Turnip Yellow Mosaic Virus RNA Research Articles

In vivo aminoacylation and ‘processing’ of turnip yellow mosaic virus RNA in Xenopus laevis oocytes

IT is now well established that several plant and animal virus RNAs possess a ‘tRNA-like’ structure, as they are recognised in vitro by many tRNA-specific enzymes. To approach the problem of the possible physiological significance of these structures for the development of the virus, it seemed important to determine whether they are also recognised in vivo. The most logical approach would have been to use plant protoplast systems to study the behaviour of viral genomes in the infected cells. However, because such systems are still difficult to control, we decided to use the oocyte system. We describe here how turnip yellow mosaic virus (TYMV) RNA, which can be aminoacylated in vitro by valine1, was microinjected into Xenopus laevis oocytes, incubated in the presence of 3H-valine and the RNA charged in vivo analysed. We conclude that TYMV RNA, the 3′ terminal sequence of which is –CC (refs 2, 3), is amino-acylated in vivo. This indicates that the viral RNA is recognised by the oocyte tRNA nucleotidyltransferase and valyl-tRNA synthetase. Furthermore, the TYMV Val-RNA recovered after the in vivo reaction is fragmented and migrates between 4S and 5S RNA markers. This implies that (an) RNase(s) split(s) the viral genome, liberating this tRNA-like structure.

Studies of virus structure by laser-Raman spectroscopy. 4. Turnip yellow mosaic virus and capsids

Laser-Raman spectroscopy of the turnip yellow mosaic virus (TYMV) and its capsid indicate the following features of the structure and assembly of the virion. The secondary structure of coat-protein molecules in TYMV is comprised of 9 +/- 5% alpha-helix, 43 +/- 6% beta-sheet, and 48 +/- 6% irregular conformation and is not altered by the removal of the RNA from the capsid. Introduction of as many as 200 chain scissions per RNA molecule also does not affect the overall secondary structure of the encapsulated RNA, which is 77 +/- 5% in the A-helix form. Tryptophan and cysteine residues of the coat protein appear to be in contact with the solvent, while only one of three tyrosines per coat protein is available for hydrogen bonding of its p-hydroxyl group with H2O molecules. Both cytosine and adenine residues of TYMV RNA are protonated in substantial numbers near pH 4.5, suggesting elevation of their respective pKa values within the virion. The Raman data are consistent with chemical evidence favoring interaction between protonated bases of RNA and amino acid side chains of coat protein in TYMV.

Reverse transcription of turnip yellow mosaic virus RNA primed with calf-thymus DNA hydrolysate: characterization of the purified cDNA product.

Complementary DNA was transcribed from turnip yellow mosaic virus RNA, using the method of Taylor et al. (1). The purified cDNA thus obtained sedimented between 2 and 4 S and was a mostly uniform transcript of template RNA. It hybridized with a sharp transition to homologous TYMV-RNA (Crt 1/2 = 2.7 x 10(-2)), but showed a low level of hybridization (less than 5%) to the RNAs of two other tymoviruses, namely Andean potato latent virus and eggplant mosaic virus.

Properties of the DNA product of reverse transcription of turnip yellow mosaic virus RNA preparations

The reverse transcriptase of avian myeloblastosis virus catalyzes the synthesis of complementary DNA in the presence of turnip yellow mosaic virus (TYMV) RNA template and oligo(dG) 6 primer. The transcription product (single-stranded cDNA) had an estimated molecular weight of 150,000; it hybridized with TYMV-RNA, eggplant mosaic virus RNA, and Andean potato latent virus RNA, which are all members of the tymovirus group, but not with tobacco mosaic virus (TMV) RNA. It is concluded that all three tymovirus RNA preparations have common nucleotide sequences not shared with TMV-RNA. Furthermore, it appears that reverse transcription of TYMV-RNA preparations occurs in the absence of poly(A) at the 3′-end of genome RNA, when primed with oligo(dG) 6.

Messenger Properties of TYMV-RNA

Turnip yellow mosaic virus (TYMV) RNA extracted from virions (“virion” TYMV-RNA) contains two types of molecules of molecular weight 2 × 10 6: one which resists heat denaturation (“intact” TYMV-RNA) and one which yields smaller fragments upon heat denaturation (TYMV-RNA containing hidden breaks). A small RNA molecule of 0.2 × 10 6 MW is also present in virions. “Virion” TYMV-RNA can therefore be separated into “intact” RNA and smaller molecules by heat denaturation followed by sucrose gradient centrifugation. The RNA from the sucrose gradient fractions were analyzed by formamide PAGE: The small RNA with a molecular weight of 0.2 × 10 6 was separated from “intact” TYMV-RNA. The amount of coat protein synthesized by the RNA fractions when added to the wheat germ cell-free system was quite proportional to the amount of small RNA present. We conclude that coat protein synthesis is not directed by “intact” TYMV-RNA but by the small RNA. In addition, infectivity studies show that “intact” TYMV-RNA is fully infectious and thus it also contains the coat protein cistron.

Studies on the Sequence of the 3′‐Terminal Region of Turnip‐Yellow‐Mosaic‐Virus RNA

A fragment representing the 3'-terminal 'tRNA-like' region of turnip yellow mosaic (TYM) virus RNA has been purified following incubation of intact TYM virus RNA with Escherichia coli 'RNase P'. This fragment, which is 112+3-nucleotides long has been completely digested with T1 RNase and pancreatic RNase and all the oligonucleotides present in such digests have been sequenced using 32P-end labelling techniques in vitro. The TYM virus RNA fragment is free of modified nucleosides and does not contain a G-U-U-C-R sequence. Using nuclease P1 from Penicillium citrinum, the sequence of 26 nucleotides from the 5' end and 16 nucleotides from the 3' end of this fragment has been deduced. The nucleotide sequence at the 5' end of the TYM virus RNA fragment indicates that this fragment includes the end of the TYM virus coat protein gene.

Nucleotide sequence (n=159) of the amino-acid-accepting 3'-OH extremity of turnip-yellow-mosaic-virus RNA and the last portion of its coat-protein cistron.

The experiments described in this paper and the following one establish the sequence of the 3'-OH terminal 159 nucleotides of turnip yellow mosaic virus RNA. Uniformly 32P-labeled turnip yellow mosaic virus RNA was partially digested with T1 ribonuclease and the fragments were fractionated by polyacrylamide gel electrophoresis. Fragments originating from the 3'-OH end of the RNA molecule were identified by testing for the 3'-terminal oligonucleotide, C-COH, after total U2 ribonuclease hydrolysis. Once identified, the 3'-OH terminal fragments were sequenced by the methods of Sanger et al. The first 51 nucleotides of the longest of the sequenced fragments (158 nucleotides) extends into the 3'-terminal part of the coat protein cistron. The coat protein cistron is followed by a stretch of 108 untranslated nucleotides whose function, though still unknown, is probably linked to the tRNA-like properties which have been attributed to the 3'-OH extremity of this viral RNA. Two possible secondary structures are proposed for the sequence and the implications of the findings with regard to the tRNA-like properties of the extremity are discussed.

Translation of turnip yellow mosaic virus RNA in vitro: a closed and an open coat protein cistron.

Sucrose gradient centrifugation of heat-denatured RNA of turnip yellow mosaic virus permitted the isolation of five RNA classes with molecular weights ranging from 2.0 to 0.25 X 10(6). The infectivity was shown to be confined to an RNA molecule of molecular weight 2.0 X 10(6). No significant increase in infectivity was obtained by combination of the latter RNA with the RNA classes of smaller size. Translation in vitro of the RNAs of different size classes in a wheat germ cell-free system revealed that the infectious RNA (molecular weight 2.0 X 10(6) does not promote the synthesis of the coat protein of turnip yellow mosaic virus. Efficient production of this coat protein was found exclusively when the smallest RNA class (molecular weight 250,000) was used as a messenger. It is concluded that RNA molecules of turnip yellow mosaic virus of molecular weight 2.0 X 10(6) contain a closed coat protein cistron and that RNA molecules of molecular weight about 2 to 3 X 10(5) with an open coat protein cistron can be isolated from the virions.

Physical and functional heterogeneity in TYMV RNA: evidence for the existence of an independent messenger coding for coat protein.

Turnip yellow mosaic virus RNA can be separated into two distinct components of 2 times 10(6) and 300 000 daltons molecular weight after moderate heat treatment in the presence of SDS or EDTA. The two species cannot have arisen by accidental in vitro degradation of a larger RNA, as they both possess capped 5' ends. Analysis of the newly synthesized proteins resulting from translation of each RNA by a wheat germ extract shows that the 300 000 molecular weight RNA can be translated very efficiently into coat protein. When translated in vitro the longer RNA gave a series of high molecular weight polypeptides but only very small amounts of a polypeptide having about the same mass as the coat protein. Thus our results suggest that the small RNA is the functional messenger for coat protein synthesis in infected cells.

In Vitro Synthesis of Turnip Yellow Mosaic Virus Coat Protein in a Wheat Germ Cell-Free System

Turnip Yellow Mosaic Virus RNA directs the synthesis in vitro of its coat protein in a wheat germ cell-free extract. Optimum conditions for synthesis have been defined, and the effect of spermine on specifically enhancing coat protein formation has been examined. Identity between the in vitro synthesized coat protein and authentic coat protein of Turnip Yellow Mosaic Virus was established by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and immunoprecipitation.

Low pH RNA-protein interactions in turnip yellow mosaic virus: II. Binding of synthetic polynucleotides of TYMV capsids and RNA

It has been previously shown that turnip yellow mosaic virus capsids and RNA obtained by incubation in 8 M urea, 1 M NaCl at pH 7 could be reassociated by dialysis against low ionic strength buffer, in the presence of MgCl 2 or spermidine. The nucleoprotein complexes differed from native virions in stability at neutral pH and in RNase-resistance at low pH, but the association is specific, as TMV-RNA or alfalfa mosaic virus-RNA was found not to interact with capsids under such conditions. We present here further data concerning the specificity of this interaction. Radioactive poly(A), poly(C), poly(G) and poly(U) were added to the dissociation mixture of TYMV in the presence of 5 × 10 −3 M MgCl 2. It was found that only poly C reassociated specifically with “nascent capsids.” Poly(A) did not interact at all with either capsids or RNA. At either neutral or acidic pH, small amounts of poly U bound to capsids, but not to RNA or virions. Poly(G) forms a hybrid with TYMV-RNA. The nature of low pH RNA-protein interaction in TYMV is discussed.

Aminoacylation of RNA from several viruses: amino acid specificity and differential activity of plant, yeast and bacterial synthetases.

SUMMARY The RNA of broad bean mottle virus (BBMV), cowpea chlorotic mottle virus (CCMV), and cucumber mosaic virus (CMV) bound tyrosine, as does brome mosaic virus (BMV) RNA. Other amino acids were not bound. Comparison of the efficiency of synthetase enzymes from Escherichia coli, bean, wheat, and Saccharomyces cerevisiae in aminoacylation of RNA from these viruses, and from tobacco mosaic virus (TMV) and turnip yellow mosaic virus (TYMV), showed that the plant enzymes were effective in tyrosylation of RNA from bromoviruses and CMV, whereas the yeast and bacterial enzymes were ineffective. The E. coli enzyme did not esterify TMV RNA with histidine, and the bean enzyme was poor in this ability. All enzymes were able to catalyse valine binding to TYMV RNA. However, 40 mm-KCl inhibited valine binding to TYMV RNA by the bacterial enzyme while its ability to catalyse binding by tRNA was not affected.

Role of Deoxyribonucleic Acid-Ribonucleic Acid Hybrids in Eukaryotes: SYNTHETIC RIBO- AND DEOXYRIBOPOLYNUCLEOTIDES AS TEMPLATE FOR YEAST RIBONUCLEIC ACID POLYMERASE B (OR II)

Abstract The template specificity of yeast RNA polymerase B (or II) has been investigated using single-stranded homopolymers, helical homopolymers, and alternated copolymers. The enzyme exhibited a remarkable base sequence specificity for single-stranded polynucleotides; only those containing pyrimdine nucleotides were transcribed with (dC)n g (dT)n. Homopolymer pairs were transcribed asymmetrically, the pyrimidine-containing strand acting as template. The best synthetic template was the alternated d(I-C)n copolymer. The results signal a preference by the enzyme for initiating in pyrimidine-rich and (dC)-rich regions of the DNA. The structure of the RNA product was studied with ribonucleases of different specificity, RNases A and Ti, RNase H, and RNase III. It appeared that the dissociation of the RNA product from the template strand was determined by the relative stabilities of the helical structures involved. Net synthesis of RNA was favored by the release of the RNA chain from the template. Among polyribonucleotides, (rC)n was a very efficient template, turnip yellow mosaic virus RNA had some activity, but no other ribopolymer tested was found to have a significant activity. Competition experiments with polyribonucleotides indicated a different specificity with respect to the binding reaction, since (rG)n and (rU)n, which had no template activity, are much better inhibitors of transcription than (rC)n.

Messenger and aminoacylation functions of brome mosaic virus RNA after chemical modification of 3' terminus.

THE ability of RNA from several plant viruses to bind a specific amino acid in a manner like tRNA is now well established. All members of the tymovirus group so far examined contain RNA which can bind valine1–3; all members of the bromovirus group bind tyrosine (ref. 4 and T.C.H., in preparation), and tobacco mosaic virus (TMV) RNA binds histidine5. The ubiquity of the reaction, and the similarity of the esterification to that of aminoacylation of tRNA indicates that a significant biological function for the property exists. A tRNA-like function such as that of a virus-specific tRNA, or a missense tRNA seems plausible. Although there is evidence that turnip yellow mosaic virus (TYMV) RNA, or a fragment containing the 3′ terminus of TYMV RNA, may donate valine to nascent polypeptides6, it has been established that tyrosine, bound to brome mosaic virus (BMV) RNA, is not donated to peptidyl material in a cell-free system from wheat embryo when directed by viral, plant, or synthetic messenger RNA (ref. 7).

Turnip yellow mosaic virus-RNA replicase: Partial purification of the enzyme from the solubilized enzyme-template complex

The turnip yellow mosaic virus (TYMV) RNA-replicase bound to the chloroplast fraction of a participate cell-free preparation isolated from TYMV-infected Chinese cabbage leaves catalyzes the synthesis of RNA of the viral type, “plus” RNA, in the absence of added “minus” RNA template. Addition of TYMV-RNA does not stimulate RNA synthesis. The complex formed by the replicase bound to “minus” RNA template can be removed from the chloroplast fraction by solubilization with a non-ionic detergent, Lubrol W, and separated from insoluble material by centrifugation. The resulting supernatant contains the template-bound replicase as well as a small proportion of template-free replicase molecules. Treatment of this preparation with a polyethylene glycol-dextran two-phase aqueous system at high salt concentration removes the replicase from the template and yields, after centrifugation, a polyethylene glycol upper phase containing the template-free replicase molecules (PEG-enzyme) and a lower dextran phase containing a small proportion of replicase molecules still bound to their template. The soluble PEG-enzyme is markedly stimulated by added RNA. With TYMV-RNA as the added RNA, the product synthesized by the enzyme is RNA complementary to TYMV-RNA, i.e., “minus” RNA, indicating the enzyme uses the viral RNA as template. The “minus”-RNA synthesized remains hydrogen bonded to the viral RNA template and forms a RNase resistant, double-stranded structure. Chromatography on cellulose CF 11 column reveals two different double-stranded structures, one, fully double-stranded, in which the synthesized “minus” RNA strand is hydrogen bonded to template RNA of equal length, and one partly double-stranded and partly single-stranded in which probably the template RNA is very much longer than the synthesized “minus” RNA. The PEG-enzyme seems to be sensitive to high ionic strength since its activity can be completely suppressed by various salts; removal of the salts restores the activity.

Specificity of TMV RNA encapsidation: in vitro coating of heterologous RNA by TMV protein

Tobacco mosaic virus (TMV) RNA and TMV coat protein were reconstituted in the presence of an excess of host RNA or turnip yellow mosaic virus (TYMV) RNA. TMV protein and its homologous RNA recognized each other specifically in the presence of an excess of host RNA, but when TMV RNA and TYMV RNA were mixed with protein, both RNAs were encapsidated. Indeed, at pH 6, TYMV RNA was recognized by TMV protein at least as well as TMV RNA itself. This suggests that TYMV RNA like TMV RNA may possess a special site recognized by TMV protein.

Possible heterogeneity of 5′-terminal of turnip yellow mosaic virus RNA

Possible heterogeneity of 5′-terminal of turnip yellow mosaic virus RNA

TYMV RNA as a Substrate of the tRNA Maturation Endonuclease

RECENT studies have shown that the RNA (mol wt 2 × 106) of turnip yellow mosaic virus (TYMV) possesses at its 3′ end a structure similar to that of a tRNA. This is deduced from the fact that in the presence of valyl-tRNA synthetase, the 3′ end of TYMV RNA can be acylated with valine1,2. This RNA is also recognized by the tRNA nucleotidyltransferase3 and by the peptidyl-tRNA hydrolase2,4. Similar observations have been reported with tobacco mosaic virus (TMV) RNA which can be charged with histidine5,6 or with either methionine or serine7, and brome mosaic virus (BMV) RNA with tyrosine (Hall, Shih and Kaesberg, in the press).

Turnip Yellow Mosaic Virus RNA as a Substrate of the Transfer RNA Nucleotidyltransferase II. Incorporation of Cytidine 5′-Monophosphate and Determination of a Short Nucleotide Sequence at the 3′ End of the RNA

Turnip yellow mosaic virus (TYMV) RNA treated with snake venom phosphodiesterase accepts cytidine 5'-monophosphate and adenosine 5'-monophosphate (AMP) when it is incubated in the presence of cytidine 5'-triphosphate (CTP), adenosine 5'-triphosphate, and Escherichia coli transfer RNA nucleotidyltransferase; untreated TYMV RNA accepts only AMP. When alpha (32)PCTP was used for terminal labeling, the nearest neighbor analyses and the anallyses after action of various nucleases showed that the sequence of five nucleotides at the 3' end of TYMV RNA is: pGpCpApCpC. A nuclease present in commerical preparations of snake venom phosphodiesterase leads to the fragmentation of TYMV RNA, the 3' end of which is found in a fragment having a sedimentation constant close to 5s.

TYMV valyl-RNA as an amino-acid donor in protein biosynthesis.

THE observation that turnip yellow mosaic virus (TYMV) RNA (of sedimentation constant 23–25S) appears to have a tRNA-like structure at or near its 3′ end1–3 has been supported by the fact that it is recognized by three different enzymes which specifically catalyse reactions involving tRNAs. The tRNA nucleotidyltransferase catalyses the positioning of a terminal adenosine residue to the 3′-CC end of the viral genome. In the presence of valyl-tRNA synthetase and ATP, valine is then esterified to the 3′-CCA terminus of TYMV RNA. The N-acylaminoacyl-tRNA hydrolase causes the release of acetylvaline from acetylvalyl-RNA of TYMV.