A functional role for the conserved protonatable hairpins in the 5' untranslated region of turnip yellow mosaic virus RNA.

The 5' untranslated region (UTR) of the RNA of several tymoviruses contains conserved hairpins with protonatable internal loops, consisting of C-C and C-A mismatches (K. Hellendoorn, P. J. A. Michiels, R. Buitenhuis, and C. W. A. Pleij, Nucleic Acids Res. 24, 4910-4917, 1996). Here, we present a functional analysis of the 5' UTR of turnip yellow mosaic virus (TYMV) RNA, which contains two protonatable hairpins with nearly identical internal loops. Mutations were introduced in an infectious cDNA clone, and T7 RNA transcripts were used to infect Chinese cabbage plants. Different symptoms were observed for the various mutants, pointing to a functional role of the C-C and C-A mismatches in the hairpins of the 5' UTR. The replication of the virus is influenced by the mutations made, while in vitro translation studies showed that the expression of the two overlapping reading frames of TYMV is not influenced by the secondary structure of the leader. Various mutants were propagated for up to five serial passages of infection, and the sequence of the 5' UTR was determined. This resulted in virus RNA with new non-wild-type sequences that produced the wild-type phenotype in infected plants. Remarkably, in all cases C-C or C-A mismatches were introduced. The internal loop of the 5'-proximal hairpin seems to be more important for the viral life cycle than that of the second hairpin. A deletion of 75% of the leader, including the two hairpins, resulted in a virus that was deficient in viral spread. Since the ratio between filled and empty capsids was changed drastically by this mutation, a role of the 5' UTR in viral packaging is proposed.

Protonatable hairpins are conserved in the 5'-untranslated region of tymovirus RNAs.

The secondary structures of the 5'-untranslated region (5'-UTR) of five different tymoviruses have been determined by structure probing, computer prediction and sequence comparison. Despite large sequence differences, there are remarkable similarities in the secondary structure. In all viruses two or four hairpins are found, most of which contain a symmetrical internal loop consisting of adjacent C-C or C-A mismatches. Since it is known that such mismatches can be protonated and protonated cytosines play an important role in RNA-protein interactions in tymoviral virions, the influence of pH on the conformation of the internal loop was studied. UV melting experiments and 1-dimensional proton NMR at varying pH values and salt concentrations confirm that the hairpins can be protonated under relatively mild conditions. The hairpin found in the 5'-UTR of erysimum latent virus, which has an asymmetrical internal loop consisting of cytosines and uridines, shows comparable behaviour. It is concluded that all tymoviral RNAs contain protonatable hairpins in the 5'-UTR. Binding experiments with empty viral capsids, however, do not yet establish a role in capsid protein binding.

Secondary structure model of the coat protein gene of turnip yellow mosaic virus RNA: long, C-rich, single-stranded regions.

The RNA of all tymoviruses, a group of ssRNA plant viruses, has a base composition that is different from that of most other viruses. The excess of cytosines (35-42%) and the low number of guanosines (15-17%) must impel an RNA structure with a relatively low amount of base pairing and a high incidence of unpaired cytosines. These unpaired cytosines probably function in RNA-protein interactions. To gain Insight into the way the RNA is positioned inside the virion, the secondary structure has been determined of a part of TYMV RNA, including the so-called tymobox, the coat protein gene, and the 3' untranslated region, by structure probing, sequence comparison, and computer predictions. Conservation of secondary structure elements in tymoviruses is not high and does not parallel the conservation of the primary structure. A combination of structure prediction and probing experiments, however, results in a model consisting of structured domains of 100-200 nucleotides interspersed by long unpaired cytosine-rich regions. The latter may interact with the coat protein inside the virion. The structure of some functionally interesting regions of the 3' part of TYMV RNA is also discussed.