Subgenomic mRNA transcription in Tombusviridae

Turnip crinkle virus (TCV) contains a structured 3= region with hairpins and pseudoknots that form a complex network of noncanonical RNA:RNA interactions supporting higher-order structure critical for translation and replication. We investigated several second-site mutations in the p38 coat protein open reading frame (ORF) that arose in response to a mutation in the asymmetric loop of a critical 3= untranslated region (UTR) hairpin that disrupts local higher-order structure. All tested second-site mutations improved accumulation of TCV in conjunction with a partial reversion of the primary mutation (TCV-rev1) but had neutral or a negative effect on wild-type (wt) TCV or TCV with the primary mutation. SHAPE (selective 2=-hydroxyl acylation analyzed by primer extension) structure probing indicated that these second-site mutations reside in an RNA domain that includes most of p38 (domain 2), and evidence for RNA:RNA interactions between domain 2 and 3=UTR-containing domain 1 was found. However, second-site mutations were not compensatory in the absence of p38, which is also the TCV silencing suppressor, or in dcl-2/dcl4 or ago1/ago2 backgrounds. One second-site mutation reduced silencing suppressor activity of p38 by altering one of two GW motifs that are required for p38 binding to double-stranded RNAs (dsRNAs) and interaction with RNA-induced silencing complex (RISC)-associated AGO1/AGO2. Another second-site mutation substantially reduced accumulation of TCVrev1 in the absence of p38 or DCL2/DCL4. We suggest that the second-site mutations in the p38 ORF exert positive effects through a similar downstream mechanism, either by enhancing accumulation of beneficial DCL-produced viral small RNAs that positively regulate the accumulation of TCV-rev1 or by affecting the susceptibility of TCV-rev1 to RISC loaded with viral small RNAs. IMPORTANCEGenomes of positive-strand RNA viruses fold into high-order RNA structures. Viruses with mutations in regions critical for translation and replication often acquire second-site mutations that exert a positive compensatory effect through reestablishment of canonical base pairing with the altered region. In this study, two distal second-site mutations that individually arose in response to a primary mutation in a critical 3= UTR hairpin in the genomic RNA of turnip crinkle virus did not directly interact with the primary mutation. Although different second-site changes had different attributes, compensation was dependent on the production of the viral p38 silencing suppressor and on the presence of silencing-required DCL and AGO proteins. Our results provide an unexpected connection between a 3= UTR primary-site mutation proposed to disrupt higher-order structure and the RNA-silencing machinery.T he structure of the genomic RNA (gRNA) of positive-strand RNA viruses participates in many basic functions, including replication, translation, and regulation of the competing processes of translation and transcription (1-3). Studies examining the secondary structure and tertiary in...

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confidence: 99%

Requirement for Host RNA-Silencing Components and the Virus-Silencing Suppressor when Second-Site Mutations Compensate for Structural Defects in the 3′ Untranslated Region

Chattopadhyay

Stupina

Gao

et al. 2015

“…Some, but not all, plus-strand RNA viruses contain a substantial number of genome-wide secondary structure elements (genome-scale ordered RNA structure [GORS]) and adopt a compact spheroid shape that correlates with viral persistence (5,6). While RNA viruses likely make extensive use of local and long-distance RNA interactions between such secondary structure elements to control basic processes, only a limited number of such interactions have been reported, with identified connections forming more readily discernible canonical base pairings (3,4,9,10,12,13,14,15,23,26,28,40,41).…”

Section: Discussionmentioning

confidence: 99%

“…For dengue virus (DENV), several sets of overlapping 5=-and 3=-interacting sequences have been identified that control the balance between linear and circular forms of the genome, which is critical for replication but not translation (13,38). Tomato bushy stunt virus (TBSV) requires a complex network of long-distance RNA-RNA interactions to promote replication, subgenomic RNA (sgRNA) synthesis, translation initiation, and translational recoding (4,14,20,40). For both examples, however, only canonical WatsonCrick interactions have been elucidated.…”

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confidence: 99%

A Local, Interactive Network of 3′ RNA Elements Supports Translation and Replication of Turnip Crinkle Virus

Yuan

Shi

Simon

2012

The majority of the 3= untranslated region (UTR) of Turnip crinkle virus (TCV) was previously identified as forming a highly interactive structure with a ribosome-binding tRNA-shaped structure (TSS) acting as a scaffold and undergoing a widespread conformational shift upon binding to RNA-dependent RNA polymerase (RdRp). Tertiary interactions in the region were explored by identifying two highly detrimental mutations within and adjacent to a hairpin H4 upstream of the TSS that reduce translation in vivo and cause identical structural changes in the loop of the 3= terminal hairpin Pr. Second-site changes that compensate for defects in translation/accumulation and reverse the structural differences in the Pr loop were found in the Pr stem, as well as in a specific stem within the TSS and within the capsid protein (CP) coding region, suggesting that the second-site changes were correcting a conformational defect and not restoring specific base pairing. The RdRp-mediated conformational shift extended upstream through this CP open reading frame (ORF) region after bypassing much of an intervening, largely unstructured region, supporting a connection between 3= elements and coding region elements. These data suggest that the Pr loop, TSS, and H4 are central elements in the regulation of translation and replication in TCV and allow for development of an RNA interactome that maps the higher-order structure of a postulated RNA domain within the 3= region of a plus-strand RNA virus.T he genomes of positive-strand RNA viruses control fundamental processes such as translation and replication through multiple RNA elements that interact dynamically with each other and with viral and host proteins. Upon entry into host cells, the viral genomic RNA (gRNA) is recognized as a template by the host translational apparatus for production of replication-associated proteins, which combine with an increasingly diverse variety of host factors to synthesize negative-strand RNAs that then serve as the templates for synthesis of progeny positive-strand RNAs (1,7,8,19). The widespread positioning of cis-elements that function in translation and/or replication requires short-range or long-range bridges within the RNA to deliver bound elements to locations where the specific processes initiate.Understanding how functional, dynamic RNA structures regulate viral processes requires a detailed knowledge of the topology of important regions of the RNA genome and the canonical and noncanonical tertiary interactions that connect various elements. Attempts to decipher networks of short-and long-distance RNA-RNA interactions have been limited to a few viruses. For dengue virus (DENV), several sets of overlapping 5=-and 3=-interacting sequences have been identified that control the balance between linear and circular forms of the genome, which is critical for replication but not translation (13,38). Tomato bushy stunt virus (TBSV) requires a complex network of long-distance RNA-RNA interactions to promote replication, subgenomic RNA (sgRNA) synthesis, tr...

“…Tombusviruses are among the best-characterized plus-strand RNA plant viruses (6), and a great deal has been learned about viral RNA synthesis in terms of both the RNA elements required and the protein factors involved (8)(9)(10)(11)(12). The prototype tombusvirus is tomato bushy stunt virus (TBSV), which encodes two viral proteins critical for viral RNA synthesis.…”

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confidence: 99%

Conserved Motifs in a Tombusvirus Polymerase Modulate Genome Replication, Subgenomic Transcription, and Amplification of Defective Interfering RNAs

Gunawardene

Jaluba

White

2015

Self Cite

The replication of plus-strand RNA virus genomes is mediated by virally encoded RNA-dependent RNA polymerases (RdRps). We have investigated the role of the C-proximal region in the RdRp of tomato bushy stunt virus (TBSV) in mediating viral RNA synthesis. TBSV is the prototype species in the genus Tombusvirus, family Tombusviridae, and its RdRp is responsible for replicating the viral genome, transcribing two subgenomic mRNAs, and supporting replication of defective interfering RNAs. Comparative sequence analysis of the RdRps of tombusvirids identified three highly conserved motifs in their C-proximal regions, and these sequences were subsequently targeted for mutational analysis in TBSV. The results revealed that these motifs are important for (i) synthesizing viral genomic RNA and subgenomic mRNAs, (ii) facilitating plus-and/or minus-strand synthesis, and (iii) modulating trans-replication of a defective interfering RNA. These motifs were also found to be conserved in other plant viruses as well as in a fungal and insect virus. The collective findings are discussed in relation to viral RNA synthesis and taxonomy. IMPORTANCELittle is currently known about the structure and function of the viral polymerases that replicate the genomes of RNA plant viruses. Tombusviruses, the prototype of the tombusvirids, have been used as model plus-strand RNA plant viruses for understanding many of the steps in the infectious process; however, their polymerases remain poorly characterized. To help address this issue, the function of the C-terminal region of the polymerase of a tombusvirus was investigated. Three conserved motifs were identified and targeted for mutational analysis. The results revealed that these polymerase motifs are important for determining what type of viral RNA is produced, facilitating different steps in viral RNA production, and amplifying subgenomic RNA replicons. Accordingly, the C-terminal region of the tombusvirus polymerase is needed for a variety of fundamental activities. Furthermore, as these motifs are also present in distantly related viruses, the significance of these results extends beyond tombusvirids. Replication of the genomes of plus-strand RNA viruses is mediated by virally encoded RNA-dependent RNA polymerases (RdRps) (1). RdRps are translated shortly after infection and associate with other viral or host proteins to assemble into RNA replication complexes (RCs) (2). These RCs are responsible for synthesizing complementary minus strands of the genomes that are then used as the templates for production of plus-strand progeny genomes (2). In some viruses, these RCs are also responsible for synthesizing viral subgenomic (sg) mRNAs that are required for expression of other viral proteins (3). Viral RdRps of animal viruses have been the focus of many functional studies, and there are atomic structures available for several viruses (4). In contrast, much less is known about the RdRps of plant viruses (5).The genus Tombusvirus (6) is the prototype member of the family Tombusviridae, whi...