tRNA-like structure regulates translation of Brome mosaic virus RNA

For various groups of plant viruses, the genomic RNAs end with a tRNA-like structure (TLS) instead of the 3' poly(A) tail of common mRNAs. The actual function of these TLSs has long been enigmatic. Recently, however, it became clear that for turnip yellow mosaic virus, a tymovirus, the valylated TLS(TYMV) of the single genomic RNA functions as a bait for host ribosomes and directs them to the internal initiation site of translation (with N-terminal valine) of the second open reading frame for the polyprotein. This discovery prompted us to investigate whether the much larger TLSs of a different genus of viruses have a comparable function in translation. Brome mosaic virus (BMV), a bromovirus, has a tripartite RNA genome with a subgenomic RNA4 for coat protein expression. All four RNAs carry a highly conserved and bulky 3' TLS(BMV) (about 200 nucleotides) with determinants for tyrosylation. We discovered TLS(BMV)-catalyzed self-tyrosylation of the tyrosyl-tRNA synthetase but could not clearly detect tyrosine incorporation into any virus-encoded protein. We established that BMV proteins do not need TLS(BMV) tyrosylation for their initiation. However, disruption of the TLSs strongly reduced the translation of genomic RNA1, RNA2, and less strongly, RNA3, whereas coat protein expression from RNA4 remained unaffected. This aberrant translation could be partially restored by providing the TLS(BMV) in trans. Intriguingly, a subdomain of the TLS(BMV) could even almost fully restore translation to the original pattern. We discuss here a model with a central and dominant role for the TLS(BMV) during the BMV infection cycle.     

Functional analysis of the tobacco mosaic virus tRNA-like structure in cytoplasmic gene regulation.

The 3'-untranslated region (UTR) of tobacco mosaic virus (TMV), which terminates in a tRNA-like structure, functionally substitutes for a poly(A) tail in both plant and animal cells. The addition of the TMV 3'-UTR to chimeric mRNA constructs increases their expression up to 100-fold, increasing both translational efficiency and mRNA stability. The domain largely responsible for the regulation maps to a 72 base region immediately upstream of the tRNA-like structure, however, the 3'-terminal, tRNA-like structure is required for full function. Its contribution is lost if separated from the upstream pseudoknot domain by as few as 5 bases or if 6 bases are removed from the 3'-terminus. Sequence addition to the 3'-terminus of the TMV 3'UTR or the upstream pseudoknot domain inhibits function in both tobacco and Chinese hamster ovary cells.     

tRNA-like properties of tobacco rattle virus RNA.

The 3' terminal forty nucleotides of tobraviral RNAs readily fold into a tertiary structure, resembling that of tymo- and tobamoviral RNAs. The latter RNAs possess a tRNA-like structure at their 3' end that is recognized by a number of tRNA-specific enzymes (Rietveld et al. (1984), EMBO J. 3, 2613-2619). Characteristic for their aminoacyl acceptor arm is the presence of a so-called pseudoknot which we now also find in a corresponding position at the 3' terminus of TRV RNA2 (PSG strain). The nucleotide sequences of all tobraviral RNAs analysed so far indicate that they all possess a similar 3' terminal structure. A domain resembling the anticodon arm of canonical tRNA is not readily recognizable. TRV RNA2 can be adenylated with CTP, ATP; tRNA nucleotidyl transferase and ATP. It is unable, however, to accept any of the twenty common amino acids when incubated with ATP and aminoacyl-tRNA synthetases from wheat germ or yeast. We conclude that TRV RNA contains a tRNA-like structure, which, in contrast to the tymo- and tobamoviral tRNA-like structures, cannot be aminoacylated. It is unlikely therefore, that aminoacylation of plant viral RNAs with a tRNA-like structure is a prerequisite for viral RNA replication.     

A new principle of RNA folding based on pseudoknotting.

Tertiary interactions involving hairpin or interior loops of RNA can lead to extended quasi-continuous double helical stem regions, consisting of coaxially stacked segments of duplex RNA, bridged by single-stranded connections. This type of compact folding plays a role in various strategic regions of RNA molecules. Their role in ribosome functioning, RNA splicing and recognition of tRNA-like structures is discussed.     

Mutational analysis of the pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. Aminoacylation efficiency and RNA pseudoknot stability.

Site-directed mutations were introduced in the connecting loops and one of the two stem regions of the RNA pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. The kinetic parameters of valylation for each mutated RNA were determined in a cell-free extract from wheat germ. Structure mapping was performed on most mutants with enzymic probes, like RNase T1, nuclease S1 and cobra venom ribonuclease. An insertion of four A residues in the four-membered connecting loop L1 that crosses the deep groove of the pseudoknot reduces aminoacylation efficiency. Deletions up to three nucleotides do not affect aminoacylation or RNA pseudoknot formation. Deletion of the entire loop abolishes aminoacylation. Although elimination of the pseudoknot is presumed, this could not be demonstrated. Unlike the mutations in loop L1, all mutations in the three-membered connecting loop L2 that crosses the shallow groove of the RNA pseudoknot decrease the aminoacylation efficiency considerably. Nonetheless, the RNA pseudoknot is still present in most mutated RNAs. These results indicate that a number of mutations can be introduced in both loops without abolishing aminoacylation. Results obtained with the introduction of mismatches and A.U base-pairs in stem S1 of the pseudoknot, containing three G.C base-pairs in wild-type RNA, indicate that the pseudoknot is only marginally stable. Our estimation of the gain of free energy due to the pseudoknot formation is at most 2.0 kcal/mol. The pseudoknot structure can, however, be stabilized upon binding the valyl-tRNA synthetase.     

The tRNA-like structure at the 3' terminus of turnip yellow mosaic virus RNA. Differences and similarities with canonical tRNA.

The 3' terminus of TYMV RNA, which possesses tRNA-like properties, has been studied. A 3' terminal fragment of 112 nucleotides was obtained by cleavage with RNase H after hybridization of a synthetic oligodeoxynucleotide to the viral RNA. The accessibility of cytidine and adenosine residues was probed with chemical modification. Enzymatic digestion studies were performed with RNase T1, nuclease S1 and the double-strand specific RNase from the venom of the cobra Naja naja oxiana. A model is proposed for the secondary structure of the 3' terminal region of TYMV RNA comprising 86 nucleotides. The main feature of this secondary structure is the absence of a conventional acceptor stem as present in canonical tRNA. However, the terminal 42 nucleotides can be folded in a tertiary structure which bears strong resemblance with the acceptor arm of canonical tRNA. Comparison of this region of TYMV RNA with that of other RNAs from both the tymovirus group and the tobamovirus group gives support to our proposal for such a three-dimensional arrangement. The consequences for the recognition by TYMV RNA of tRNA-specific enzymes is discussed.     

Electroporation-mediated infection of tobacco leaf protoplasts with tobacco mosaic virus RNA and cucumber mosaic virus RNA

Conditions were established for the introduction of both tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) RNAs into tobacco mesophyll protoplasts by electroporation. The proportion of infected protoplasts was quantified by staining with viral coat protein-specific antibodies conjugated to fluorescein isothiocyanate. Approximately 30–40% of the protoplasts survived electroporation. Under optimal conditions, up to 75% of these were infected with TMV-RNA. Successful infection was demonstrated in 19 out of 20 experiments. Optimal infection was achieved with several direct current pulses of 90 sec at a field strength of 5 to 10 kV/cm. Changing the position of the protoplasts within the chamber between electric pulses was essential for achievement of high rates of infection. Optimal viral RNA concentration was about 10 g/ml in a solution of 0.5 M mannitol without buffer salts.     

Replication of tobacco mosaic virus RNA

The replication of tobacco mosaic virus (TMV) RNA involves synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template, followed by the synthesis of positive-strand RNA on the negative-strand RNA templates. Intermediates of replication isolated from infected cells include completely double-stranded RNA (replicative form) and partly double-stranded and partly single-stranded RNA (replicative intermediate), but it is not known whether these structures are double-stranded or largely single-stranded in vivo. The synthesis of negative strands ceases before that of positive strands, and positive and negative strands may be synthesized by two different polymerases. The genomic-length negative strand also serves as a template for the synthesis of subgenomic mRNAs for the virus movement and coat proteins. Both the virus-encoded 126-kDa protein, which has amino-acid sequence motifs typical of methyltransferases and helicases, and the 183-kDa protein, which has additional motifs characteristic of RNA-dependent RNA polymerases, are required for efficient TMV RNA replication. Purified TMV RNA polymerase also contains a host protein serologically related to the RNA-binding subunit of the yeast translational initiation factor, eIF3. Study of Arabidopsis mutants defective in RNA replication indicates that at least two host proteins are needed for TMV RNA replication. The tomato resistance gene Tm-1 may also encode a mutant form of a host protein component of the TMV replicase. TMV replicase complexes are located on the endoplasmic reticulum in close association with the cytoskeleton in cytoplasmic bodies called viroplasms, which mature to produce 'X bodies'. Viroplasms are sites of both RNA replication and protein synthesis, and may provide compartments in which the various stages of the virus mutiplication cycle (protein synthesis, RNA replication, virus movement, encapsidation) are localized and coordinated. Membranes may also be important for the configuration of the replicase with respect to initiation of RNA synthesis, and synthesis and release of progeny single-stranded RNA.     

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.     

Satellite tobacco mosaic virus RNA: structure and implications for assembly

The initial appearance of 45% of the single-stranded RNA of satellite tobacco mosaic virus in electron density maps suggested the entire RNA conformation could be delineated. Subsequent work has localized nearly 80% of the RNA as stem-loop elements. Connection of the stem-loops in the most efficient manner produces a persuasive model for the encapsidated RNA. This arrangement has significant implications for virus assembly and for the essential role of RNA.     

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.     

Nucleotide sequence of the 3''-terminal tRNA-like structure in barley stripe mosaic virus genome.

This paper describes the sequence of 257 nucleotides from the 3' end of RNA 2 of barley stripe mosaic virus ( BSMV , strain Argentina Mild) including an internal oligo (A) tract localized at a distance of 236 nucleotides from the 3' end, and the 3' terminal tRNA-like structure accepting tyrosine. This sequence is shown to be the same with RNAs 1,2 and 3 of another BSMV strain, Norwich , for at least the first 106 nucleotides from the 3' end. The 3' extremity of BSMV RNA bears some resemblance to tRNATyr from yeast in its primary structure. The possible secondary structures of the tRNA-like sequence in BSMV genome are discussed.     

Minus-strand initiation by brome mosaic virus replicase within the 3' tRNA-like structure of native and modified RNA templates

An RNA-dependent RNA polymerase (replicase) extract from brome mosaic virus-infected barley leaves has been shown to initiate synthesis of (-) sense RNA from (+) sense virion RNA. Initiation occurred de novo, as demonstrated by the incorporation of [gamma-32P]GTP into the product. Sequencing using cordycepin triphosphate to terminate (-) strands during their synthesis by the replicase generated sequence ladders that confirmed that copying was accurate, and that initiation occurred very close to the 3' end. The precise site of initiation was further defined by testing the replicase template activity after stepwise removal of 3'-terminal nucleotides. Whereas removal of the terminal A did not decrease template activity, removal of the next nucleotide (C-2) did. Thus, initiation almost certainly occurs opposite the penultimate 3'-nucleotide (C-2) in vitro. The structure of the double-stranded replicative form of RNA isolated from brome mosaic virus-infected leaves was consistent with such a mechanism occurring in vivo, in that it lacked the 3'-terminal A found on virion RNAs. The specific site of (-) strand initiation and normal template activity were retained for RNAs with as many as 15 to 30 A residues added to the 3' end. However, only limited oligonucleotide 3' extensions can be present on active templates. In order to assess the 5' extent of sequences required for an active template, a 134-nucleotide-long fragment of brome mosaic virus RNA, corresponding to the tRNA-like structure, was generated. This RNA had high template activity, but a shorter 3' (85-nucleotide) fragment was inactive. RNAs with various heterologous sequences 5' to position 134 also showed high template activity. Thus, the 3'-terminal tRNA-like structure common to all four brome mosaic virus virion RNAs contains all of the signals required for initiation of replication, and sequences 5' to it do not play a role in template selection.     

The region of tobacco mosaic virus RNA involved in the nucleation of assembly.

The interaction of TMV RNA with the disk aggregate of TMV protein at the initiation of assembly has been studied by using the techniques of RNA sequencing. The 5' end group has been identified, and shown not to be protected in the early stages of assembly from accessibility to nuclease digestion. A population of RNA fragments of average length 250 nucleotides, originating from a unique region of TMV RNA, is encapsidated by limited assembly, and sufficient sequence information is available to identify certain unusual features. The protected region does not contain highly reiterated simple repeating sequences, but may contain more complicated repeats. The length and complexity of the nucleation region may reflect adaptation to the efficient mediation of the conformational change from disk to helix of TMV protein, besides a requirement for binding to the disk, and this may be an important part of the mechanism of specificity in the nucleation of assembly.     

Secondary structure of tobacco mosaic virus protein

A set of rules is proposed for the prediction of α-helices in proteins. These rules lead to the correct assignment to either helical or non-helical regions of over 80% of the amino acid residues in the proteins from which they are derived. Applied to tobacco mosaic virus protein these rules lead to the prediction of five α-helical regions which may be consistent with other data.