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 313:68-70, 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.

Efficient In Vitro System of Homologous Recombination in Brome Mosaic Bromovirus

ABSTRACT Recent in vivo studies have revealed that the subgenomic promoter (sgp) in brome mosaic bromovirus (BMV) RNA3 supports frequent homologous recombination events (R. Wierzchoslawski, A. Dzianott, and J. Bujarski, J. Virol. 78:8552-8564, 2004). In this paper, we describe an sgp-driven in vitro system that supports efficient RNA3 crossovers. A 1:1 mixture of two (−)-sense RNA3 templates was copied with either a BMV replicase (RdRp) preparation or recombinant BMV protein 2a. The BMV replicase enzyme supported a lower recombination frequency than 2a, demonstrating a role of other viral and/or host factors. The described in vitro system will allow us to study the mechanism of homologous RNA recombination.

Homologous Crossovers among Molecules of Brome Mosaic Bromovirus RNA1 or RNA2 Segments In Vivo

ABSTRACT Previously we demonstrated frequent homologous crossovers among molecules of the RNA3 segment in the tripartite brome mosaic bromovirus (BMV) RNA genome (A. Bruyere, M. Wantroba, S. Flasinski, A. Dzianott, and J. J. Bujarski, J. Virol. 74:4214-4219, 2000). To further our knowledge about mechanisms of viral RNA genome variability, in this paper we have studied homologous recombination in BMV RNA1 and RNA2 components during infection. We have found that basal RNA-RNA crossovers could occur within coding regions of both RNAs, although recombination frequencies slightly varied at different RNA sections. In all cases, the frequencies were much lower than the rate observed for the intercistronic recombination hot spot in BMV RNA3. Probability calculations accounted for at least one homologous crossover per RNA molecule per replication cycle. In addition, we have demonstrated an efficient repair of mutations within the conserved 3′ and 5′ noncoding regions, most likely due to error-prone BMV RNA replication. Overall, our data verify that homologous crossovers are common events a during virus life cycle, and we discuss their importance for viral RNA genetics.

A Transcriptionally Active Subgenomic Promoter Supports Homologous Crossovers in a Plus-Strand RNA Virus

ABSTRACT Genetic RNA recombination plays an important role in viral evolution, but its molecular mechanism is not well understood. In this work we describe homologous RNA recombination activity that is supported by a subgenomic promoter (sgp) region in the RNA3 segment of brome mosaic bromovirus (BMV), a tripartite plus-strand RNA virus. The crossover frequencies were determined by coinoculations with pairs of BMV RNA3 variants that carried a duplicated sgp region flanked by marker restriction sites. A region composed of the sgp core, a poly(A) tract, and an upstream enhancer supported homologous exchanges in 25% of the analyzed RNA3 progeny. However, mutations in the sgp core stopped both the transcription of the sgp RNA and homologous recombination. These data provide evidence for an association of RNA recombination with transcription.

RNA Recombination in Brome Mosaic Virus: Effects of Strand-Specific Stem-Loop Inserts

ABSTRACT A model system of a single-stranded trisegment Brome mosaic bromovirus (BMV) was used to analyze the mechanism of homologous RNA recombination. Elements capable of forming strand-specific stem-loop structures were inserted at the modified 3′ noncoding regions of BMV RNA3 and RNA2 in either positive or negative orientations, and various combinations of parental RNAs were tested for patterns of the accumulating recombinant RNA3 components. The structured negative-strand stem-loops that were inserted in both RNA3 and RNA2 reduced the accumulation of RNA3-RNA2 recombinants to a much higher extent than those in positive strands or the unstructured stem-loop inserts in either positive or negative strands. The use of only one parental RNA carrying the stem-loop insert reduced the accumulation of RNA3-RNA2 recombinants even further, but only when the stem-loops were in negative strands of RNA2. We assume that the presence of a stable stem-loop downstream of the landing site on the acceptor strand (negative RNA2) hampers the reattachment and reinitiation processes. Besides RNA3-RNA2 recombinants, the accumulation of nontargeted RNA3-RNA1 and RNA3-RNA3 recombinants were observed. Our results provide experimental evidence that homologous recombination between BMV RNAs more likely occurs during positive- rather than negative-strand synthesis.

Frequent Homologous Recombination Events between Molecules of One RNA Component in a Multipartite RNA Virus

ABSTRACT Brome mosaic bromovirus (BMV), a tripartite plus-sense RNA virus, has been used as a model system to study homologous RNA recombination among molecules of the same RNA component. Pairs of BMV RNA3 variants carrying marker mutations at different locations were coinoculated on a local lesion host, and the progeny RNA3 in a large number of lesions was analyzed. The majority of doubly infected lesions accumulated the RNA3 recombinants. The distribution of the recombinant types was relatively even, indicating that both RNA3 counterparts could serve as donor or as acceptor molecules. The frequency of crossovers between one pair of RNA3 variants, which possessed closely located markers, was similar to that of another pair of RNA3 variants with more distant markers, suggesting the existence of an internal recombination hot spot. The majority of crossovers were precise, but some recombinants had minor sequence modifications, possibly marking the sites of imprecise homologous crossovers. Our results suggest discontinuous RNA replication, with the replicase changing among the homologous RNA templates and generating RNA diversity. This approach can be easily extended to other RNA viruses for identification of homologous recombination hot spots.

Defective Movement of Viruses in the Family Bromoviridae Is Differentially Complemented in Nicotiana benthamiana Expressing Tobamovirus or Dianthovirus Movement Proteins

Taxonomically distinct tobacco mosaic tobamovirus (TMV), red clover necrotic mosaic dianthovirus (RCNMV), cucumber mosaic cucumovirus (CMV), brome mosaic bromovirus (BMV), and cowpea chlorotic mottle bromovirus (CCMV) exhibit differences in their host range. Each of these viruses encodes a functionally similar nonstructural movement protein (MP) that is essential for cell-to-cell movement of a progeny virus. Despite the lack of significant amino acid identity among the MPs of CMV, TMV, and RCNMV, movement-defective CMV (CMVFnyΔMP-ΔKPN) was able to move locally and systemically in transgenic Nicotiana benthamiana expressing either TMV MP (NB-TMV-MP(+)) or RCNMV MP (NB-RCNMV-MP(+)). These observations contrast with those of previous studies in which transgenic N. tabacum cv. Xanthi plants expressing TMV MP supported only the cell-to-cell movement of CMVFnyΔMP-ΔKPN. To verify whether similar complementation could be observed for movement-defective bromoviruses, NB-TMV-MP(+) and NB-RCNMV-MP(+) plants were inoculated independently with movement-defective variants of BMV (B3ΔMP) and CCMV (CC3ΔMP). Neither NB-TMV-MP(+) nor NB-RCNMV-MP(+) was able to rescue the defective cell-to-cell and long-distance movement of B3ΔMP. In contrast, NB-RCNMV-MP(+) complemented the cell-to-cell, but not the long-distance, movement of CC3ΔMP. Taken together, these studies suggest that virus movement is a complex process and that, in some cases, the host species plays a major role in determining the long-distance movement function of a virus.