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.     

mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase from vaccinia virions. Donor and acceptor substrate specificites.

Characterization of the donor and acceptor specificities of mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase isolated from vaccinia virus cores has enabled us to discriminate between alternative reaction sequences leading to the formation of the 5'-terminal m7G(5')pppN-structure. The mRNA guanylyltransferase catalyzes the transfer of a residue of GMP from GTP to acceptors which possess a 5'-terminal diphosphate. A diphosphate-terminated polyribonucleotide is preferred to a mononucleoside diphosphate as an acceptor suggesting that the guanylyltransferase reaction occurs after initiation of RNA synthesis. Although all of the homopolyribonucleotides tested (pp(A)n, pp(G)n, pp(I)n, pp(U)n, and pp(C)n) are acceptors for the mRNA guanylyltransferase indicating lack of strict sequence specificity, those containing purines are preferred. Only GTP and dGTP are donors in the reaction; 7-methylguanosine (m7G) triphosphate specifically is not a donor indicating that guanylylation must precede guanine-7-methylation. The preferred acceptor of the mRNA (guanine-7-)-methyltransferase is the product of the guanylyltransferase reaction, a polyribonucleotide with the 5'-terminal sequence G(5')pppN-. The enzyme can also catalyze, but less efficiently methylation of the following: dinucleoside triphosphates with the structure G(5')pppN, GTP, dGTP, ITP, GDP, GMP, and guanosine. The enzyme will not catalyze the transfer of methyl groups to ATP, XTP, CTP, UTP, or to guanosine-containing compounds with phosphate groups in either positions 2' or 3' or in 3'-5' phosphodiester linkages. The latter specificity provides an explanation for the absence of internal 7-methylguanosine in mRNA. In the presence of PPi, the mRNA guanylyltransferase catalyzes the pyrophosphorolysis of the dinucleoside triphosphate G(5')pppA, but not of m7G(5')pppA. Since PPi is generated in the process of RNA chain elongation, stabilization of the 5'-terminal sequences of mRNA is afforded by guanine-7-methylation.     

Mechanism of interferon action. Effect of double-stranded RNA and the 5'-O-monophosphate form of 2',5'-oligoadenylate on the inhibition of reovirus mRNA translation in vitro

The effect of reovirus double-stranded RNA (dsRNA) and 5'-O-monophosphate form of 2',5'-oligoadenylate (pA(2'p5'A)2) on the translation and degradation of reovirus messenger RNA and on protein phosphorylation was examined in extracts prepared from interferon-treated mouse L fibroblasts. The following results were obtained. 1) The enhanced degradation of reovirus [3H]mRNA observed in the presence of either dsRNA or the 5'-O-triphosphate form of 2',5'-oligoadenylate (pppA(2'p5'A)3) was completely blocked by pA(2'p5'A)2. 2) The dsRNA-dependent phosphorylation of protein P1 and the alpha subunit of eukaryotic initiation factor (eIF-2) depended in a similar manner upon the concentration of dsRNA and was optimal at low dsRNA concentrations (0.1 to 1 microgram/ml). However, high concentrations of dsRNA (greater than 100 micrograms/ml) drastically reduced the phosphorylation of both P1 and eIF-2 alpha. Neither P1 nor eIF-2 alpha phosphorylation was affected by either pA(2'p5'A)2 or pppA(2'p5'A)3. 3) The translation of reovirus mRNA in vitro was inhibited by the addition of either low concentrations of dsRNA or pppA(2'p5'A)3. Whereas pA(2'p5'A)2 completely reversed the pppA(2'p5'A)3-mediated inhibition of translation, the inhibition mediated by low concentrations of dsRNA was only partially reversed by pA(2'p5'A)2. Under conditions where the pppA-(2'p5'A)3mediated degradation of reovirus mRNA was blocked, the translation of reovirus mRNA was still inhibited by low but not by high concentrations of dsRNA in a manner that correlated with the activation of P1 and eIF-2 alpha phosphorylation. These results suggest that the pppA(2'p5'A)n-dependent ribonuclease is not required and that protein phosphorylation may indeed be sufficient for the dsRNA-dependent inhibition of reovirus mRNA translation in cell-free systems derived from interferon-treated mouse fibroblasts.     

Requirement for 7-methylguanosine in translation of globin mRNA in vivo.

The 7-methylguanosine (m7G) residue present in the m7G5' ppp5'X-"CAP" structure of rabbit globin mRNA was removed quantitatively by periodate oxidation followed by beta-elimination in the presence of cyclohexylamine. The RNA thus treated was intact and exhibited no signs of degradation as examined by polyacrylamide gel electrophoresis in formamide. Assay for protein synthesis using a wheat germ cell-free system showed that the globin mRNA lacking m7G had lost most of its messenger activity. Identical treatment, of satellite tobacco necrosis virus (STNV) RNA, which does not contain the 5'-terminal "CAP" structure, resulted in no loss of its mRNA activity. Since the importance of the m7G residue in eukaryotic mRNA has not yet been shown essential for translation in vivo, both untreated and treated globin mRNAs were injected into frog oocytes and their translation into globin was measured at intervals over a ninety-six hour period. Globin mRNA either treated with periodate alone or lacking in m7g altogether were both found to have lost more than 90% of their activity in vivo.     

Effect of capping upon the mRNA properties of satellite tobacco necrosis virus ribonucleic acid.

The mRNA guanyltransferase-mRNA methyltransferases of vaccinia virions can be used to introduce a 5'-terminal m7g(5')pp(5')Apm... capping group onto the RNA of satellite tobacco necrosis virus (STNV RNA) to yield intact capped STNV RNA. Studies with an in vitro system from wheat germ and limiting quantities of capped and uncapped STNV RNA show that the rates and extents of formation of initiation complexes of protein synthesis by intact capped and uncapped STNV RNA are identical, suggesting that 5'-terminal cap groups cannot function in the translation of STNV RNA. Also, the cap analogue pm7G equally inhibits the initiation and the translation of limiting quantities of both capped and uncapped STNV RNA. These contrasting observations suggest that the wheat germ system contains a pm7G sensitive protein and that STNV RNA has a tertiary structure that restricts the function of an added 5'-terminal capping group. This theory is supported by observations that fragmented capped STNV RNA is better at forming initiation complexes than is equally fragmented uncapped STNV RNA.     

Two methyltransferase activities in the purified virions of vesicular stomatitis virus.

In addition to an RNA-dependent RNA polymerase, purified vesicular stomatitis virus contains a methyltransferase activity which transfers the methyl group from the methyl donor, S-adenosyl-L-methionine, to two positions in the 5'-terminal capped structure of the nascent mRNA's synthesized in vitro as 7mG-(5)'ppp(5')Apm... In the present study it is shown that two distinct methyltransferase activities are discernible in the purified virus. The in vitro concentrations of the methyl donor specify the number and location of the methyl groups transferred to the capped 5'-termini of VSV mRNA's. Limited concentrations of the methyl donor result in a single methylation of the penultimate base in the 2'-hydroxyl position, that is, G(5')ppp(5')Apm..., whereas saturating concentrations of the methyl donor methylate the blocking guanosine residue at the 7-position, resulting in the dimethylated cap, 7mG(5')ppp(5')Apm... Pulse-chase experiments demonstrate that the monomethylated cap structure is the precursor substrate for the dimethylated cap. In this respect, vesicular stomatitis virus system is quite distinct from the vaccinia and reovirus systems. Virus purified from different host cells including hamster, mouse, and human contain both methyltransferase activities. The mRNA's containing monomethylated capped structures are poor templates for protein synthesis in vitro.     

Specific inhibition of capped mRNA translation in vitro by m7G5''pppp5''G and m7G5''pppp5''m7G.

A unique set of diguanosine cap analogues containing a 5'-5' tetraphosphate linkage instead of the normal triphosphate was synthesized by chemical methylation of G5'pppp5'G. Both 7-methylguanosine products, m7G5'pppp5'G and m7G5'pppp5'm7G, acted as potent inhibitors of capped brome mosaic virus (BMV) RNA translation in the homologous wheat germ protein synthesis system. Inhibition of in vitro protein synthesis required the presence of the 7-methyl group on guanosine and was specific for capped mRNA. In comparison with the partial cap analogue, m7GTP, the methylated diguanosine tetraphosphate structures were 25-50 fold more potent inhibitors of in vitro protein synthesis. Analysis of the in vitro translation products of the four species of BMV RNA showed a differential sensitivity to inhibition by m7G5'pppp5'm7G.     

Methyl group analysis of virion-associated high-molecular-weight RNA synthesized in vitro by purified vaccinia virus.

The methylation pattern of virion-associated high-molecular-weight RNA synthesized in vitro by purified vaccinia virus has been determined. Analysis of purified high-molecular-weight RNA synthesized with S-[methyl-3H]-adenosylmethionine and alpha[32P]UTP as precursors gave the following results. (i) Eessentially all molecules contained blocked and methylated structures of the type m7G(5')ppp(5')Gm and m7G(5')ppp(5')Am. (ii) There was no detectable methylation at internal sites. (iii) Under several different conditions of synthesis, the ratio of molecules containing m7G(5')ppp(5')Gm to those containing m7G(5')ppp(5')Am was imilar for both the virion-associated high-molecular-weight RNA and the virion-released 8-12S mRNA.     

Structural requirements of (2'-5') oligoadenylate for protein synthesis inhibition in human fibroblasts.

The structural requirements of (2'-5')-oligoadenylic acid (pppA(2'p5'A)x, X greater than or equal to 1 or (2'-5'An) for inhibition of protein synthesis in cells were examined with a modified calcium-coprecipitation technique, using a series of trinucleotide analogs (pppA2'p5'A2'p5'N, N=rC, rG, rU, T, dC, dG, dA). In this system both the degree and the duration of the inhibition of protein synthesis were dependent on the added concentration of (2'-5')A3. Of all the heterotrimers, only the deoxy A derivative was active as an inhibitor of protein synthesis, while the other members of the analog series were found to have no inhibitory effects. In competition experiments between (2'-5')A3 and the non-active analogs, three heterotrimers were shown to reduce the activity of (2'-5')A3 in protein inhibition. In contrast, the dephosphorylated (2'-5')A3 had no inhibitory effect and was not effective in blocking (2'-5')A3. These results indicate that the 5'-terminal triphosphate is important for binding of (2'-5')A3 to the site of (2'-5')An action and the adenine base at the 2'-terminus is important for activating the machinery responsible for protein synthesis inhibition in the cells, most likely the (2'-5')An-activated nuclease.     

The respective roles of the protein kinase and pppA2'' p5'' A2'' p5 A-activated endonuclease in the inhibition of protein synthesis by double stranded RNA in rabbit reticulocyte lysates.

Double-stranded RNA (dsRNA) inhibits protein synthesis in rabbit reticulocyte lysates by activating the synthesis of the endonuclease effector pppA2' p5' A2' p5' A(2-5A) and a protein kinase which phosphorylates the protein synthesis initiation factor eIF-2. Under certain assay conditions, high concentrations of dsRNA are without inhibitory effect in many lysates (high dsRNA "reversible" lysates). In these lysates natural dsRNA at low concentrations stimulated protein kinase activity to a greater extent than did the synthetic dsRNA poly rI.rC. Synthesis of 2--5A was greater when poly rI.rC was used. However, a number of factors, including the salt concentration and messenger RNA used, combine to determine the overall effect of dsRNA on protein synthesis under any given set of experimental conditions.     

Chemical synthesis of the 5'-terminal part bearing cap structure of messenger RNA of cytoplasmic polyhedrosis virus (CPV): m7G5'pppAmpG and m7G5'pppAmpGpU.

The 5'-terminal structures of mRNA bearing the so-called 'cap' from cytoplasmic polyhedrosis virus (CPV), m7G5' pppAmpG and m7G5' pppAmpGpU, were first chemically synthesized. S,S-Di(4-methoxyphenyl) N6-benzoyl-2'-O-methyladenosine 5'-phosphorodithioate ((ArS) 2pAbmz) was prepared by phosphorylation of the 5'-hydroxyl group of N6-benzoyl-2'-O-methyladenosine with S,S-di(4-methoxyphenyl) phosphorodithioate by TPS. By the triester approach using (ArS) 2pAbmz as starting material, the protected dinucleotide and trinucleotide bearing 5'-phosphate group were synthesized. The protective groups of the dinucleotide and trinucleotide were removed to obtain pAmpG and pAmpGpU, respectively. By the reaction of a capping agent ((PhS) ppm7G) with pAmpG and pAmpGpU in the presence of silver nitrate or iodine. The 5'-terminal structure of the messenger RNA strand of CPV which was labelled isotopically, was confirmed completely as m7G5' pppAmGpU by cochromatography with the materials chemically synthesized here.     

Characterization and translation of methylated and unmethylated vesicular stomatitis virus mRNA synthesized in vitro by ribonucleoprotein particles from vesicular stomatitis virus-infected L cells.

Ribonucleoprotein particles isolated from extracts of vesicular stomatitis virus (VSV) -infected L cells synthesized in vitro four classes of polyadenylated RNA sedimenting at 29S, 19S, 17S, and 13S. When synthesized in vitro in the presence of the methyl donor S-adenosyl methionine, these RNA species contained the following 5'-terminal structures: (i) m7G5ppp5'AmpAp(70%) ; (ii) m7G5'ppp5'AmpAmpNp (20%) and (iii) pppAp (10%). In the presence of the methylation inhibitor S-adenosylhomocysteine, however, the mRNA contained the 5'-terminal structures G5'ppp5'Ap (80%) and pppAp (20%). The mRNA's synthesized in vitro were translated in the homologous ascites and the heterologous wheat embryo cell-free systems. In both, the products were shown by sodium dodecyl sulfate gel electrophoresis and by immunoprecipitation to contain all five viral proteins, L, G, N, NS, and M. The presumed precursor to the G protein (G*) was also identified by fingerprint analysis. Methylated VSV mRNA was more active in protein synthesis than unmethylated mRNA in both the ascites system and the wheat embryo systems. Addition of S-adenosylmethionine stimulated translation of unmethylated mRNA in the wheat embryo but not in the ascites extract. S-adenosylhomocysteine, however, by preventing mRNA methylation inhibited the translation of unmethylated VSV mRNA in both systems. The mRNA methylating activity present in wheat embryo S-30 extracts was recovered in the ribosome-free supernatant fraction (S-150) and was insensitive to the protein synthesis inhibitor pactamycin.     

Influence of potassium salt concentration and temperature on inhibition of mRNA translation by 7-methylguanosine5'-monophosphate.

The effect of 7-methylguanosine 5'-monophosphate (pm7G) on mRNA translation was examined in the wheat germ and rabbit reticulocyte cell-free systems. Differences between the two cell extracts with respect to inhibition of translation by pm7G can be attributed to different conditions commonly used for in vitro protein synthesis. Inhibition of globin mRNA translation by pm7G is strongly influenced by the concentration of potassium salt and to a lesser extent by incubation temperature. The effectiveness of the inhibitor increases with potassium salt concentration and diminishes with increasing temperature. Translation is inhibited by pm7G at physiological K+ concentration in both cell-free systems in that only the rate of binding of mRNA to ribosomes is affected by the inhibitor, not the extent of binding. Translation of different capped mRNAs is affected differently by pm7G, but this appears to be property of the mRNA rather than the translation system. These results indicate that while the 5'-terminal cap structure may be more important for translation of some mRNA's than others, this structure functions in translation of capped mRNAs in all types of cells.     

On the base-stacking in the 5'-terminal cap structure of mRNA: a fluorescence study.

The fluorescence at 370 nm of the 7-methylguanosine residue (m7G) is found to be quenched when the base residue is involved in a stacking interaction with the adenosine residue in the cap structure m7G5' pppA of an eukaryotic mRNA. On the basis of the observed degree of quenching, the amounts of the stacked and unstacked forms in the cap structure have been determined at various temperatures and pH's. It has been found that at pH 6.2 effective enthalpy and entropy in the unstacked leads to stacked change are delta H degrees = 4.4 +/- 0.1 kcal/mole and delta S degrees = - 14.3 +/- 0.2 e.u., respectively. The pka value for the m7G residue is found to be 7.7 at 10 degrees C and 7.3 at 30 degrees C. The stacked structure seems to be less favourable in the deprotonated form that occurs in the higher pH solution. A similar analysis of some other cap structures indicates that the stacked form in m7G5' pppN structure is favourable if N is a purine nucleoside or a 2'-O-methylpyrimidine nucleoside but not for an unmethylated pyrimidine nucleoside.     

Partial purification and characterization of rat-liver messenger RNA coding for phosphoenolpyruvate carboxykinase (GTP).

The mRNA coding for the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was partially purified from the liver of cyclic-AMP-treated rats by a procedure involving multiple oligo(dT)-cellulose chromatographies and sucrose gradient fractionations. The purification was monitored by translational assay using a wheat germ extract. Relative to RNA bound once to oligo(dT)-cellulose, the final material was enriched 20-fold in template activity for phosphoenolpyruvate carboxykinase synthesis. With this RNA preparation, cell-free enzyme synthesis amounted to 5% of total mRNA-directed protein synthesis. The apparent sedimentation coefficient of phosphoenolpyruvate carboxykinase mRNA in sucrose gradients was between 20 and 22 S, corresponding to an average molecular weight of 0.93 X 10(6). By formamide/polyacrylamide gel electrophoresis the molecular weight of the enzyme mRNA was estimated at between 0.91 X 10(6) and 1.12 X 10(6). From these estimates, it was concluded that considerable non-coding sequence(s) are present in the mRNA. Approximately 20% of the enzyme mRNA in rat liver failed to bind to oligo(dT)-cellulose, presumably because of the absence of a poly(A) segment. The translation of phosphoenolpyruvate carboxykinase mRNA by the wheat germ extract was inhibited in the presence of 7-methylguanosine 5'-phosphate. The enzyme mRNA appears therefore to have a 'cap' at the 5' end.     

Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity

During mRNA synthesis, the polymerase of vesicular stomatitis virus (VSV) copies the genomic RNA to produce five capped and polyadenylated mRNAs with the 5'-terminal structure 7mGpppA(m)pApCpApGpNpNpApUpCp. The 5' mRNA processing events are poorly understood but presumably require triphosphatase, guanylyltransferase, [guanine-N-7]- and [ribose-2'-O]-methyltransferase (MTase) activities. Consistent with a role in mRNA methylation, conserved domain VI of the 241-kDa large (L) polymerase protein shares sequence homology with a bacterial [ribose-2'-O]-MTase, FtsJ/RrmJ. In this report, we generated six L gene mutations to test this homology. Individual substitutions to the predicted MTase active-site residues K1651, D1762, K1795, and E1833 yielded viruses with pinpoint plaque morphologies and 10- to 1,000-fold replication defects in single-step growth assays. Consistent with these defects, viral RNA and protein synthesis was diminished. In contrast, alteration of residue G1674 predicted to bind the methyl donor S-adenosylmethionine did not significantly perturb viral growth and gene expression. Analysis of the mRNA cap structure revealed that alterations to the predicted active site residues decreased [guanine-N-7]- and [ribose-2'-O]-MTase activity below the limit of detection of our assay. In contrast, the alanine substitution at G1674 had no apparent consequence. These data show that the predicted MTase active-site residues K1651, D1762, K1795, and E1833 within domain VI of the VSV L protein are essential for mRNA cap methylation. A model of mRNA processing consistent with these data is presented.     

"Cap" sequences in poly(A)-rich RNA from dry wheat embryos

Although template-active RNA in dry seeds and embryos has attracted widespread interest, there have been no published reports about 5'-terminal "capping" sequences in such RNA. Boro[3H]hydride labeling of periodate-oxidized termini and high performance liquid chromatography of cap oligonucleotides have been used to compare terminal sequences in poly(A)-rich RNA from dry and germinating embryos. As is the case in germinating embryos, poly(A)-rich RNA from dry embryos contains only "type 0" cap sequences, i.e., m7G(5')ppp(5')N, in which m7G is the 7-methylguanosine cap and N is any of the classical ribonucleosides: adenosine (A), guanosine (G), cytidine (C),a nd uridine (U). Striking differences between the cell-free translational capacities of bulk messenger RNA (mRNA) populations from dry and germinating embryos are not reflected in signal differences in their proportions of "type 0" cap structures: in general, there is approximately 40% m7G(5')ppp(5')A, with roughly equivalent amounts of m7G(5')ppp(5')G and m7G(5')ppp(5')C accounting for most of the remaining sequences. The findings with mRNA from dry plant embryos serve to emphasize interesting differences between patterns of methylation in the capped and uncapped RNA molecules in higher plants and animals; the differences have not been previously noted in the literature and are the subject of brief comment in this paper.