Second-site suppressor mutations assist in studying the function of the 3'' noncoding region of turnip yellow mosaic virus RNA.

The 3' noncoding region of turnip yellow mosaic virus RNA includes an 82-nucleotide-long tRNA-like structure domain and a short upstream region that includes a potential pseudoknot overlapping the coat protein termination codon. Genomic RNAs with point mutations in the 3' noncoding region that result in poor replication in protoplasts and no systemic symptoms in planta were inoculated onto Chinese cabbage plants in an effort to obtain second-site suppressor mutations. Putative second-site suppressor mutations were identified by RNase protection and sequencing and were then introduced into genomic cDNA clones to permit their characterization. A C-57----U mutation in the tRNA-like structure was a strong suppressor of the C-55----A mutation which prevented both systemic infection and in vitro valylation of the viral RNA. Both of these phenotypes were rescued in the double mutant. An A-107----C mutation was a strong second-site suppressor of the U-96----G mutation, permitting the double mutant to establish systemic infection. The C-107 and G-96 mutations are located on opposite strands of one helix of a potential pseudoknot, and the results support a functional role for the pseudoknot structure. A mutation near the 5' end of the genome (G + 92----A), at position -3 relative to the initiation codon of the essential open reading frame 206, was found to be a general potentiator of viral replication, probably as a result of enhanced expression of open reading frame 206. The A + 92 mutation enhanced the replication of mutant TYMC-G96 in protoplasts but was not a sufficiently potent suppressor to permit systemic spread of the A + 92/G-96 double mutant in plants.     

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.     

The turnip yellow mosaic virus tRNA-like structure cannot be replaced by generic tRNA-like elements or by heterologous 3' untranslated regions known to enhance mRNA expression and stability.

The tRNA-like structure (TLS) at the 3' end of the turnip yellow mosaic virus genome was replaced with heterologous tRNA-like elements, and with a poly(A) tail, in order to assess its role. Replacement with the valylatable TLSs from two closely related tymoviruses resulted in infectious viruses. In contrast, no systemic symptoms on plants, and only low viral accumulations in protoplasts, were observed for three chimeric genomes with 3' sequences known to enhance mRNA stability and translatability. One of these chimeras had a poly(A) tail, and the others had the TLS with associated upstream pseudoknot tracts from the 3' ends of brome mosaic and tobacco mosaic viruses. The latter two chimeric RNAs were shown to be appropriately folded by demonstrating their aminoacylation in vitro with tyrosine and histidine, respectively. The results show that enhancement of genome stability or gene expression is not the major role of the turnip yellow mosaic virus TLS. The major role is likely to be replicational, dependent on features present in tymoviral TLSs but not in generic tRNA-like structures.     

Contributions of the brome mosaic virus RNA-3 3'-nontranslated region to replication and translation.

Sequences upstream of the 3'-terminal tRNA-like structure of brome mosaic virus RNAs have been predicted to fold into several stem-loop and pseudoknot structures. To elucidate the functional role of this upstream region, a series of deletions was made in cDNA clones of RNA-3, a genomic component not required for replication. These deletion mutants were transcribed in vitro and cotransfected with RNA-1 and RNA-2 into barley protoplasts. Deletion of single stem-loop structures gave progeny retaining near-wild-type accumulation levels. Constructions representing deletion of two or three stem-loops substantially lowered the accumulation of progeny RNA-3 relative to wild-type levels. RNA-3 mutants bearing deletions of longer sequences or of the entire region (delta PsKs RNA-3) replicated poorly, yielding no detectable RNA-3 or RNA-4 progeny. Levels of RNA-1 and RNA-2, in the presence of a mutant RNA-3, were found to increase relative to the accumulation observed in a complete wild-type transfection. The stability of delta PsKs RNA-3 in protoplasts was somewhat lower than that of wild-type RNA during the first 3 h postinoculation. Little difference in translatability in vitro of wild-type and RNA-3 constructs bearing deletions within the stem-loop region was observed, and Western immunoblot analysis of viral coat protein produced in transfected protoplasts showed that protein accumulation paralleled the amount of RNA-4 message produced from the various sequences evaluated. These results indicate that the RNA-3 pseudoknot region plays a minor role in translational control but contributes substantially to the overall replication of the brome mosaic virus genome.     

Eukaryotic elongation factor 1A interacts with the upstream pseudoknot domain in the 3' untranslated region of tobacco mosaic virus RNA

The genomic RNA of tobacco mosaic virus (TMV), like that of other positive-strand RNA viruses, acts as a template for both translation and replication. The highly structured 3' untranslated region (UTR) of TMV RNAs plays an important role in both processes; it is not polyadenylated but ends with a tRNA-like structure (TLS) preceded by a conserved upstream pseudoknot domain (UPD). The TLS of tobamoviral RNAs can be specifically aminoacylated and, in this state, can interact with eukaryotic elongation factor 1A (eEF1A)/GTP with high affinity. Using a UV cross-linking assay, we detected another specific binding site for eEF1A/GTP, within the UPDs of TMV and crucifer-infecting tobamovirus (crTMV), that does not require aminoacylation. A mutational analysis revealed that UPD pseudoknot conformation and some conserved primary sequence elements are required for this interaction. Its possible role in the regulation of tobamovirus gene expression and replication is discussed.     

Mutational analysis of the sequence and structural requirements in brome mosaic virus RNA for minus strand promoter activity

An RNA-dependent RNA polymerase (replicase) activity that specifically copies brome mosaic virus (BMV) RNAs in vitro can be prepared from BMV-infected barley leaves. The signals directing complementary (minus) strand synthesis reside within the 3' 134-nucleotide-long tRNA-like structure that is common to each of the virion RNAs. By studying the influence of minus strand synthesis of numerous mutations introduced throughout this region of the RNA, we have mapped in detail the sequence and structural elements necessary for minus strand promoter activity. Sequence alterations (either substitutions or small, structurally discrete deletions) in most parts of the tRNA-like structure resulted in decreased minus strand synthesis. This suggests that BMV replicase is a large enzyme, possibly composed of several subunits. The lowest activities, 5 to 8% of wild type, were observed for mutants with substitutions at three separate loci, identifying one structural and two sequence-specific elements essential for optimal promoter activity. (1) Destabilization of the pseudoknot structure in the aminoacyl acceptor stem resulted in low promoter activity, demonstrating the importance of a tRNA-like conformation. (2) Substitution of the C residue adjacent to the 3' terminus resulted in low promoter activity, probably by interfering with strand initiation. (3) The low activities resulting from substitutions and a small deletion in arm C suggest this region of the RNA to be a major feature involved in replicase binding. In particular, nucleotides within the loop of arm C appear to be involved in a sequence-specific interaction with the replicase.     

Characterization of the RNA components of a putative molecular switch in the 3' untranslated region of the murine coronavirus genome

RNA virus genomes contain cis-acting sequence and structural elements that participate in viral replication. We previously identified a bulged stem-loop secondary structure at the upstream end of the 3' untranslated region (3' UTR) of the genome of the coronavirus mouse hepatitis virus (MHV). This element, beginning immediately downstream of the nucleocapsid gene stop codon, was shown to be essential for virus replication. Other investigators discovered an adjacent downstream pseudoknot in the 3' UTR of the closely related bovine coronavirus (BCoV). This pseudoknot was also shown to be essential for replication, and it has a conserved counterpart in every group 1 and group 2 coronavirus. In MHV and BCoV, the bulged stem-loop and pseudoknot are, in part, mutually exclusive, because of the overlap of the last segment of the stem-loop and stem 1 of the pseudoknot. This led us to hypothesize that they form a molecular switch, possibly regulating a transition occurring during viral RNA synthesis. We have now performed an extensive genetic analysis of the two components of this proposed switch. Our results define essential and nonessential components of these structures and establish the limits to which essential parts of each element can be destabilized prior to loss of function. Most notably, we have confirmed the interrelationship of the two putative switch elements. Additionally, we have identified a pseudoknot loop insertion mutation that appears to point to a genetic interaction between the pseudoknot and a distant region of the genome.     

A conformational switch at the 3' end of a plant virus RNA regulates viral replication.

3' untranslated regions of alfamo- and ilar-virus RNAs fold into a series of stem-loop structures to which the coat protein binds with high affinity. This binding plays a role in initiation of infection ('genome activation') and has been thought to substitute for a tRNA-like structure that is found at the 3' termini of related plant viruses. We propose the existence of an alternative conformation of the 3' ends of alfamo- and ilar-virus RNAs, including a pseudoknot. Based on (i) phylogenetic comparisons, (ii) in vivo and in vitro functional analyses of mutants in which the pseudoknot has been disrupted or restored by compensatory mutations, (iii) competition experiments between coat protein and viral replicase, and (iv) investigation of the effect of magnesium, we demonstrate that this pseudoknot is required for replication of alfalfa mosaic virus. This conformation resembles the tRNA-like structure of the related bromo- and cucumo-viruses. A low but specific interaction with yeast CCA-adding enzyme was found. The existence of two mutually exclusive conformations for the 3' termini of alfamo- and ilar-virus RNAs could enable the virus to switch from translation to replication and vice versa. The role of coat protein in this modulation and in genome activation is discussed.     

Characterization of an essential RNA secondary structure in the 3' untranslated region of the murine coronavirus genome

We have previously identified a functionally essential bulged stem-loop in the 3' untranslated region of the positive-stranded RNA genome of mouse hepatitis virus. This 68-nucleotide structure is composed of six stem segments interrupted by five bulges, and its structure, but not its primary sequence, is entirely conserved in the related bovine coronavirus. The functional importance of individual stem segments of this stem-loop was characterized by genetic analysis using targeted RNA recombination. We also examined the effects of stem segment mutations on the replication of mouse hepatitis virus defective interfering RNAs. These studies were complemented by enzymatic and chemical probing of the stem-loop. Taken together, our results confirmed most of the previously proposed structure, but they revealed that the terminal loop and an internal loop are larger than originally thought. Three of the stem segments were found to be essential for viral replication. Further, our results suggest that the stem segment at the base of the stem-loop is an alternative base-pairing structure for part of a downstream, and partially overlapping, RNA pseudoknot that has recently been shown to be necessary for bovine coronavirus replication.     

Temperature dependent chemical and enzymatic probing of the tRNA-like structure of TYMV RNA

In this paper we report on the thermal unfolding of the tRNA-like structure present at the 3' end of turnip yellow mosaic virus (TYMV) RNA. Diethyl pyrocarbonate (DEP), sodium bisulphite, nuclease S1 and ribonuclease T1 were used as structure probes at a broad range of temperatures. In this way most of the nucleotides present in the tRNA-like moiety were analysed. The melting behaviour of both secondary and tertiary interactions could be followed on the basis of the temperature dependent accessibility of the individual nucleotides or bases towards the various probes. The three-dimensional model of the tRNA-like domain (Dumas et al., J. Biomol. Struct. and Dyn. 4, 707 (1987] was supported by the results to a large extent. The interactions occurring between the T- and D-loop appear to be more complex than proposed in the latter model. Additional evidence for the presence of the RNA pseudoknot (Rietveld et al., Nucleic Acids Res. 10, 1929 (1982] was derived from the fact that the three coaxially stacked helical segments in the aminoacylacceptor arm displayed different melting transitions under certain experimental conditions. Aspects of melting behaviour and thermal stability of double helical regions within the tRNA-like structure are discussed, as well as the applicability of nucleases and modifying reagents at various temperatures in the analysis of RNA structure.     

Multi-domain Packing in the Aminoacylatable 3′ End of a Plant Viral RNA

Turnip yellow mosaic virus (TYMV) contains a tRNA-like structure (TLS) in its 3′ untranslated region (3′ UTR).  This highly structured element induces valylation of the viral RNA by host cell enzymes and is important for virus proliferation. Directly upstream of the TYMV TLS is an upstream pseudoknot domain (UPD) that has been considered to be structurally distinct from the TLS.  However, using a combination of functional, biochemical, and biophysical assays, we show that the entire 3′ UTR of the viral genome is a single structured element in the absence of cellular protein.  This packing architecture stabilizes the RNA structure and creates a better substrate for aminoacylation, and thus the UPD and TLS are functionally and structurally coupled.  It has been proposed that the TYMV TLS acts as a molecular switch between translation and replication. Our results suggest that this putative switch could be based on structural changes within the global architecture of the UTR induced by interactions with the ribosome. The TYMV TLS·UPD might demonstrate how RNA structural plasticity can play a role in regulation of biological processes.     

3-D graphics modelling of the tRNA-like 3'-end of turnip yellow mosaic virus RNA: structural and functional implications

The tRNA-like structure of the aminoacylatable 3'-end of turnip yellow mosaic virus (TYMV) RNA was submitted to 3-D graphics modelling. A model of this structure has been inferred previously from both biochemical results and sequence comparisons which presents a new RNA folding feature, the "pseudoknot". It has been verified that this structure can be constructed without compromising accepted RNA stereochemical rules, namely base stacking and preferential 3'-endo sugar pucker. The model has aided interpretation of previous structural mapping experiments using chemical and enzymatic probes, and new accessibilities of residues could be predicted and tested. Pseudoknots have been considered as potential splice sites because they form antiparallel helical segments in a single RNA molecule. We have examined this possibility with the constructed 3-D model and could verify the hypothesis on a structural basis. The model presents a striking similarity with canonical tRNA and allows a valuable comparison between the protection patterns of yeast tRNA(Val) and tRNA-like viral RNA by cognate yeast valyl-tRNA synthetase against structural probes.     

Recognition of the core RNA promoter for minus-strand RNA synthesis by the replicases of Brome mosaic virus and Cucumber mosaic virus

Replication of viral RNA genomes requires the specific interaction between the replicase and the RNA template. Members of the Bromovirus and Cucumovirus genera have a tRNA-like structure at the 3' end of their genomic RNAs that interacts with the replicase and is required for minus-strand synthesis. In Brome mosaic virus (BMV), a stem-loop structure named C (SLC) is present within the tRNA-like region and is required for replicase binding and initiation of RNA synthesis in vitro. We have prepared an enriched replicase fraction from tobacco plants infected with the Fny isolate of Cucumber mosaic virus (Fny-CMV) that will direct synthesis from exogenously added templates. Using this replicase, we demonstrate that the SLC-like structure in Fny-CMV plays a role similar to that of BMV SLC in interacting with the CMV replicase. While the majority of CMV isolates have SLC-like elements similar to that of Fny-CMV, a second group displays sequence or structural features that are distinct but nonetheless recognized by Fny-CMV replicase for RNA synthesis. Both motifs have a 5'CA3' dinucleotide that is invariant in the CMV isolates examined, and mutational analysis indicates that these are critical for interaction with the replicase. In the context of the entire tRNA-like element, both CMV SLC-like motifs are recognized by the BMV replicase. However, neither motif can direct synthesis by the BMV replicase in the absence of other tRNA-like elements, indicating that other features of the CMV tRNA can induce promoter recognition by a heterologous replicase.     

3-D Graphics Modelling of the tRNA-Like 3′-End of Turnip Yellow Mosaic Virus RNA: Structural and Functional Implications

Abstract

The tRNA-like structure of the aminoacylatable 3′-end of turnip yellow mosaic virus (TYMV) RNA was submitted to 3-D graphics modelling. A. model of this structure has been inferred previously from both biochemical results and sequence comparisons which presents a new RNA folding feature, the “pseudoknot”. It has been verified that this structure can be constructed without compromising accepted RNA stereochemical rules, namely base stacking and preferential 3′-endo sugar pucker. The model has aided interpretation of previous structural mapping experiments using chemical and enzymatic probes, and new accessibilities of residues could be predicted and tested.

Pseudoknots have been considered as potential splice sites because they form antiparallel helical segments in a single RNA molecule. We have examined this possibility with the constructed 3-D model and could verify the hypothesis on a structural basis.

The model presents a striking similarity with canonical tRNA and allows a valuable comparison between the protection patterns of yeast tRNA^Val and tRNA-like viral RNA by cognate yeast valyl-tRNA synthetase against structural probes.