[show abstract][hide abstract] ABSTRACT: The genomes of plus-strand RNA viruses contain many regulatory sequences and structures that direct different viral processes. The traditional view of these RNA elements are as local structures present in non-coding regions. However, this view is changing due to the discovery of regulatory elements in coding regions and functional long-range intra-genomic base pairing interactions. The ∼4.8 kb long RNA genome of the tombusvirus tomato bushy stunt virus (TBSV) contains these types of structural features, including six different functional long-distance interactions. We hypothesized that to achieve these multiple interactions this viral genome must utilize a large-scale organizational strategy and, accordingly, we sought to assess the global conformation of the entire TBSV genome. Atomic force micrographs of the genome indicated a mostly condensed structure composed of interconnected protrusions extending from a central hub. This configuration was consistent with the genomic secondary structure model generated using high-throughput selective 2'-hydroxyl acylation analysed by primer extension (i.e. SHAPE), which predicted different sized RNA domains originating from a central region. Known RNA elements were identified in both domain and inter-domain regions, and novel structural features were predicted and functionally confirmed. Interestingly, only two of the six long-range interactions known to form were present in the structural model. However, for those interactions that did not form, complementary partner sequences were positioned relatively close to each other in the structure, suggesting that the secondary structure level of viral genome structure could provide a basic scaffold for the formation of different long-range interactions. The higher-order structural model for the TBSV RNA genome provides a snapshot of the complex framework that allows multiple functional components to operate in concert within a confined context.
[show abstract][hide abstract] ABSTRACT: Certain plus-strand RNA plant viruses that are uncapped and non-polyadenylated rely on RNA elements in their 3' untranslated region, termed 3' cap-independent translational enhancers (3' CITEs), for efficient translation of their proteins. Here, we have investigated the properties of the Y-shaped class of 3' CITE present in the tombusvirus Carnation Italian ringspot virus (CIRV). While some types of 3' CITE have been found to function through recruitment of translation initiation factors to the viral genome, no trans-acting translation-related factors have yet been identified for the Y-shaped 3' CITE. Our results indicate that the CIRV 3' CITE complexes with eIF4F and eIFiso4F, with the former mediating translation more efficiently than the latter. In nature, some classes of 3' CITE are present in several different viral genera, suggesting that these elements hold a high degree of modularity. Here, we test this concept by engineering chimeric viruses containing heterologous 3' CITEs, and show that the Y-shaped class of 3' CITE in CIRV can be replaced by two alternative types of 3' CITE, i.e. a Panicum mosaic virus-like or I-shaped 3' CITE, without any major loss in translation in vitro or replication efficiency in protoplasts. The heterologous 3' CITEs also mediated whole plant infections of Nicotiana benthamiana, where distinct symptoms were observed for each of the alternative 3' CITEs and 3' CITE evolution occurred during serial passaging. Our results supply new information on Y-shaped 3' CITE function and provide insights into 3' CITE-virus-host compatibilities.
[show abstract][hide abstract] ABSTRACT: In addition to its central role as a template for replication and translation, the viral plus-strand RNA genome also has nontemplate functions, such as recruitment to the site of replication and assembly of the viral replicase, activities that are mediated by cis-acting RNA elements within viral genomes. Two noncontiguous RNA elements, RII(+)-SL (located internally in the tombusvirus genome) and RIV (located at the 3'-terminus), are involved in template recruitment into replication and replicase assembly; however, the importance of each of these RNA elements for these two distinct functions is not fully elucidated. We used an in vitro replicase assembly assay based on yeast cell extract and purified recombinant tombusvirus replication proteins to show that RII(+)-SL, in addition to its known requirement for recruitment of the plus-strand RNA into replication, is also necessary for assembly of an active viral replicase complex. Additional studies using a novel two-component RNA system revealed that the recruitment function of RII(+)-SL can be provided in trans by a separate RNA and that the replication silencer element, located within RIV, defines the template that is used for initiation of minus-strand synthesis. Collectively, this work has revealed new functions for tombusvirus cis-acting RNA elements and provided insights into the pioneering round of minus-strand synthesis.
Journal of Virology 01/2012; 86(1):156-71. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Positive-strand RNA plant viruses that are neither 5'-capped nor 3'-polyadenylated use nontraditional mechanisms to recruit ribosomes to the 5'-end of their viral genomes. One strategy employed by some of these viruses involves a type of RNA element, termed the 3' cap-independent translation enhancer (3'CITE), located in or near the 3'-untranslated region of viral RNA genomes. 3'CITEs function to mediate efficient translation of 5'-proximally encoded viral proteins and function by recruiting either translation initiation factors or the 60S ribosomal subunit to the viral RNA. Recent mechanistic and structural studies have revealed important new insights and details of how 3'CITEs are able to facilitate viral translation and allow these viruses to compete efficiently against cellular mRNAs for the host translational machinery.
Current opinion in virology. 11/2011; 1(5):373-80.
[show abstract][hide abstract] ABSTRACT: Tobacco necrosis virus-D (TNV-D), a positive-strand RNA Necrovirus in the family Tombusviridae, transcribes two subgenomic (sg) mRNAs during infections. We have investigated the strategy used by TNV-D in this process and uncovered evidence that it employs a premature termination (PT) mechanism for the transcription of its sg mRNAs. Structural and mutational analysis of the TNV-D genome identified local RNA structures upstream from transcriptional initiation sites that functioned in the plus-strand as attenuation structures and mediated the production of sg mRNA-sized minus-strands. Other evidence in support of a PT mechanism included the ability to uncouple minus-strand sg RNA production from plus-strand sg mRNA synthesis and the sequence similarities observed between the sg mRNA promoter and that for the viral genome. Accordingly, our results indicate that the necrovirus TNV-D, like several other genera in the family Tombusviridae, uses a PT mechanism for transcription of its sg mRNAs.
[show abstract][hide abstract] ABSTRACT: The transcriptional mechanism utilized by turnip crinkle carmovirus to synthesize subgenomic (sg) mRNAs was investigated by analyzing viral RNAs and their associated regulatory RNA elements. In vivo analyses revealed the following: (i) that minus-strand sg RNAs are detectable in infections, (ii) that minus-strand sg RNA accumulation can be partially uncoupled from that of their plus-strand sg mRNA counterparts, and (iii) that an RNA secondary structure located upstream of the sg mRNA start site mediates transcription by functioning in the plus strand of the viral genome. Collectively, these observations are consistent with this carmovirus using a premature termination mechanism for sg mRNA transcription.
Journal of Virology 08/2010; 84(15):7904-7. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: RNA viruses recruit the host translational machinery by different mechanisms that depend partly on the structure of their genomes. In this regard, the plus-strand RNA genomes of several different pathogenic plant viruses do not contain traditional translation-stimulating elements, i.e., a 5'-cap structure and a 3'-poly(A) tail, and instead rely on a 3'-cap-independent translational enhancer (3'CITE) located in their 3' untranslated regions (UTRs) for efficient synthesis of viral proteins. We investigated the structure and function of the I-shaped class of 3'CITE in tombusviruses--also present in aureusviruses and carmoviruses--using biochemical and molecular approaches and we determined that it adopts a complex higher-order RNA structure that facilitates translation by binding simultaneously to both eukaryotic initiation factor (eIF) 4F and the 5' UTR of the viral genome. The specificity of 3'CITE binding to eIF4F is mediated, at least in part, through a direct interaction with its eIF4E subunit, whereas its association with the viral 5' UTR relies on complementary RNA-RNA base-pairing. We show for the first time that this tripartite 5' UTR/3'CITE/eIF4F complex forms in vitro in a translationally relevant environment and is required for recruitment of ribosomes to the 5' end of the viral RNA genome by a mechanism that shares some fundamental features with cap-dependent translation. Notably, our results demonstrate that the 3'CITE facilitates the initiation step of translation and validate a molecular model that has been proposed to explain how several different classes of 3'CITE function. Moreover, the virus-host interplay defined in this study provides insights into natural host resistance mechanisms that have been linked to 3'CITE activity.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Virology 08/2009; 83(19):10096-105. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA-RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA-RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA-based interaction spanning approximately 3000 nts. In vivo and in vitro analyses suggest that the discontinuous RNA platform formed by the interaction facilitates efficient assembly of the viral RNA replicase. This finding has allowed us to build an integrated model for the role of global RNA structure in regulating the reproduction of a eukaryotic RNA virus, and the insights gained have extended our understanding of the multifunctional nature of viral RNA genomes.
[show abstract][hide abstract] ABSTRACT: Tomato bushy stunt virus (TBSV) possesses a positive-strand RNA genome that is not 5'-capped or 3'-polyadenylated. Previous analysis revealed that the TBSV genome contains a 3'-cap-independent translational enhancer (3'CITE) in its 3'-untranslated region (3'UTR) that facilitates translation of viral mRNAs in vivo. A long-range 5'-3' RNA-RNA interaction between the 3'CITE and the 5'UTR of viral mRNAs is necessary for function, and this RNA bridge has been proposed to mediate delivery of translation-related factors bound to the 3'CITE to the 5'-end of the message. Although fully functional when assayed in plant protoplasts, the TBSV 3'CITE was previously found to be unable to activate translation in vitro in wheat germ extract (wge). In the current report we have determined that (i) another Tombusvirus, Carnation Italian ringspot virus (CIRV), contains a TBSV-like 3'CITE that is active in wge; (ii) the CIRV 3'CITE functions in vitro in a manner analogous to the TBSV 3'CITE in vivo; (iii) the TBSV 3'CITE is able to competitively inhibit CIRV 3'CITE-dependent translation in wge and (iv) the TBSV 3'CITE can enhance translation in wge when present in short viral messages. These results reveal the contrasting activities of different TBSV-like 3'CITEs in vitro and shed light on the nature of the defect in TBSV.
[show abstract][hide abstract] ABSTRACT: Subgenomic (sg) mRNAs are small viral messages that are synthesized by polycistronic positive-strand RNA viruses to allow for the translation of certain viral proteins. Tombusviruses synthesize two such sg mRNAs via a premature termination mechanism. This transcriptional process involves the viral RNA-dependent RNA polymerase terminating minus-strand RNA synthesis prematurely at internal RNA signals during copying of the viral genome. The 3'-truncated minus-strand RNAs generated by the termination events then serve as templates for sg mRNA transcription. A higher-order RNA structure in the viral genome, located just upstream from the termination site, is a critical component of the RNA-based polymerase attenuation signal. Here, we have analyzed the role of this RNA structure in mediating efficient sg mRNA2 transcription. Our results include the following: (i) we define the minimum overall thermodynamic stability required for an operational higher-order RNA attenuation structure; (ii) we show that the distribution of stability within an attenuation structure affects its function; (iii) we establish that an RNA quadruplex structure can act as an effective attenuation structure; (iv) we prove that the higher-order RNA structure forms and functions in the plus strand; (v) we provide evidence that a specific attenuation structure-binding protein factor is not required for transcription; (vi) we demonstrate that sg mRNA transcription can be controlled artificially through small-molecule activation using RNA aptamer technology. These findings provide important new insights into the premature termination mechanism and present a novel approach to regulate the transcriptional process.
Journal of Virology 05/2008; 82(8):3864-71. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Single-stranded RNA plant viruses not only code for viral proteins within their RNA genomes, they often maintain elaborate RNA secondary structures. These structures can be integral to a variety of viral processes, such as viral translation, genome replication, subgenomic mRNA transcription, and genome encapsidation. RNA secondary structures may function to recruit and bind trans-acting protein factors, or may become part of higher order tertiary RNA structures, which themselves may be functionally relevant. To fully understand such viral RNA elements and their mechanisms of action, it is necessary to first determine their secondary structures.Computer modeling based on free energy minimizing principles is generally used as an initial approach to predict potential RNA secondary structures in a sequence. The most popular program is mfold, which is available for free to the public at http://www.bioinfo.rpi.edu/applications/mfold/old/rna/form1.cgi (1). Though useful starting points, the mfold-predicted structures must be confirmed by more direct experimental approaches. Solution structure probing of RNA is a commonly utilized technique that provides information regarding the secondary structure of an RNA primary sequence in solution. This process involves treating the RNA of interest with enzymes or chemicals that modify RNA differentially based on its secondary structure. The modified sites, at either single- or double-stranded regions, can be subsequently identified by primer extension and gel electrophoresis. Data from solution structure probing experiments can be superimposed onto a computer-predicted structure to further help confirm or refine the predicted RNA secondary structure model.
Methods in molecular biology (Clifton, N.J.) 02/2008; 451:243-50.
[show abstract][hide abstract] ABSTRACT: Many eukaryotic positive-strand RNA viruses transcribe subgenomic (sg) mRNAs that are virus-derived messages that template the translation of a subset of viral proteins. Currently, the premature termination (PT) mechanism of sg mRNA transcription, a process thought to operate in a variety of viruses, is best understood in tombusviruses. The viral RNA elements involved in regulating this mechanism have been well characterized in several systems; however, no corresponding protein factors have been identified yet. Here we show that tombusvirus genome replication can be effectively uncoupled from sg mRNA transcription in vivo by C-terminal modifications in its RNA-dependent RNA polymerase (RdRp). Systematic analysis of the PT transcriptional pathway using viral genomes harboring mutant RdRps revealed that the C-terminus functions primarily at an early step in this mechanism by mediating both efficient and accurate production of minus-strand templates for sg mRNA transcription. Our results also suggest a simple evolutionary scheme by which the virus could gain or enhance its transcriptional activity, and define global folding of the viral RNA genome as a previously unappreciated determinant of RdRp evolution.
The EMBO Journal 01/2008; 26(24):5120-30. · 9.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: The analysis of viral RNA is a fundamental aspect of plant RNA virus research. Studies that focus on viral RNAs often involve virus infections of plant protoplasts (see UNITS 16D.1-16D.4). Protoplast offer the advantage of simultaneous initiation of infections, which allows for superior temporal and quantitative analyses of viral RNAs. The efficient isolation of intact viral RNA is key to any such investigations. This unit describes two basic protocols for extracting viral RNAs from plant protoplasts. An approach for preparing double-stranded viral RNA from total RNA pools is also provided. The viral RNA prepared by using these techniques can be used for further analyses such as primer extension, reverse transcription-PCR, and northern blotting.
Current protocols in microbiology 09/2007; Chapter 16:Unit 16E.1.
[show abstract][hide abstract] ABSTRACT: Positive-strand RNA viruses direct different virus-specific processes during their infection of host cells. Fundamental events such as viral RNA genome replication are controlled by viral regulatory RNA elements (REs). Here, we have investigated the possibility of specifically modulating the action of a viral RE using RNA aptamer technology. Through rational design, a tombusvirus RE, which has the structure of a perfect RNA stem loop in the plus-strand RNA genome, was replaced with a theophylline-binding RNA aptamer sequence, an imperfect stem loop. The aptamer-RE hybrid was designed so that, upon binding theophylline, it would become more stable and structurally mimic the functional RE (i.e., represent a ligand-inducible RE riboswitch). Initial experiments were conducted with a small noncoding virus genome-derived RNA replicon, and the results showed that replication was inducible, up to approximately 10-fold, in a theophylline-specific and dose-dependent manner. A similar level of theophylline-dependent induction was also observed when a full-length viral genome containing an RE riboswitch was tested. Analysis of this engineered viral genome revealed that this RE, located in the 5' untranslated region, specifically mediates efficient accumulation of plus-strands of the virus genome. Therefore, in addition to allowing for modulation of virus reproduction, the RE riboswitch system also provided insight into RE function. The ability to chemically induce a viral process via modulation of virus genome structure could be useful for basic and applied aspects of research.
Proceedings of the National Academy of Sciences 07/2007; 104(25):10406-11. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Virology 04/2007; 81(5):2429-39. · 5.08 Impact Factor