The 5 UTR of turnip yellow mosaic virus RNA contains two conserved hairpins with internal loops consisting of C⅐C and C⅐A mismatches. In this article, evidence is presented indicating that the 5 proximal hairpin functions as an encapsidation initiation signal. Extensive mutagenesis studies on this hairpin and sequencing of virus progeny showed a clear preference for C⅐C and C⅐A mismatches within the internal loop. The importance of these mismatches lies in their pH-dependent protonation and stable base pair formation. Encapsidation efficiency was found to be severely affected for several mutants lacking the protonatable mismatches in the internal loop of the 5 proximal hairpin. Furthermore, gel mobility-shift assays were performed with various RNA hairpins and empty capsids with a hole. Protonatable hairpins containing C⅐C and͞or C⅐A pairs were found to bind specifically to the interior of the protein shell under acidic conditions (pH 4.5) in the presence of spermidine. Based on these results we propose that this binding of protonated cytosines to the coat protein of turnip yellow mosaic virus may represent a new motif in RNA-protein interactions.T urnip yellow mosaic virus (TYMV) is a nonenveloped plant virus and the type member of the genus Tymovirus. The virion is an icosahedral particle with T ϭ 3 symmetry, and the protein shell consists of 180 identical subunits (1). The genomic RNA is 6,318 nt long and has an unusually high cytosine content (38%) and a rather low guanine content (18%). The genome carries three ORFs coding for the overlapping protein, which is thought to be involved in cell-to-cell transport, the RNAdependent RNA polymerase polyprotein, and the coat protein, which is expressed from a subgenomic messenger RNA. The 3Ј UTR has been shown to contain a tRNA-like structure whose function is still unclear (2).The 5Ј UTR of TYMV RNA is 90 nt long and has been shown, by means of structure probing and sequence comparison, to harbor two simple hairpins containing symmetric internal loops, which consist of C⅐C and C⅐A mismatches (ref. 3; see Fig. 1A). Under slightly acidic conditions these mismatches can form rather stable base pairs. NMR and UV melting experiments have confirmed that protonation of the C⅐C and C⅐A mismatches in hairpin 2 (HP2) stabilizes the structure when the pH is lowered from 7 to 5 (refs. 3 and 4; see Fig. 1B). A preliminary study of the possible function of the protonatable cytosines in the two 5Ј UTR hairpins suggested a dependence of TYMV on these nucleotides to establish a successful infection (5). A role for these hairpins in encapsidation was proposed. However, conclusions about the apparent requirement of C⅐C and͞or C⅐A mismatches were preliminary, as this study described one single mutant for which only a few revertants were recovered after passaging in Chinese cabbage. Moreover, this construct could not solve the question of whether an A⅐C pair instead of C⅐A was also allowed, because the chosen mutation rather forced the recovery of C⅐C and C⅐A mismatches through single ...
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.
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.
The 5 untranslated region (UTR) of the RNA of several tymoviruses contains conserved hairpins with protonatable internal loops, consisting of CC 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 CC 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 CC 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.
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