tRNA-Like Structure Regulates Translation of Brome Mosaic Virus RNA

Thirty years ago, the exciting discovery was made that the RNAs of certain plant-infecting viruses can be aminoacylated. For the genera Tymovirus, Bromovirus, Tobamovirus, and Furovirus of the family Bromoviridae, the 3′ ends of the viral RNAs can be charged with either valine, tyrosine, or histidine in a way that is comparable to the aminoacylation of the corresponding canonical tRNAs (for reviews, see references 8, 10, and 22). On the basis of chemical and enzymatic probing experiments and functional assays, these 3′ untranslated regions (UTRs) can all be folded into structures more or less resembling those of canonical tRNAs. The full functional meaning of these tRNA-like structures (TLSs) has remained enigmatic. For the tymovirus turnip yellow mosaic virus (TYMV), it was recently discovered, however, that valylated TLSTYMV entraps ribosomes and directs them to the second open reading frame (ORF) of the single genomic RNA for synthesis of the replicase domain-containing polyprotein (3). Removal of the TLSTYMV from the native TYMV RNA completely abolished polyprotein synthesis, whereas translation of the first ORF into movement protein and that of the subgenomic RNA (sgRNA) into coat protein were unchanged. The 3′-linked valine was found to become incorporated at the N terminus of the polyprotein in a cap-independent and initiator-tRNA-independent fashion. Polyprotein synthesis could also start, however, by the action of a nonaminoacylated or 3′-truncated TLSTYMV. This discovery prompted us to investigate whether the much larger TLSs of a different genus of viruses have a comparable function in translation, and in the present study we focus on the TLSBMV of Brome mosaic virus (BMV), the type species of the genus Bromovirus. The TLSTYMV-mediated initiation of TYMV polyprotein synthesis is exceptional, since eukaryotic translation normally starts with initiation-factor-dependent recognition of the 5′ m7G(5′)pppN cap and recruitment of the 40S ribosomal subunit. Then, by numerous events that are catalyzed and regulated by at least 12 initiation factors, the 40S ribosomal subunit with initiator tRNA is prepared for scanning towards the first AUG as a start codon. After 60S subunit association, the ribosome can enter the elongation phase of the translation cycle. Efficient translation depends on 5′- to 3′-end communication of the mRNA, established by a 3′ poly(A) stretch that communicates via the poly(A) binding protein (PABP) and eIF4G with eIF4E bound to the capped 5′ end. This circular form is believed to assess the mRNA integrity and to recycle ribosomes for multiple rounds of translation. Indeed, the 5′ cap binding factors and PABP synergistically stimulate translation (33). As intracellular parasites, viruses need the host translation apparatus for their reproduction, and their RNAs compete with the host mRNAs for ribosomes to start translation. Remarkably, viral RNAs do not always contain the usual 5′ cap and 3′ poly(A) entities, although they are efficiently translated. For TYMV RNA, this circularization appears to be established by 3′-TLSTYMV communication with the start of ORF2 (3). For BMV, with a different genomic organization, the 3′ TLSBMV may also contribute to communication with the 5′ end in a somehow related way. BMV has a tripartite genome (Fig. ​(Fig.1A):1A): RNA1 codes for a protein with domains homologous to methyltransferases and helicases (Me/He), RNA2 codes for an RNA-dependent RNA polymerase (RdRp), and RNA3 codes for the viral movement protein (MP) and the coat protein (CP). Because of the downstream location of the CP cistron, the latter is exclusively expressed from an sgRNA synthesized from RNA3. All of these RNAs carry a 5′ cap and a highly conserved 3′ TLSBMV, a bulky structure of about 200 nucleotides (Fig. ​(Fig.1B).1B). Though not strongly mimicking a canonical tRNATyr, the TLS is a substrate for tyrosylation both in vivo and in vitro (9, 11, 16, 21), and it functions as a promoter for minus-strand synthesis (25, 31). It also functions as a nucleation site for coat protein assembly and encapsidation of the BMV RNAs into virions (7). FIG. 1. Genomic organization of BMV. (A) Schematic view of genomic RNAs 1 to 3 with ORFs for the following proteins (molecular masses are shown in parentheses in the figure): methyltransferase/helicase (Me/He), RdRp, MP, and CP, which is only translated from ... It is not self-evident to see an analogy between the TLSBMV and the TLSTYMV with respect to the latter's functioning as an initiator of translation of a second and overlapping ORF, since the genomic organization of BMV is quite different, without overlapping ORFs. Our experimental strategy was as follows. We first studied the effect of TLSBMV disruption on the translation of the BMV RNAs in an in vitro wheat germ system and discovered clear effects on genomic RNAs 1 to 3, but no effect on the sgRNA. We looked for TLSBMV-mediated tyrosine incorporation in BMV translation products but did not get a final answer due to the discovery of an unexpectedly high level of self-tyrosylation of the tyrosyl-tRNA synthetase (TyrRS) induced by the TLSBMV. Finally, we did complementation studies with the TLSBMV added in trans to BMV RNAs with 3′-disrupted TLSs and localized the complementation activity to a specific TLSBMV subdomain. On the basis of our results, we discuss a model with a prime role for the TLSBMV during the BMV infection cycle.