tRNA elements mediate the assembly of an icosahedral RNA virus

Abstract

tRNAs, the adapter molecules in protein synthesis, also serve as metabolic cofactors and as primers for viral RNA-directed DNA synthesis. The genomic and subgenomic RNAs of some plant viruses have a 3-terminal tRNA-like structure (TLS) that can accept a specific amino acid and serve as a site for initiation of replication and as a simple telomere. We report a previously undescribed role for the TLS of brome mosaic virus (BMV), and potentially for cellular tRNA, in mediating the assembly of its icosahedral virions. BMV genomic RNAs and subgenomic RNA lacking the TLS failed to assemble into virions when incubated with purified BMV coat protein. Assembly was restored by addition of a 201-nt RNA containing the BMV TLS. TLSs from two other plant viruses as well as tRNAs from wheat germ and yeast were similarly active in the BMV virion assembly reaction, but ribosomal RNA and polyadenylate did not facilitate assembly. Surprisingly, virions assembled from TLS-less BMV RNA in the presence of tRNAs or TLS-containing short RNA did not incorporate the latter molecules. Consistent with a critical role for the BMV TLS in virion assembly, mutations in the BMV genomic RNAs that were designed to disrupt the folding of the TLS also abolished virion assembly. We discuss the likely roles of the TLS in early stages of virion assembly.tRNA-like structure ͉ bromoviruses ͉ virus assembly ͉ RNA packaging T ransfer RNAs (tRNAs) are multifunctional. Their primary role is to be an adapter molecule that translates the codon sequences in mRNA into the amino acid sequence of a protein. tRNAs also participate in specialized functions in cellular metabolism such as biosynthesis of the bacterial cell wall (1), chlorophyll, and heme (2). tRNAs and tRNA-related activities are associated with a variety of RNA viruses, including several plant viruses and the retroviruses (3, 4). Host tRNAs found in the virions of retroviruses function as primers for RNA-directed DNA synthesis (3). The tRNA-like structures (TLSs) found at the 3Ј end of the genomes of some plant viruses serve as efficient origins of replication and as primitive telomeres to ensure that the 3Ј-terminal CCA nucleotides are not lost during replication (4, 5). It has been suggested that these viral TLSs are molecular fossils that may relate to a primordial role for tRNA in RNA replication in the ancient RNA world (6).Brome mosaic virus (BMV) is an example of an RNA virus that has an Ϸ170-nt-long TLS covalently bound to the 3Ј end of its genomic and subgenomic RNAs. BMV is a member of the plant virus family Bromoviridae (7) and the alphavirus-like superfamily of human-, animal-, and plant-infecting positive-strand RNA viruses (8). Mature BMV virions encapsidate three genomic RNAs (B1-B3) and a single subgenomic RNA, B4 (7). Physical and biochemical data suggest that B1-B4 are packaged into three morphologically indistinguishable virus particles: B1 (3.2 kb) and B2 (2.9 kb) are packaged individually into separate particles, whereas the genomic B3 (2.1 kb) and the subgenomic B4 (0.9 kb) are...

Deletion of Highly Conserved Arginine-Rich RNA Binding Motif in Cowpea Chlorotic Mottle Virus Capsid Protein Results in Virion Structural Alterations and RNA Packaging Constraints

“…Bromovirus CP and RNA are amenable for assembly in vitro (2,5,8,37). Previous studies with BCP demonstrated that in vitro assembly is ideal for confirming the packaging defects observed in vivo (5).…”

cis -Acting Elements Required for Efficient Packaging of Brome Mosaic Virus RNA3 in Barley Protoplasts

“…This suggests that the SL-II structure in BMV RNA3 functions as a cofactor for RNA packaging and also functions in concert with other cis-acting elements that may be present in BMV RNA3 regions other than the 3a ORF or within the 3a ORF. In BMV, the 3Ј-terminal TLS that is conserved in all BMV RNAs also plays a crucial role in RNA packaging in vitro (6). TLS and SL-II in B3PE may cooperate in RNA3 packaging in vivo through direct or indirect interactions between these regions.…”

“…RNA regions or elements involved in the packaging of BMV RNAs have been assigned to the coding region of BMV RNA1 by UV cross-linking and band-shift assays (11) as well as to the 3Ј-proximal region of the 3a open reading frame (ORF) in RNA3 (9). The tRNA-like structures (TLS) in the 3Ј-untranslated regions of BMV RNAs also play a crucial role in BMV RNA packaging in vitro (6). In the present study, we delimit a nucleotide sequence required for the efficient packaging of BMV RNA3 and show that 69 nucleotides (nt) in the 3Ј-proximal region of the BMV 3a ORF, especially a predicted stem-loop structure (30 nt), is essential for the efficient packaging of BMV RNA3.…”

Use of Spring beauty latent virus to identify compatible interactions between bromovirus components required for virus infection

“…Moreover, BMV 1a protein must interact with the intercistronic region of BMV RNA 3 for efficient amplification of RNA 3 Sullivan & Ahlquist, 1999). Recent studies have identified sequences in the CP and viral RNAs that are required for bromovirus encapsidation (Choi et al, 2002;Damayanti et al, 2002). However, the interactions between bromovirus components required for movement have not been examined closely.…”