Role of tRNA-like structures in controlling plant virus replication

Abstract

Transfer RNA-like structures (TLSs) that are sophisticated functional mimics of tRNAs are found at the 3'-termini of the genomes of a number of plant positive strand RNA viruses. Three natural aminoacylation identities are represented: valine, histidine, and tyrosine. Paralleling this variety in structure, the roles of TLSs vary widely between different viruses. For Turnip yellow mosaic virus, the TLS must be capable of valylation in order to support infectivity, major roles being the provision of translational enhancement and down-regulation of minus strand initiation. In contrast, valylation of the Peanut clump virus TLS is not essential. An intermediate situation seems to exist for Brome mosaic virus, whose RNAs 1 and 2, but not RNA 3, need to be capable of tyrosylation to support infectivity. Other known roles for certain TLSs include: (i) the recruitment of host CCA nucleotidyltransferase as a telomerase to maintain intact 3' CCA termini, (ii) involvement in the encapsidation of viral RNAs, and (iii) presentation of minus strand promoter elements for replicase recognition. In the latter role, the promoter elements reside within the TLS but are not functionally dependent on tRNA mimicry. The phylogenetic distribution of TLSs indicates that their evolutionary history includes frequent horizontal exchange, as has been observed for protein-coding regions of plant positive strand RNA viruses.

Fig. 1. The TYMV TLS is a highly efficient mimic of tRNA^Val

A Sequence and structure of the 82-nt-long TLS of TYMV RNA (Rietveld et al., 1982). The pseudoknot-containing amino acid acceptor stem and the T-, D- and anticodon (A/C) domains are clear structural analogs of the four arms of canonical tRNAs. A CAC valine anticodon in present. The nucleotides that serve as valine identity elements are indicated with adjacent dots (black strongest, light grey weakest). Note that it is conventional to number the nucleotides in TLSs from the 3′-end. B. Diagram of the TYMV genome showing the three coding regions for expressing the movement protein/RNAi suppressor (MP/RNAi); replication protein, with methyltranserase (MTR), proteinase (PRO), helicase (HEL) and RNA-dependent RNA polymerase (RdRp) domains; and coat protein (CP). The asterisk signifies a 5′-cap structure; the arrowhead indicates the site of proteolytic maturation cleavage. The cartoon of the 3′-UTR features the TLS and the 3′-terminus, which lacks the A residue in most virion RNAs; the TLS serves as translational enhancer and the 3′-CC(A) serves as the initiation box controlling minus strand synthesis (see text). C. Summary of in vitro tRNA-like properties of the TYMV TLS, assayed with wheat germ enzymes and eEF1A (Dreher and Goodwin, 1998), compared to a lupine tRNA^Val transcript. The TLS was studied in 83-nt long RNA (TLS), 264-nt long RNA (TY-264) or in the 6.3 kb virion RNA (vRNA). TM buffer has low ionic strength, while IV buffer has higher ionic strength and is similar to the conditions used for in vitro translation. Plateau refers to the extent of aminoacylation (moles valine per mole RNA). D. Contributions to valine identity by anticodon loop nucleotides, studied with wheat germ ValRS (Dreher et al., 1992), and by A4, studied with yeast ValRS (Florentz et al., 1991).

Fig. 2. The histidine-specific TMV TLS

A Sequence and structure of the 104-nt-long TLS of TMV RNA (Rietveld et al., 1984) and the adjacent upstream pseudoknots (UPSK 1–3) (van Belkum et al., 1985). The TMV TLS has obvious acceptor and T stem analogs and an anticodon (A/C) domain with a GUU histidine anticodon; 98-UGGA-95 is thought to serve as a D-loop analog (Felden et al., 1996). Nucleotide A18 in the acceptor stem is thought to be the major histidine identity element (Rudinger et al., 1997). B. Comparison of histidylation efficiency between TMV-U5 RNA and a yeast tRNA^His transcript, determined with yeast HisRS (Felden et al., 1994). C. Diagram of the TMV genome with domains labeled as in Fig. 1. The dot indicates the location of a suppressible stop codon that allows RdRp expression by read-through. The major domains in the 3′-UTR are the TLS and the upstream pseudoknot domain (UPSK). The minus strand promoter overlaps the TLS; the UPSK provides translational enhancement (see text).

Fig. 3. The tyrosine-specific BMV TLS

A, B Sequence of the 133-nt-long TLS of BMV RNA3 and adjacent upstream stem-loops. The structure in (A) was originally proposed by (Rietveld et al., 1983), who suggested that arm C with its AUA tyrosyl anticodon serves as the analog of the anticodon domain of tRNA. The current structural model proposed by (Fechter et al., 2001a) in (B) shows arm B2 as the analog of the anticodon. The major tyrosine identity elements are present at the 3′-end of the acceptor stem helix, and only minor tyrosine identity is contributed by the B2 loop (Fechter et al., 2001a). Stems C and D can be considered as insertions into the basic tRNA-like structure; the terminal AUA loop of stem C serves as the core element of the minus strand promoter, and stem D is absent from some bromoviral TLSs. Stems B3 and E (shaded) are outside the core TLS, but mutations in B3 impair tyrosylation (see text). C. Comparison of in vitro tyrosylation by yeast TyrRS of two forms of BMV RNA with an in vitro-generated yeast tRNA^{Tyr; 1} BMV-161 is a 161 nt-long 3′-fragment (Ω161) produced from RNA4 by limited ribonuclease cleavage (Perret et al., 1989); ² BMV-195 is a 195 nt RNA3 transcript (Fechter et al., 2001a). D. Contributions to tyrosine identity by nucleotides at the end of the acceptor stem helix, and by the B2 loop, studied with yeast TyrRS (Fechter et al., 2001a). E. Diagram of the tripartite BMV genome with domains labeled as in Fig. 1. The major domains in the 3′-UTR are the TLS and the upstream domain (UPD), both of which are strongly conserved among the three genomic RNAs. The core minus strand promoter resides in stem C within the TLS; the 3′-UTR serves as a translational enhancer, but the responsible domain has not been identified (see text).

Fig. 4. Phylogenetic distribution of TLSs

The occurrence of distinct classes of 3′-termini is shown for selected genera of the three lineages of viruses that have been classified to Supergroup 3 on the basis of their RdRp sequences (based on Koonin and Dolja, 1993). The 3′-termini are indicated as: pA, poly(A) tail; TLS with aminoacyl specificity indicated; hemi-TLS, to indicate an acceptor-T arm not capable of aminoacylation; no indication for viruses whose genomes have none of the above types of termini. Branch lengths are approximate.

Fig. 5. Varied functions of TLSs and significance of tRNA mimicry during infection

A Plant viral TLSs have a number of properties and provide varied and different functions in different viral RNAs. B. The role of TLS aminoacylation differs between the three viruses in which its importance for infectivity has been assessed.