Methylation of TMV RNA

Several plant and animal viral RNAs contain a tRNA like structure at their 3′ ends. In this communication we show that tobacco mosaic virus (TMV) RNA is an acceptable substrate for a specific tRNA methyltransferase. Using a crude preparation of $\text{E. coli}$ ribothymidine (rT) forming uracil methylase and (methyl ³H) S-adenosyl-L-methionine (SAM) as a methyl donor, 0.7 moles of methyl group is incorporated per mole of TMV RNA in 10 hours at 30°C. Upon T₂ RNAse digestion of the labeled RNA, all of the radioactivity was found to be in TMP. T₁ RNAse digestion of ³H methylated TMV RNA showed that all of the label was located in a tetranucleotide which co-migrated with authentic TpψpCpGp, an oligonucleotide characteristically found in normal cellular tRNA.The use of this specific methyl transferase reaction may provide a simple assay for the detection of tRNA like structures in large RNAs.     

Replication and packaging of Turnip yellow mosaic virus RNA containing Flock house virus RNA1 sequence

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. In this study, RNA1 sequence of Flock house virus (FHV) was inserted into the TYMV genome to test whether TYMV can accommodate and express another viral entity. In the resulting construct, designated TY-FHV, the FHV RNA1 sequence was expressed as a TYMV subgenomic RNA. Northern analysis of the Nicotiana benthamiana leaves agroinfiltrated with the TY-FHV showed that both genomic and subgenomic FHV RNAs were abundantly produced. This indicates that the FHV RNA1 sequence was correctly expressed and translated to produce a functional FHV replicase. Although these FHV RNAs were not encapsidated, the FHV RNA having a TYMV CP sequence at the 3’-end was efficiently encapsidated. When an eGFP gene was inserted into the B2 ORF of the FHV sequence, a fusion protein of B2-eGFP was produced as expected. [BMB Reports 2014; 47(6): 330-335]     

Crystal structure of an empty capsid of turnip yellow mosaic virus

Empty capsids (artificial top component) of turnip yellow mosaic virus were co-crystallized with an encapsidation initiator RNA hairpin. No clear density was observed for the RNA, but there were clear differences in the conformation of a loop of the coat protein at the opening of the pentameric capsomer (formed by five A-subunits) protruding from the capsid, compared to the corresponding loop in the intact virus. Further differences were found at the N terminus of the A-subunit. These differences have implications for the mechanism of decapsidation of the virus, required for infection.     

A Turnip yellow mosaic virus infection system in Arabidopsis suspension cell culture

Turnip yellow mosaic virus (TYMV) is a positive-strand RNA virus able to infect Arabidopsis thaliana. To establish a TYMV infection system in Arabidopsis cell culture, TYMV replicons with the capsid protein gene replaced by a reporter gene expressing the Sh ble protein conferring zeocin resistance were used to transfect Arabidopsis cells. Zeocin-resistant Arabidopsis calli were used to generate a suspension cell culture. Detection of viral proteins and RNAs after 18 months in culture demonstrated persistent replication of the replicon. The Arabidopsis cell culture yielded soluble, active replication complexes, providing a useful tool to study host factors involved in TYMV replication.     

Detection of periodic patterns in RNA sequences: the first encapsidated region of the TMV RNA

A method is developed to study the periodic properties of nucleotide sequences allowing the favoured pattern of the repeating unit, as well as the length and localization of this periodic segment to be determined simultaneously. The degree of periodicity is evaluated calculating the probabilities for random occurrence of the maximal deviations of the nucleotide composition in each phase, making use of the binomial formula.The nucleotide sequence of the tobacco mosaic virus (TMV) RNA responsible for recognition of the homologous protein (“assembly origin”, AO) (Zimmern & Butler, 1977) was investigated in order to find periodic regions of primary structure which might be essential in the recognition process. As a result the most periodic segments of the AO consisting of 31 and 17 nucleotides corresponding to the schemes GAU or GA1 have been found. However, the periodicities in these regions do not exceed that expected for random sequences. It can be considered as an evidence that in addition to peculiarities of primary structure, some other features such as RNA secondary or tertiary structure are essential in this interaction.For comparison the nucleotide sequences of the other fragments of TMV RNA as well as MS2 RNA, TYMV RNA, 16S rRNA and phage fd DNA were investigated by the same method.     

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.     

Modification of Turnip yellow mosaic virus coat protein and its effect on virion assembly

Turnip yellow mosaic virus (TYMV) is a positive strand RNA virus. We have modified TYMV coat protein (CP) by inserting a c-Myc epitope peptide at the N- or C-terminus of the CP, and have examined its effect on assembly. We introduced the recombinant CP constructs into Nicotiana benthamiana leaves by agroinfiltration. Examination of the leaf extracts by agarose gel electrophoresis and Western blot analysis showed that the CP modified at the N-terminus produced a band co-migrating with wild-type virions. With C-terminal modification, however, the detected bands moved faster than the wild-type virions. To further examine the effect, TYMV constructs producing the modified CPs were prepared. With N-terminal modification, viral RNAs were protected from RNase A. In contrast, the viral RNAs were not protected with C-terminal modification. Overall, the results suggest that virion assembly and RNA packaging occur properly when the N-terminus of CP is modified, but not when the C-terminus is modified. [BMB Reports 2013; 46(10): 495-500]     

Two histidines of the coat protein of turnip yellow mosaic virus at the capsid interior are crucial for viability

RNA-coat protein interactions in turnip yellow mosaic virus (TYMV) have been shown to involve low pK proton-donating groups. Two different types of interaction have been proposed. In the so-called type I interaction, protonated C-residues interact with acidic amino acids at low pH, thereby providing a rationale for the high C-content (38%) of the genomic RNA. The type II interaction involves charged histidines interacting with phosphates of the RNA backbone. Site-directed mutagenesis of the TYMV coat protein and subsequent in vivo analysis were performed to distinguish between these two types of RNA-protein interaction. The results reveal a prominent role for the histidines H68 and H180, since mutation to an alanine residue inhibits symptom development on secondary leaves, indicating that spreading of the virus in the plant is blocked. Viral RNA and coat protein synthesis are not altered, showing that these two histidines may play a role in the process of RNA encapsidation. Overexpression of the TYMV coat protein in Escherichia coli leads to the formation of bona fide capsids, showing that the two histidines are not critical in capsid assembly. Mutagenesis of the acidic amino acids D11, E135, and D143 to alanine apparently did not interfere with virus viability. The functional role of the histidines during the infection cycle is discussed in terms of the structure of the coat protein, both at the level of amino acid sequence conservation among the members of the Tymoviridae family and as the three-dimensional structure of the coat protein.