the RNA. In addition, these analyses showed that this reduced activity must reflect a reduction in the rate or frequency of translation rather than a decrease in the length of the EMC RNA translated, for the same polypeptides were synthesized in response to the RNA with material from interferon-treated and control cells. Interferon added directly to the cell-free system was without effect. Although suggestive, these results do not provide definitive evidence for or against the hypothesis that virus protein synthesis is inhibited at the translational level in the interferon-treated cell. Possible alternative interpretations of the data are discussed.     

Protein-Synthetic Activity of Ribosomes from Interferon-Treated Cells

Cell-free protein-synthetic systems from normal and interferon-treated chick cells were compared. No difference was found in the amino acid incorporation activities of such ribosome-cell sap systems or in their response to polyuridylic acid. Throughout a variety of experiments we failed to detect the formation of a discrete peak of virus-specific polysomes, when ribosome monomers and subunits (from interferon-treated or control cells) were incubated with labeled Sindbis or Semliki Forest virus ribonucleic acid (RNA). Some binding of viral RNA did occur, but the complexes formed were evident in sucrose gradients as a broad, rapidly sedimenting shoulder on the ribosome monomer peak. Interferon pretreatment of cells did not affect the formation of these complexes in vitro, nor did it alter their rate of breakdown on incubation under amino acid incorporation conditions. Experiments with inhibitors of protein synthesis showed that such "breakdown" was not dependent upon amino acid incorporation and was not an index of translation. In these respects, our results are in marked contrast to those of Marcus and Salb. These results, together with our failure to detect any significant change in the protein composition of ribosomes from interferon-treated cells, suggest that such treatment does not result in a modification of the ribosome per se. They do not, however, rule out the involvement of a factor(s) required for ribosomes and viral RNA to function in viral protein synthesis. Indeed, it remains likely that interferon acts through such a mechanism, although the precise level at which the inhibition occurs remains to be elucidated.     

Inhibition of viral mRNA translation in interferon-treated L cells infected with reovirus.

Murine L cells were treated with interferon (IFN) concentrations which reduced by 75 to 80% the synthesis of viral mRNA after infection with reovirus. Protein synthesis was not inhibited in these cells up to 6 h after infection, but a large fraction of the viral mRNA was not associated with polyribosomes and sedimented at about 50S. In contrast, most of the reovirus mRNA was associated with polyribosomes in control infected cells. This mRNA was of similar size to non-polyribosomal mRNA from IFN-treated cells when analyzed by Northern blot hybridization with a cloned cDNA for the s2 reovirus mRNA, indicating that the non-polyribosomal mRNA was not appreciably degraded. Viral mRNA was labeled with [3H]uridine and the non-polyribosomal mRNA was isolated from IFN-treated cells. This mRNA could quantitatively bind to 80S initiation complexes when incubated in a rabbit reticulocyte cell-free system. These findings indicated that the non-polyribosomal RNA was translatable, but that its binding to functional initiation complexes was inhibited in IFN-treated cells by a discriminatory mechanism, which did not affect translation of cellular mRNA. Previous experiments showed that mRNA is blocked in 48S complexes when the alpha subunit of initiation factor eIF-2 is phosphorylated by the double-stranded RNA-dependent protein kinase induced by IFN. A localized activation of this kinase could explain the block of viral mRNA in 48S complexes. By labeling the phosphoproteins of IFN-treated cells with 32P, eIF-2 (alpha P) was shown to cosediment with non-polyribosomal mRNA, presumably in 48S complexes.     

Inhibition of protein synthesis directed by added viral and cellular messenger RNAs in extracts of interferon-treated Ehrlich ascites tumor cells. Location and dominance of the inhibitor(s)

Protein synthesis directed by exogenous (viral or cellular) messengers is impaired, but endogenous protein synthesis is not affected in an extract of interferon-treated Ehrlich ascites tumor cells (INT-extract). Protein synthesis directed by exogenous messengers is also impaired in a mixture of an INT-extract with an extract from control cells. This reveals that the impairment is due to one or more inhibitors in the INT-extract. The nondialyzability of the inhibitor(s) is probably an indication of large molecular size. In a not incubated INT-extract much of the inhibitory activity is in the high speed sediment fraction i.e., is presumably bound directly or indirectly to ribosomes. During incubation of the extract most of the inhibitory activity is released into the high speed supernatant fraction. The dose-response curve shows that in our conditions the translation of cellular messengers (from mouse L cells) is as sensitive to impairment by the inhibitor(s) as that of viral messengers (from reovirus or from encephalomyocarditis virus).     

Mechanism of interferon action: stability and translation of simian virus-40 early mRNA coding for T-antigen is comparable in untreated and interferon-treated monkey cells

The effect of interferon treatment on the translation and the stability of simian virus 40 (SV40) early mRNA coding for T-antigen was examined in tsA-infected monkey kidney BSC-1 cells at 40°. Neither the translation nor the stability of SV40 early mRNA was altered by interferon under cellular conditions where the synthesis of reovirus polypeptides was significantly inhibited by interferon. SV40 early mRNA decayed with a half-life of about 3 hours as measured by T-antigen synthesis; the decay rate was indistinguishable between untreated and interferon-treated cells.     

In vitro translation of polyadenylated RNA isolated from control and interferon-treated human promyelocytic HL-60 leukemic cells

Polyadenylated RNA has been isolated from control and interferon-treated HL-60 cells by centrifugation through cesium chloride and oligo(dT)-cellulose column chromatography. The affinity column-purified RNA is poorly translated in the mRNA-dependent rabbit reticulocyte lysates but is an excellent template for in vitro protein synthesis using the wheat germ cell extracts. The discrepancy in the efficiency of HL-60 mRNA utilization in the two commonly used cell-free protein synthesizing systems is attributable to an inhibitory component present in the polyadenylated RNA. This contaminant is most likely double-stranded RNA based on (i) the ability of 2-aminopurine (3-5 mM) or high concentrations of penicillium chrysogenum double-stranded RNA (10-15 micrograms/ml) to overcome the inhibition exerted by the component, and (ii) the ability of the component to promote the enzymatic conversion of ATP into 2-5A by the highly purified rabbit reticulocyte 2-5A synthetase.     

Impairment of reovirus mRNA 'cap' methylation in interferon-treated mouse L929 cells.

Reovirus mRNAs synthesized in vitro by the virionassociated enzyme have a 5' 'cap 1' structure (m7G(5')ppp(5')GmpCp...). However, about one third to one half of the reovirus mRNAs formed in mouse L929 cells have a 5' 'cap 2' structure (m7G(5')ppp(5')GmpCmp...) and the rest have a 5' 'cap 1' structure. The finding that virus mRNA 'cap' methylation is impaired in extracts of interferon-treated cells prompted us to study the effect of interferon on virus mRNA 'cap' methylation in vivo. Using labeling with [3H]-guanosine and dual labeling with [3H]methionine and [14C]uridine we compared the 5' structures of reovirus mRNAs accumulating between 5 and 11 h after infection in: L929 cells treated with 390 to 2600 U/ml of a partially purified mouse interferon preparation and untreated L929 cells. The treatment resulted in a 70 to 98% decrease in the 24 h virus yield and in a 50 to 55% decrease in the label accumulated in virus mRNAs. The 'capping' of virus mRNAs and the methylation of their 5' terminal and adjacent G residues were not diminished in interferon-treated cells. However, the percent of 'cap 2' termini was 36 to 47% lower in virus mRNAs from interferon-treated cells than in virus mRNAs from control cells. The interferon treatment did not result in the appearance of additional methylated nucleotides in the virus mRNAs.     

Specific protein phosphorylation in interferon-treated uninfected and virus-infected mouse L929 cells: enhancement by double-stranded RNA.

The enhanced phosphorylation of specific protein(s) observed in extracts from interferon-treated cells (in the presence of ATP and double-stranded [ds] RNA) was also seen in intact mouse L929 cells upon treatment with dsRNA, polyriboinosinic.polyribocytidylic acid [poly(rI.rC)] or reovirus dsRNA, using 32Pi as radiolabel. Labeling of a 65,000-dalton protein(s) with 32P was greatly increased in interferon-treated cells in the presence of added dsRNA, suggesting that the expression in vivo of the kinase activity involved is regulated by dsRNA. This was used as a test system to investigate whether the activity of interferon-induced enzyme(s) is stimulated following virus infection, possibly owing to the accumulation of dsRNA. No obvious increase in 32P-labeling of 65,000-dalton protein(s) was observed upon infection of interferon-treated cells with mengovirus or vesicular stomatitis virus. A basal level of 32P-labeling of the 65,000-dalton protein(s) was detected in interferon-treated cells in the absence of added dsRNA, indicating a basal level of expression of the kinase activity involved. The possible implications of these results are discussed.     

Purification and translation of murine mammary tumor virus mRNA's

We have studied the functions of the intracellular RNAs of mouse mammary tumor virus (MMTV) by purification and translation in vitro. Two major size classes of MMTV RNA, 35S and 24S RNA, were isolated from MMTV-infected rat (XC) cells and cultured mammary tumor cells by preparative hybridization of whole cell or polyadenylated RNA to cloned MMTV DNA covalently bound to chemically activated paper disks (diazobenzyloxymethyl paper). Genomic-length (35S) RNA was prepared free of 24S RNA by rate zonal sedimentation in sucrose gradients. Experiments using [3H]uridine-labeled cellular RNA indicated that the preparative annealing method was highly specific and capable of effecting a 300-fold enrichment for viral RNA; the recovered RNA appeared to be intact under denaturing conditions and directed synthesis of full-length gag and env polypeptides in vitro. The products of in vitro translation were identified by gel mobility, immunoprecipitation tests with antisera against gag and env products, and partial digestion with Staphylococcus V8 protease. The 35S RNA species directed synthesis of several gag-related polypeptides, including three previously reported in extracts of infected cells; 24S RNA directed synthesis of two polypeptides closely related to env proteins from infected cells. Therefore, 35S RNA includes mRNA's for gag and gag-pol, whereas 24S RNA is the mRNA for env. These results help establish the position of env on the physical map of the MMTV genome and bear upon the coding potential of the genome.     

Cellular integrity is required for inhibition of initiation of cellular DNA synthesis by reovirus type 3.

Synchronized HeLa cells, primed for entry into the synthesis phase by amethopterin, were prevented from initiating DNA synthesis 9 h after infection with reovirus type 3. However, nuclei isolated from synchronized cells infected with reovirus for 9 or 16 h demonstrated a restored ability to synthesize DNA. The addition of enucleated cytoplasmic extracts from infected or uninfected cells did not affect this restored capacity for synthesis. The addition of ribonucleotide triphosphates to nuclei isolated from infected cells stimulated additional DNA synthesis, suggesting that these nuclei were competent to initiate new rounds of DNA replication. Permeabilization of infected cells did not restore the ability of these cells to synthesize DNA. Nucleoids isolated from intact or permeabilized cells, infected for 9 or 16 h displayed an increased rate of sedimentation when compared with nucleoids isolated from uninfected cells. Nucleoids isolated from the nuclei of infected cells demonstrated a rate of sedimentation similar to that of nucleoids isolated from the nuclei of uninfected cells. The inhibition of initiation of cellular DNA synthesis by reovirus type 3 appears not to have been due to a permanent alteration of the replication complex, but this inhibition could be reversed by the removal of that complex from factors unique to the structural or metabolic integrity of the infected cell.