Translational readthrough of stop codons by ribosomes is a recoding event used by a variety of viruses, including plus-strand RNA tombusviruses. Translation of the viral RNA-dependent RNA polymerase (RdRp) in tombusviruses is mediated using this strategy and we have investigated this process using a variety of in vitro and in vivo approaches. Our results indicate that readthrough generating the RdRp requires a novel long-range RNA-RNA interaction, spanning a distance of ∼3.5 kb, which occurs between a large RNA stem-loop located 3'-proximal to the stop codon and an RNA replication structure termed RIV at the 3'-end of the viral genome. Interestingly, this long-distance RNA-RNA interaction is modulated by mutually-exclusive RNA structures in RIV that represent a type of RNA switch. Moreover, a different long-range RNA-RNA interaction that was previously shown to be necessary for viral RNA replicase assembly was also required for efficient readthrough production of the RdRp. Accordingly, multiple replication-associated RNA elements are involved in modulating the readthrough event in tombusviruses and we propose an integrated mechanistic model to describe how this regulatory network could be advantageous by (i) providing a quality control system for culling truncated viral genomes at an early stage in the replication process, (ii) mediating cis-preferential replication of viral genomes, and (iii) coordinating translational readthrough of the RdRp with viral genome replication. Based on comparative sequence analysis and experimental data, basic elements of this regulatory model extend to other members of Tombusviridae, as well as to viruses outside of this family.
The replication of positive-strand RNA viral genomes involves various cis-acting RNA sequences. Generally, regulatory RNA sequences are present at or near genomic termini; however, internal replication elements (IREs) also exist. Here we report the structural and functional characterization of an IRE present in the readthrough portion of the p92 polymerase gene of Tomato bushy stunt virus. Analysis of this element in the context of a noncoding defective interfering RNA revealed a functional core structure composed of two noncontiguous segments of sequence that interact with each other to form an extended helical conformation. IRE activity required maintenance of several base-paired sections as well as two distinct structural features: (i) a short, highly conserved segment that can potentially form two different and mutually exclusive structures and (ii) an internal loop that contains a critical CC mismatch. The IRE was also shown to play an essential role within the context of the viral genome. In vivo analysis with novel RNA-based temperature-sensitive genomic mutants and translationally active subgenomic viral replicons revealed the following about the IRE: (i) it is active in the positive strand, (ii) it is dispensable late in the viral RNA replication process, and (iii) it is functionally inhibited by active translation over its sequence. Together, these results suggest that IRE activity is required in the cytosol at an early step in the viral replication process, such as template recruitment and/or replicase complex assembly.Positive-strand RNA viruses replicate their genomes via a negative-strand RNA intermediate (2). This process is catalyzed by a virally encoded RNA-dependent RNA polymerase that is the core subunit of a viral replicase complex. Replicase activity is regulated in part by cis-acting RNA elements in the viral genome (2). The core promoters for the synthesis of positive and negative strands are found at the termini of RNA templates, while other important RNA elements that modulate replicase function are more internally located. These auxiliary RNA elements can function directly by either enhancing or silencing promoter activity (18,20,24,28,29,41) or indirectly by influencing replicase assembly, template recruitment, or primer synthesis functions (4,22,23,26,33).RNA elements involved in genome replication have been studied extensively in the genus Tombusvirus (37). The type species of this genus, Tomato bushy stunt virus (TBSV), is an icosahedral virus that contains a 4.8-kb-long nonsegmented positive-strand RNA genome (11) (Fig. 1A). Replication of the TBSV genome requires two 5Ј-proximally encoded proteins, p33, an auxiliary replication protein, and p92, the RNA-dependent RNA polymerase (19) (Fig. 1A). p92 is produced by translational readthrough of the p33 stop codon and accumulates at about 5% the level of p33 in infected tissue (31).Most studies investigating the role of RNA elements in TBSV replication have been carried out by using TBSV defective interfering (DI) RNAs (34, 37). Typica...
During infections, positive-strand RNA tombusviruses transcribe two subgenomic (sg) mRNAs that allow for the expression of a subset of their genes. This process is thought to involve an unconventional mechanism involving the premature termination of the virally encoded RNA-dependent RNA polymerase while it is copying the virus genome. The 3 truncated minus strands generated by termination are then used as templates for sg mRNA transcription. In addition to requiring an extensive network of long-distance RNA-RNA interactions (H.-X. Lin and K. A. White, EMBO J. 23:3365-3374, 2004), the transcription of tombusvirus sg mRNAs also involves several additional RNA structures. In vivo analysis of these diverse RNA elements revealed that they function at distinct steps in the process by facilitating the formation or stabilization of the long-distance interactions, modulating minus-strand template production, or promoting the initiation of sg mRNA transcription. All of the RNA elements characterized could be readily incorporated into a premature termination model for sg mRNA transcription. Overall, the analyses revealed a complex system that displays a high level of structural integration and functional coordination. This multicomponent RNA-based control system may serve as a useful paradigm for understanding related transcriptional processes in other positive-sense RNA viruses.Most positive-strand RNA genomes that are polycistronic rely on the transcription of smaller genome-derived mRNAs, termed subgenomic (sg) mRNAs, to mediate the translation of their 3Ј proximally positioned genes (17). Although these viruses use different mechanisms to produce sg mRNAs, the involvement of their virally encoded RNA-dependent RNA polymerase (RdRp) is common to all (1). In viruses such as Brome mosaic virus, sg mRNAs are generated by the internal initiation of transcription by the RdRp on full-length negative strands of the viral genomes (16). Alternatively, corona-and arteriviruses transcribe their sg mRNAs from noncontiguous, minus-strand RNA templates that are produced by discontinuous RdRp copying of their genomes (10,24,25,30,36,38). A third mechanism, suggested by studies of Red clover necrotic mosaic virus (RCNMV), involves the premature termination (PT) of the RdRp while copying viral RNA genomes and the subsequent use of the 3Ј truncated minus strands as templates for sg mRNA transcription (33). This PT mechanism has also been proposed to function in a variety of viruses (e.g., Torovirus, Nodavirus, Closterovirus, and Tombusvirus) (7,14,27,35,39,41,48). Despite its prevalence and importance, many mechanistic aspects of PT-based sg mRNA transcription are poorly understood and comprehensive functional models that include all of the cis-and trans-acting factors involved are lacking (41).Tombusviruses represent one of the most advanced systems for studying viral RNA synthesis and gene expression (43). The prototype of this genus, Tomato bushy stunt virus (TBSV), possesses a small, 4.8-kb, positive-strand RNA genome that carries fi...
The genus Aureusvirus is composed of a group of positive-strand RNA plant viruses that belong to the family Tombusviridae. Expression of certain aureusvirus genes requires the transcription of two subgenomic (sg) mRNAs. Interestingly, the level of sg mRNA2 accumulation in aureusvirus infections is considerably lower than that of sg mRNA1. The nature of this difference was investigated using the aureusvirus Cucumber leaf spot virus (CLSV). Analysis of sg mRNA2 transcription indicated that it is synthesized by a premature termination mechanism. The results also implicated the transcriptional promoter, the attenuation signal, and global RNA folding of the viral genome as mediators of sg mRNA2 suppression. Additionally, evaluation of the transcriptional regulatory RNA elements in aureusviruses and related tombusviruses revealed alternative strategies for building functionally-equivalent stem-loop structures and showed that sequences encoding a critical and invariant amino acid can be successfully incorporated into essential long-distance tertiary RNA-RNA interactions.
Cucumber leaf spot virus (CLSV) is an aureusvirus (family Tombusviridae) that has a positive-sense RNA genome encoding five proteins. During infections, CLSV transcribes two subgenomic (sg) mRNAs and the larger of the two, sg mRNA1, encodes coat protein. Here, the viral RNA sequences and structures that regulate transcription and translation of CLSV sg mRNA1 were investigated. A medium-range RNA-RNA interaction in the CLSV genome, spanning 148 nucleotides, was found to be required for the efficient transcription of sg mRNA1. Further analysis indicated that the structure formed by this interaction acted as an attenuation signal required for transcription of sg mRNA1 via a premature termination mechanism. Translation of coat protein from sg mRNA1 was determined to be facilitated by a 5-terminal stem-loop structure in the message that resembled a tRNA anticodon stem-loop. The results from mutational analysis indicated that the 5-terminal stem-loop mediated efficient base pairing with a 3-cap-independent translational enhancer at the 3 end of the message, leading to efficient translation of coat protein from sg mRNA1. Comparison of the regulatory RNA structures for sg mRNA1 of CLSV to those used by the closely related tombusviruses and certain cellular RNAs revealed interesting differences and similarities that provide evolutionary and mechanistic insights into RNA-based regulatory strategies.Positive-strand RNA viruses use a variety of mechanisms for expressing their genes. For viruses in the family Tombusviridae, subgenomic (sg) mRNA transcription is utilized to mediate the expression of viral proteins encoded 3Ј proximally in their genomes (28, 50). These smaller, 3Ј-coterminal messages are efficient templates for translation and allow for expression of a subset of viral proteins. Interestingly, the plus-strand RNA genomes of the members of Tombusviridae are not 5Ј capped or 3Ј polyadenylated, and several have been shown to contain 3Ј-cap independent translational enhancers (3ЈCITEs) in their 3Ј-untranslated regions (3ЈUTRs) (1,6,9,10,19,30,31,33,38,39,41,43,45,46,51). Some of these 3ЈCITEs are known to function by recruiting translational machinery to the message (13, 41, 44), and several have been shown to communicate with their cognate 5ЈUTRs via RNA-RNA interactions (9,10,14,16,17). In addition, since sg mRNAs are 3Ј-coterminal with their genomes, they also contain 3ЈCITEs and can utilize them for efficient translation of their encoded proteins (18,29).Cucumber leaf spot virus (CLSV) is a member of the genus Aureusvirus (family Tombusviridae) (27) and it is very closely related to Tomato bushy stunt virus (TBSV; genus Tombusvirus, family Tombusviridae) (15). These viruses share a similar coding strategy in which RNA replication-related proteins are encoded 5Ј proximally in the viral genome, followed by coat protein (CP) and, finally, overlapping cell-to-cell movement and gene silencing suppressor proteins that are coded in different reading frames (Fig. 1) (26,27,35,37,50). In both cases, the CP and the 3Ј-pr...
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