Exploring the architecture of viral RNA genomes.

The genomes of RNA viruses contain local structural elements and long-range interactions that control various steps in virus replication. While many individual RNA elements have been characterized, it remains less clear how the structure and activity of such elements are integrated and regulated within the complex context of complete viral genomes. Recent technical advances, particularly the development of high-throughput solution structure mapping methods, have made secondary structural analysis of entire viral RNA genomes feasible. As a consequence, whole-genome structural models have been deduced for a number of plus-strand RNA viruses and retroviruses and these structures have provided intriguing functional and evolutionary insights into global genome architecture.

Functional long-range RNA-RNA interactions in positive-strand RNA viruses.

Positive-strand RNA viruses are important human, animal and plant pathogens that are defined by their single-stranded positive-sense RNA genomes. In recent years, it has become increasingly evident that interactions that occur between distantly positioned RNA sequences within these genomes can mediate important viral activities. These long-range intragenomic RNA-RNA interactions involve direct nucleotide base pairing and can span distances of thousands of nucleotides. In this Review, we discuss recent insights into the structure and function of these intriguing genomic features and highlight their diverse roles in the gene expression and genome replication of positive-strand RNA viruses.

Translational readthrough in Tobacco necrosis virus-D.

The plus-strand RNA genome of Tobacco necrosis virus-D (TNV-D) expresses its polymerase via translational readthrough. The RNA signals involved in this readthrough process were characterized in vitro using a wheat germ extract translation system and in vivo via protoplast infections. The results indicate that (i) TNV-D requires a long-range RNA-RNA interaction between an extended stem-loop (SL) structure proximal to the readthrough site and a sequence in the 3'-untranslated region of its genome; (ii) stability of the extended SL structure is important for its function; (iii) TNV-D readthrough elements are compatible with UAG and UGA, but not UAA; (iv) a readthrough defect can be rescued by a heterologous readthrough element in vitro, but not in vivo; and (v) readthrough elements can also mediate translational frameshifting. These results provide new information on determinants of readthrough in TNV-D and further support the concept of a common general mechanism for readthrough in Tombusviridae.

Tombusvirus Y-shaped translational enhancer forms a complex with eIF4F and can be functionally replaced by heterologous translational enhancers.

Certain plus-strand RNA plant viruses that are uncapped and nonpolyadenylated 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 3'CITE or an I-shaped 3'CITE, without any major loss in in vitro translation 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.

Internal RNA Replication Elements are Prevalent in Tombusviridae.

Internal replication elements (IREs) are RNA structures that are present at internal positions in the genomes of different types of plus-strand RNA viruses. Members of the genus Tombusvirus (family Tombusviridae) contain an IRE within the polymerase coding region of their genomes and this RNA element participates in both genome targeting to sites of replication and replicase complex assembly. Here we propose that other members of the virus family Tombusviridae also possess comparable IREs. Through sequence and structural analyses, candidate IREs in several genera of this family were identified, including aureusviruses, necroviruses, carmoviruses, and pelarspoviruses. The results from subsequent mutational analysis of selected proposed IREs were consistent with a critical role for these structures in viral genome accumulation during infections. Our study supports the existence of IREs in several genera in Tombusviridae and points to previously unappreciated similarities in genome replication strategies between members of this virus family.

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.

3' Cap-independent translation enhancers of positive-strand RNA plant viruses.

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.

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.

Context-influenced cap-independent translation of Tombusvirus mRNAs in vitro.

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.