Characterisation of the 5'-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro

Uncapped messenger RNAs (mRNAs) encoding calf preprochymosin, chicken prelysozyme, or Escherichia coli beta-glucuronidase (GUS) were synthesized in vitro, with or without a 5'-terminal 67-nucleotide sequence (omega') derived from the untranslated 5'-leader (omega) of tobacco mosaic virus (TMV) RNA. Messenger RNAs were translated in vitro, in messenger-dependent systems derived from rabbit reticulocytes (MDL), wheat-germ (WG) or E. coli (EC). The omega' sequence enhanced expression of each mRNA in almost every translation system. While MDL was the least responsive to omega', this sequence proved particularly efficient in permitting translation of the eukaryotic mRNAs in EC, despite the absence of a consensus Shine-Dalgarno sequence in either the mRNAs or omega'. The local context of the initiation codon (AUG) in two GUS mRNA constructs did not influence the relative enhancement caused by the omega' sequence. These findings extend the utility of omega' as a general enhancer of translation for both prokaryotic and eukaryotic mRNAs in either 80S- or 70S-ribosome-based systems.     

The 5'-leader of tobacco mosaic virus promotes translation through enhanced recruitment of eIF4F

The 5′-leader sequence (called Ω) of tobacco mosaic virus (TMV) functions as a translational enhancer in plants. A poly(CAA) region within Ω is responsible for the translation enhancement and serves as a binding site for the heat shock protein, HSP101, which is required for the translational enhancement. Genetic analysis of the HSP101-mediated enhancement of translation from Ω-containing mRNA suggested that two eukaryotic initiation factors (eIFs), i.e. eIF4G and eIF3, were necessary. In this study, the functional interaction between Ω and other RNA elements known to participate in the recruitment of eIF4G, i.e. the 5′-cap and the poly(A) tail, was examined. Ω exhibited functional overlap with the 5′-cap and the poly(A) tail but not with the native TMV 3′-UTR which contains an independent translational enhancer. Consistent with the role of HSP101 in mediating the translational function of Ω, the enhancement afforded by Ω increased following a heat shock, which elevates expression of HSP101. The use of a fractionated translation lysate revealed that of the two eIF4F proteins present in plants, eIF4F was specifically required for the activity of Ω. The data suggest that Ω is functionally similar to a 5′-cap and a poly(A) tail in that it serves to recruit eIF4F in order to enhance translation from an mRNA.     

Cap-independent translation of tobacco etch virus is conferred by an RNA pseudoknot in the 5'-leader

The tobacco etch virus (TEV) 5'-leader promotes cap-independent translation in a 5'-proximal position and promotes internal initiation when present in the intercistronic region of a dicistronic mRNA, indicating that the leader contains an internal ribosome entry site. The TEV 143-nucleotide 5'-leader folds into a structure that contains two domains, each of which contains an RNA pseudoknot. Mutational analysis of the TEV 5'-leader identified pseudoknot (PK) 1 within the 5'-proximal domain and an upstream single-stranded region flanking PK1 as necessary to promote cap-independent translation. Mutations to either stem or to loops 2 or 3 of PK1 substantially disrupted cap-independent translation. The sequence of loop 3 in PK1 is complementary to a region in 18 S rRNA that is conserved throughout eukaryotes. Mutations within L3 that disrupted its potential base pairing with 18 S rRNA reduced cap-independent translation, whereas mutations that maintained the potential for base pairing with 18 S rRNA had little effect. These results indicated that the TEV 5'-leader functionally substitutes for a 5'-cap and promotes cap-independent translation through a 45-nucleotide pseudoknot-containing domain.     

Complementary oligodeoxynucleotide mediated inhibition of tobacco mosaic virus RNA translation in vitro.

Two different "antisense" oligodeoxynucleotides and their RNA analogues, each complementary to non-overlapping sequences of 51 bases near the 5' end of TMV RNA, inhibit in vitro translation of the genomic RNA in a rabbit reticulocyte lysate. Inhibition is dependent upon complementarity, concentration, and hybridization of the oligomers with TMV RNA. Inhibition is observed at molar ratios of TMV RNA to antisense oligomers as low as 1:1.5. A plateau of inhibition at which 10-25% of the control signal remains is achieved by molar ratios of TMV RNA:antisense DNA or RNA greater than or equal to 1:15. The extent of inhibition is not increased by the simultaneous presence of both complementary fragments. Oligodeoxynucleotides and their RNA analogues identical to the same regions of TMV RNA have no direct effect on translation, however, they can block inhibition by the antisense fragments. Translation of BMV RNA is not affected by any of the oligodeoxynucleotides. Polyacrylamide gel electrophoresis shows translation of TMV p126 is selectively inhibited. We conclude that the observed inhibition of translation is due to direct interference with ribosome function.     

Biosynthesis of tobacco mosaic virus RNA in tobacco protoplasts

Changes in the number of protoplasts, viability, protein and chlorophyll contents and ribonucleases activity were studied in tobacco mesophyll protoplastsin vitro inoculated with tobacco mosaic virus (TMV). The number of protoplasts slowly increased during the cultivation period and the viability decreased from 95 to 67% in the control noninoculated protoplasts, and to 55% in the infected protoplasts. 30 h after inoculation the protein and chlorophyll contents strongly decreased to 25–30% and 17–19%, respectively, in comparison with contents 3 h after inoculation. The chlorophylla/b ratio decreased from 2.11 and 2.02 to 0.79 and 0.60 in healthy and infected protoplasts, respectively. The activities of ribonucleases in protoplasts quickly decreased during experiment but they were higher in infected than in noninfected protoplasts (between 20 to 30 h after inoculation they were 132 to 146% higher than that in healthy controls). These activities corresponded to the multiplication curve of TMV.     

Complete nucleotide sequence of alfalfa mosaic virus RNA 4.

Alfalfa mosaic virus RNA 4, the subgenomic messenger for viral coat protein, was partially digested with RNase T1 or RNase A and the sequence of a number of fragments was deduced by in vitro labeling with polynucleotide kinase and application of RNA sequencing techniques. From overlapping fragments, the complete primary sequence of the 881 nucleotides of RNA 4 was constructed: the coding region of 660 nucleotides (not including the initiation and termination codon) is flanked by a 5' noncoding region of 39 nucleotides and a 3' noncoding region of 182 nucleotides. The RNA sequencing data completely confirm the amino acid sequence of the coat protein as deduced by Van Beynum et al. (Fur.J. Biochem. 72, 63-78, 1977).     

Structure of the RNA in tobacco mosaic virus

A model of the RNA of tobacco mosaic virus has been built using computer model-building techniques. The model has good stereochemistry, and fits the electron density map of the virus obtained by fiber diffraction methods considerably better than did earlier models. The three sugar rings in the asymmetric unit all have the A (3′-endo) conformation, One of the bases is in the syn conformation, a conformation observed only rarely in nucleic acid structures.     

Role of the 5' leader sequence of alfalfa mosaic virus RNA 3 in replication and translation of the viral RNA.

RNA 3 of alfalfa mosaic virus (AIMV) encodes the movement protein P3 and the viral coat protein which is translated from the subgenomic RNA 4. The 5'-leader sequences of RNA 3 of AIMV strains S, A, and Y differ in length from 314 to 392 nucleotides and contain a variable number of internal control regions of type 2 (ICR2 motifs) each located in a 27 nt repeat. Infectious cDNA clones were used to exchange the leader sequences of the three strains. This revealed that the leader sequence controls the specific ratio in which RNAs 3 and 4 are synthesized for each strain. In addition, it specifies strain specific differences in the kinetics of P3 accumulation in plants. Subsequent deletion analysis revealed that a 5'-sequence of 112 nt containing one ICR2 motif was sufficient for a 10 to 20% level of RNA 3 accumulation in protoplasts and a delayed accumulation in plants. An additional leader sequence of maximally 114 nt, containing two ICR2 motifs, was required to permit wildtype levels of RNA 3 accumulation. The effect of deletions in the leader sequence on P3 synthesis in vitro and in vivo was investigated.     

The nucleotide sequence of cowpea mosaic virus B RNA

The complete sequence of the bottom component RNA (B RNA) of cowpea mosaic virus (CPMV) has been determined. Restriction enzyme fragments of double-stranded cDNA were cloned in M13 and the sequence of the inserts was determined by a combination of enzymatic and chemical sequencing techniques. Additional sequence information was obtained by primed synthesis on first strand cDNA. The complete sequence deduced is 5889 nucleotides long excluding the 3' poly(A), and contains an open reading frame sufficient to code for a polypeptide of mol. wt. 207 760. The coding region is flanked by a 5' leader sequence of 206 nucleotides and a 3' non-coding region of 82 residues which does not contain a polyadenylation signal.     

Expression of tobacco mosaic virus RNA in transgenic plants

Summary Tobacco mosaic virus (TMV) is a message-sense, single-stranded RNA virus that infects many Solanaceae plants. A full-length cDNA copy of TMV genomic RNA was constructed and introduced into the genomic DNA of tobacco plants using a disarmed Ti plasmid vector. Transformed plants showed typical symptoms of TMV infection, and their leaves contained infectious TMV particles. This is the first example of the expression of RNA virus genomic RNAs in planta.     

The ribosomal fraction mediates the translational enhancement associated with the 5''-leader of tobacco mosaic virus.

The omega sequence at the 5'-terminus of tobacco mosaic virus (TMV) RNA acts as a translational enhancer. The differential in omega-associated translational enhancement between the in vitro translation system derived from wheat germ (WG) and that from rabbit reticulocytes (MDL) was exploited to identify that lysate component which was responsible for a lysate's characteristic response to omega. Using fractionated MDL and WG lysates, which were reconstituted in various combinations, the high salt-washed ribosomal fraction was determined to be the responsive element in a lysate. Analysis of omega's ability to enhance translation was greatest at low mRNA and high ribosomal concentrations and to occur in the early phase of an in vitro translation assay. Translation of omega-containing CAT mRNA was more sensitive to the presence of micrococcal nuclease than CAT mRNA without an omega. In substitution experiments, WG ribosomes functioned at much reduced efficiency in MDL as did MDL ribosomes in WG lysate. The initiation factor-containing fraction of one system could not, as a whole, functionally replace that of the other and actually acted to inhibit translation in the heterologous system.