Investigation of Novel RNA Elements in the 3'UTR of Tobacco Necrosis Virus-D.

RNA elements in the untranslated regions of plus-strand RNA viruses can control a variety of viral processes including translation, replication, packaging, and subgenomic mRNA production. The 3' untranslated region (3'UTR) of Tobacco necrosis virus strain D (TNV-D; genus Betanecrovirus, family Tombusviridae) contains several well studied regulatory RNA elements. Here, we explore a previously unexamined region of the viral 3'UTR, the sequence located upstream of the 3'-cap independent translation enhancer (3'CITE). Our results indicate that (i) a long-range RNA-RNA interaction between an internal RNA element and the 3'UTR facilitates translational readthrough, and may also promote viral RNA synthesis; (ii) a conserved RNA hairpin, SLX, is required for efficient genome accumulation; and (iii) an adenine-rich region upstream of the 3'CITE is dispensable, but can modulate genome accumulation. These findings identified novel regulatory RNA elements in the 3'UTR of the TNV-D genome that are important for virus survival.

Biologically-supported structural model for a viral satellite RNA.

Satellite RNAs (satRNAs) are a class of small parasitic RNA replicon that associate with different viruses, including plus-strand RNA viruses. Because satRNAs do not encode a polymerase or capsid subunit, they rely on a companion virus to provide these proteins for their RNA replication and packaging. SatRNAs recruit these and other required factors via their RNA sequences and structures. Here, through a combination of chemical probing analysis of RNA structure, phylogenetic structural comparisons, and viability assays of satRNA mutants in infected cells, the biological importance of a deduced higher-order structure for a 619 nt long tombusvirus satRNA was assessed. Functionally-relevant secondary and tertiary RNA structures were identified throughout the length of the satRNA. Notably, a 3'-terminal segment was found to adopt two mutually-exclusive RNA secondary structures, both of which were required for efficient satRNA accumulation. Accordingly, these alternative conformations likely function as a type of RNA switch. The RNA switch was also found to engage in a required long-range kissing-loop interaction with an upstream sequence. Collectively, these results establish a high level of conformational complexity within this small parasitic RNA and provide a valuable structural framework for detailed mechanistic studies.

Global organization of a positive-strand RNA virus genome.

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.

Multifaceted regulation of translational readthrough by RNA replication elements in a tombusvirus.

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.

Subgenomic mRNA transcription in tobacco necrosis virus.

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.

Evidence for a premature termination mechanism of subgenomic mRNA transcription in a carmovirus.

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.

Tombusvirus recruitment of host translational machinery via the 3' UTR.

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.

A discontinuous RNA platform mediates RNA virus replication: building an integrated model for RNA-based regulation of viral processes.

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.

Uncoupling RNA virus replication from transcription via the polymerase: functional and evolutionary insights.

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.

A second functional RNA domain in the 5' UTR of the Tomato bushy stunt virus genome: intra- and interdomain interactions mediate viral RNA replication.

The 5' untranslated regions (UTRs) of (+)-strand RNA viruses play a variety of roles in the reproductive cycles of these infectious agents. Tomato bushy stunt virus (TBSV) belongs to this class of RNA virus and is the prototype member of the genus Tombusvirus. Previous studies have demonstrated that a T-shaped domain (TSD) forms in the 5' half of the TBSV 5' UTR and that it plays a central role in viral RNA replication. Here we have extended our structure-function analysis to the 3' half of the 5' UTR. Investigation of this region in the context of a model viral replicon (i.e., a TBSV-derived defective interfering [DI] RNA) revealed that this segment contains numerous functionally relevant structural features. In vitro solution structure probing along with comparative and computer-aided RNA secondary structure analyses predicted the presence of a simple stem loop (SL5) followed by a more complex downstream domain (DSD). Both structures were found to be essential for efficient DI RNA accumulation when tested in a plant protoplast system. For SL5, maintenance of the base of its stem was the principal feature required for robust in vivo accumulation. In the DSD, both helical and unpaired regions containing conserved sequences were necessary for efficient DI RNA accumulation. Additionally, optimal DI RNA accumulation required a TSD-DSD interaction mediated by a pseudoknot. Modifications that reduced accumulation did not appreciably affect DI RNA stability in vivo, indicating that the DSD and SL5 act to facilitate viral RNA replication.