Characterization of a Novel 5′ Subgenomic RNA3a Derived from RNA3 of Brome Mosaic Bromovirus

Abstract

The synthesis of 3 subgenomic RNA4 (sgRNA4) by initiation from an internal sg promoter in the RNA3 segment was first described for Brome mosaic bromovirus (BMV), a model tripartite positive-sense RNA virus (W. A. Miller, T. W. Dreher, and T. C. Hall, Nature 313: [68][69][70] 1985). In this work, we describe a novel 5 sgRNA of BMV (sgRNA3a) that we propose arises by premature internal termination and that encapsidates in BMV virions. Cloning and sequencing revealed that, unlike any other BMV RNA segment, sgRNA3a carries a 3 oligo(A) tail, in which respect it resembles cellular mRNAs. Indeed, both the accumulation of sgRNA3a in polysomes and the synthesis of movement protein 3a in in vitro systems suggest active functions of sgRNA3a during protein synthesis. Moreover, when copied in the BMV replicase in vitro reaction, the minus-strand RNA3 template generated the sgRNA3a product, likely by premature termination at the minus-strand oligo(U) tract. Deletion of the oligo(A) tract in BMV RNA3 inhibited synthesis of sgRNA3a during infection. We propose a model in which the synthesis of RNA3 is terminated prematurely near the sg promoter. The discovery of 5 sgRNA3a sheds new light on strategies viruses can use to separate replication from the translation functions of their genomic RNAs.Single-stranded positive-sense RNA viruses utilize various strategies for expression of their RNA genomes (36), most notably via subgenomic RNAs (sgRNAs). The Coronaviridae and Arteriviridae families of the order Nidovirales express 6 to 7 proteins via sgRNAs, while the Closteroviridae generate between 6 and 11 sgRNAs. In some viruses, e.g., in Brome mosaic bromovirus (BMV), sgRNAs arise via internal initiation by the viral RNA polymerase (RdRp) on genomic minus-sense RNAs (20,30,33,38). It is also likely that other viruses, e.g., red clover necrotic mosaic virus (54), tomato bushy stunt virus (10), and flock house virus (17), copy their sgRNAs from prematurely terminated minus strands (62). In Nidovirales (46, 61, 72) the noncontiguous RNA leaders are joined to variously located sequences during minus-strand synthesis, followed by sgRNA transcription, whereas toroviruses combine discontinuous and nondiscontinuous processes to produce their sgRNAs (63). The formation of sgRNAs can also result from premature termination of positive-strand synthesis. In closteroviruses, a highly structured sequence region produces 5Ј-terminal sgRNAs by pretermination (20) and additionally serves as a promoter for synthesis of another downstream sgRNA, with possible overlapping of termination and initiation signals (20).The previously described 3Ј sgRNA4 of BMV is transcribed from an intergenic 100-nucleotide (nt) promoter (sgp) that consists of the core domain, a transcription enhancer, and the poly(A) tract (18,38,57). The more-upstream 150-nt sequence functions in cis as an internal replication enhancer (IRE) for positive-strand RNA3 amplification and carries a conserved B-box motif (18). In addition, we recently reported that the poly(A) tract of the sgp...

RNA-RNA Recombination in Plant Virus Replication and Evolution

“…Sgp core hairpin was shown to act as a landing pad for RdRp during de novo initiation of sgRNA4 synthesis (2,145). Additionally, the sgp region leads to the formation of the 5 sgRNA3a (147). A recent study confirmed the high efficiency of the sgRNA3a-mediated homologous crossovers with BMV RNA3 (Figure 5a) (131).…”

The Tripartite Virions of the Brome Mosaic Virus Have Distinct Physical Properties That Affect the Timing of the Infection Process

“…The RNAs were identified in Northern blots treated with a riboprobe that recognizes the conserved 3= UTR of the BMV RNAs. Bands identified by the asterisks likely correspond to prematurely terminated RNA2 and/or degradation products (37). The relative amounts of the four full-length BMV RNAs within each sample are quantified below the image of the Northern blots.…”

“…The fulllength BMV RNAs were identified by RNAs extracted from WT BMV virions. The bands highlighted with asterisks likely correspond to prematurely terminated RNA3 (37). (C) Quantification of the accumulation of plus-strand BMV RNA1 and RNA2 over time.…”

Insights into the Single-Cell Reproduction Cycle of Members of the Family Bromoviridae : Lessons from the Use of Protoplast Systems

“…In order to synthesize sgRNA4, the replicase enzyme recognizes the core promoter sequence by an induced-fit mechanism (115). Recently, a novel 5Ј sgRNA3a in BMV that arose by premature termination of genomic RNA3 synthesis has been discovered (132). Studies with barley and A. thaliana protoplasts have shown that both the length of the oligo(A) tract and the stability of the sgp core hairpin affected sgRNA3a synthesis (J. Sztuba-Solińska and J. J. Bujarski, unpublished data).…”

“…4. Two models of the crossing-over mechanism within the sgp region (27,132). The top part shows the organization of sgp, while the sgp sequence is shown on the bottom.…”