Plant Positive-strand RNA Viruses Research Articles

Novel 3' Proximal Replication Elements in Umbravirus Genomes.

The 3' untranslated regions (UTRs) of positive-strand RNA plant viruses commonly contain elements that promote viral replication and translation. The ~700 nt 3'UTR of umbravirus pea enation mosaic virus 2 (PEMV2) contains three 3' cap-independent translation enhancers (3'CITEs), including one (PTE) found in members of several genera in the family Tombusviridae and another (the 3'TSS) found in numerous umbraviruses and several carmoviruses. In addition, three 3' terminal replication elements are found in nearly every umbravirus and carmovirus. For this report, we have identified a set of three hairpins and a putative pseudoknot, collectively termed "Trio", that are exclusively found in a subset of umbraviruses and are located just upstream of the 3'TSS. Modification of these elements had no impact on viral translation in wheat germ extracts or in translation of luciferase reporter constructs in vivo. In contrast, Trio hairpins were critical for viral RNA accumulation in Arabidopsis thaliana protoplasts and for replication of a non-autonomously replicating replicon using a trans-replication system in Nicotiana benthamiana leaves. Trio and other 3' terminal elements involved in viral replication are highly conserved in umbraviruses possessing different classes of upstream 3'CITEs, suggesting conservation of replication mechanisms among umbraviruses despite variation in mechanisms for translation enhancement.

Turnip Mosaic Virus Transcriptional Slippage Dynamics and Distribution in RNA Subpopulations.

Potyviruses comprise the largest and most important group of plant positive-strand RNA viruses. The potyviral cell-to-cell movement protein P3N-PIPO is expressed via transcriptional slippage at a conserved GAAAAAA sequence, leading to insertion of an extra 'A' in a proportion of viral transcripts. Transcriptional slippage is determined by the potyviral replicase, the conserved slippery site, and its flanking nucleotides. Here, we investigate the dynamics of transcriptional slippage at different slip-site sequences, infection stages, and environmental conditions. We detect a modest increase in the level of transcripts with insertion towards later timepoints. In addition, we investigate the fate of transcripts with insertion by separately looking at different RNA subpopulations: (+)RNA, (-)RNA, translated RNA, and virion RNA. We find differences in insertional slippage between (+)RNA and (-)RNA but not other subpopulations. Our results suggest that there can be selection against the use of (-)RNAs with insertions as templates for transcription or replication and demonstrate that insertional slippage can occur at high frequency also during (-)RNA synthesis. Since transcripts with insertions are potential targets for degradation, we investigate the connection to nonsense-mediated decay (NMD). We find that these transcripts are targeted to NMD, but we only observe an impact on the level of transcripts with insertion when the insertional slippage rate is high. Together, these results further our understanding of the mechanism and elucidate the dynamics of potyviral transcriptional slippage. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Host-Induced Gene Silencing Using BPMV on Soybean to Study Genes in the Soybean Rust Fungus Phakopsora pachyrhizi.

To obtain direct evidence for the influence of an effector on the virulence or pathogenicity of a pathogen, it is necessary to knock out, knock down, or silence the respective gene. Since genetic transformation is not yet possible for rust fungi, silencing the gene is the only option. Posttranscriptional gene silencing uses RNAi. RNAi in plant pathogens can be accomplished by introducing dsRNA either by direct application of in vitro synthesized dsRNA or through positive-strand or double-strand RNA plant viruses. For studying effectors in Phakopsora pachyrhizi, we have implemented a host-induced silencing procedure based on virus-induced gene silencing using the bean pod mottle virus system. Here, procedures and interpretations of results are described and limitations of the system are discussed.

Modulation of disease severity by plant positive-strand RNA viruses: The complex interplay of multifunctional viral proteins, subviral RNAs and virus-associated RNAs with plant signaling pathways and defense responses.

Plant viruses induce a range of symptoms of varying intensity, ranging from severe systemic necrosis to mild or asymptomatic infection. Several evolutionary constraints drive virus virulence, including the dependence of viruses on host factors to complete their infection cycle, the requirement to counteract or evade plant antiviral defense responses and the mode of virus transmission. Viruses have developed an array of strategies to modulate disease severity. Accumulating evidence has highlighted not only the multifunctional role that viral proteins play in disrupting or highjacking plant factors, hormone signaling pathways and intracellular organelles, but also the interaction networks between viral proteins, subviral RNAs and/or other viral-associated RNAs that regulate disease severity. This review focusses on positive-strand RNA viruses, which constitute the majority of characterized plant viruses. Using well-characterized viruses with different genome types as examples, recent advances are discussed as well as knowledge gaps and opportunities for further research.

Structural and Functional Diversity of Plant Virus 3'-Cap-Independent Translation Enhancers (3'-CITEs).

Most of the positive-strand RNA plant viruses lack the 5′-cap and/or the poly(A)-tail that act synergistically to stimulate canonical translation of cellular mRNAs. However, they have RNA elements in the 5′- or 3′-untranslated regions of their RNAs that are required for their cap-independent translation. Cap-independent translation enhancers (CITEs) have been identified in the genomic 3′-end of viruses belonging to the family Tombusviridae and the genus Luteovirus. Seven classes of 3′-CITEs have been described to date based on their different RNA structures. They generally control the efficient formation of the translation initiation complex by varying mechanisms. Some 3′-CITEs bind eukaryotic translation initiation factors, others ribosomal subunits, bridging these to the 5′-end by different mechanisms, often long-distance RNA–RNA interactions. As previously proposed and recently found in one case in nature, 3′-CITEs are functionally independent elements that are transferable through recombination between viral genomes, leading to potential advantages for virus multiplication. In this review, the knowledge on 3′-CITEs and their functioning is updated. We also suggest that there is local structural conservation in the regions interacting with eIF4E of 3′-CITEs belonging to different classes.

Plant Virus Infection and the Ubiquitin Proteasome Machinery: Arms Race along the Endoplasmic Reticulum.

The endoplasmic reticulum (ER) is central to plant virus replication, translation, maturation, and egress. Ubiquitin modification of ER associated cellular and viral proteins, alongside the actions of the 26S proteasome, are vital for the regulation of infection. Viruses can arrogate ER associated ubiquitination as well as cytosolic ubiquitin ligases with the purpose of directing the ubiquitin proteasome system (UPS) to new targets. Such targets include necessary modification of viral proteins which may stabilize certain complexes, or modification of Argonaute to suppress gene silencing. The UPS machinery also contributes to the regulation of effector triggered immunity pattern recognition receptor immunity. Combining the results of unrelated studies, many positive strand RNA plant viruses appear to interact with cytosolic Ub-ligases to provide novel avenues for controlling the deleterious consequences of disease. Viral interactions with the UPS serve to regulate virus infection in a manner that promotes replication and movement, but also modulates the levels of RNA accumulation to ensure successful biotrophic interactions. In other instances, the UPS plays a central role in cellular immunity. These opposing roles are made evident by contrasting studies where knockout mutations in the UPS can either hamper viruses or lead to more aggressive diseases. Understanding how viruses manipulate ER associated post-translational machineries to better manage virus–host interactions will provide new targets for crop improvement.

Ins and Outs of Multipartite Positive-Strand RNA Plant Viruses: Packaging versus Systemic Spread.

Viruses possessing a non-segmented genome require a specific recognition of their nucleic acid to ensure its protection in a capsid. A similar feature exists for viruses having a segmented genome, usually consisting of viral genomic segments joined together into one viral entity. While this appears as a rule for animal viruses, the majority of segmented plant viruses package their genomic segments individually. To ensure a productive infection, all viral particles and thereby all segments have to be present in the same cell. Progression of the virus within the plant requires as well a concerted genome preservation to avoid loss of function. In this review, we will discuss the “life aspects” of chosen phytoviruses and argue for the existence of RNA-RNA interactions that drive the preservation of viral genome integrity while the virus progresses in the plant.

Tombusvirus-Host Interactions: Co-Opted Evolutionarily Conserved Host Factors Take Center Court.

Plant positive-strand (+)RNA viruses are intracellular infectious agents that reorganize subcellular membranes and rewire the cellular metabolism of host cells to achieve viral replication in elaborate replication compartments. This review describes the viral replication process based on tombusviruses, highlighting common strategies with other plant and animal viruses. Overall, the works on Tomato bushy stunt virus (TBSV) have revealed intriguing and complex functions of co-opted cellular translation factors, heat shock proteins, DEAD-box helicases, lipid transfer proteins, and membrane-deforming proteins in virus replication. The emerging picture is that many of the co-opted host factors are from highly expressed and conserved protein families. By hijacking host proteins, phospholipids, sterols, and the actin network, TBSV exerts supremacy over the host cell to support viral replication in large replication compartments. Altogether, these advances in our understanding of tombusvirus-host interactions are broadly applicable to many other viruses, which also usurp conserved host factors for various viral processes.

Efficient Translation of Pelargonium line pattern virus RNAs Relies on a TED-Like 3´-Translational Enhancer that Communicates with the Corresponding 5´-Region through a Long-Distance RNA-RNA Interaction.

Cap-independent translational enhancers (CITEs) have been identified at the 3´-terminal regions of distinct plant positive-strand RNA viruses belonging to families Tombusviridae and Luteoviridae. On the bases of their structural and/or functional requirements, at least six classes of CITEs have been defined whose distribution does not correlate with taxonomy. The so-called TED class has been relatively under-studied and its functionality only confirmed in the case of Satellite tobacco necrosis virus, a parasitic subviral agent. The 3´-untranslated region of the monopartite genome of Pelargonium line pattern virus (PLPV), the recommended type member of a tentative new genus (Pelarspovirus) in the family Tombusviridae, was predicted to contain a TED-like CITE. Similar CITEs can be anticipated in some other related viruses though none has been experimentally verified. Here, in the first place, we have performed a reassessment of the structure of the putative PLPV-TED through in silico predictions and in vitro SHAPE analysis with the full-length PLPV genome, which has indicated that the presumed TED element is larger than previously proposed. The extended conformation of the TED is strongly supported by the pattern of natural sequence variation, thus providing comparative structural evidence in support of the structural data obtained by in silico and in vitro approaches. Next, we have obtained experimental evidence demonstrating the in vivo activity of the PLPV-TED in the genomic (g) RNA, and also in the subgenomic (sg) RNA that the virus produces to express 3´-proximal genes. Besides other structural features, the results have highlighted the key role of long-distance kissing-loop interactions between the 3´-CITE and 5´-proximal hairpins for gRNA and sgRNA translation. Bioassays of CITE mutants have confirmed the importance of the identified 5´-3´ RNA communication for viral infectivity and, moreover, have underlined the strong evolutionary constraints that may operate on genome stretches with both regulatory and coding functions.

Cell-Free and Cell-Based Approaches to Explore the Roles of Host Membranes and Lipids in the Formation of Viral Replication Compartment Induced by Tombusviruses.

Plant positive strand RNA viruses are intracellular infectious agents that take advantage of cellular lipids and membranes to support replication and protect viral RNA from degradation by host antiviral responses. In this review, we discuss how Tomato bushy stunt virus (TBSV) co-opts lipid transfer proteins and modulates lipid metabolism and transport to facilitate the assembly of the membrane-bound viral replicase complexes within intricate replication compartments. Identification and characterization of the proviral roles of specific lipids and proteins involved in lipid metabolism based on results from yeast (Saccharomyces cerevisiae) model host and cell-free approaches are discussed. The review also highlights the advantage of using liposomes with chemically defined composition to identify specific lipids required for TBSV replication. Remarkably, all the known steps in TBSV replication are dependent on cellular lipids and co-opted membranes.

Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis

Reverse genetics systems have been established for all major groups of plant DNA and positive-strand RNA viruses, and our understanding of their infection cycles and pathogenesis has benefitted enormously from use of these approaches. However, technical difficulties have heretofore hampered applications of reverse genetics to plant negative-strand RNA (NSR) viruses. Here, we report recovery of infectious virus from cloned cDNAs of a model plant NSR, Sonchus yellow net rhabdovirus (SYNV). The procedure involves Agrobacterium-mediated transcription of full-length SYNV antigenomic RNA and co-expression of the nucleoprotein (N), phosphoprotein (P), large polymerase core proteins and viral suppressors of RNA silencing in Nicotiana benthamiana plants. Optimization of core protein expression resulted in up to 26% recombinant SYNV (rSYNV) infections of agroinfiltrated plants. A reporter virus, rSYNV-GFP, engineered by inserting a green fluorescence protein (GFP) gene between the N and P genes was able to express GFP during systemic infections and after repeated plant-to-plant mechanical passages. Deletion analyses with rSYNV-GFP demonstrated that SYNV cell-to-cell movement requires the sc4 protein and suggested that uncoiled nucleocapsids are infectious movement entities. Deletion analyses also showed that the glycoprotein is not required for systemic infection, although the glycoprotein mutant was defective in virion morphogenesis. Taken together, we have developed a robust reverse genetics system for SYNV that provides key insights into morphogenesis and movement of an enveloped plant virus. Our study also provides a template for developing analogous systems for reverse genetic analysis of other plant NSR viruses.

Tobacco etch virus protein P1 traffics to the nucleolus and associates with the host 60S ribosomal subunits during infection.

The genus Potyvirus comprises a large group of positive-strand RNA plant viruses whose genome encodes a large polyprotein processed by three viral proteinases. P1 protein, the most amino-terminal product of the polyprotein, is an accessory factor stimulating viral genome amplification whose role during infection is not well understood. We infected plants with Tobacco etch virus (TEV; genus Potyvirus) clones in which P1 was tagged with a fluorescent protein to track its expression and subcellular localization or with an affinity tag to identify host proteins involved in complexes in which P1 also takes part during infection. Our results showed that TEV P1 exclusively accumulates in infected cells at an early stage of infection and that the protein displays a dynamic subcellular localization, trafficking in and out of the nucleus and nucleolus during infection. Inside the nucleolus, P1 particularly targets the dense granular component. Consistently, we found functional nucleolar localization and nuclear export signals in TEV P1 sequence. Our results also indicated that TEV P1 physically interacts with the host 80S cytoplasmic ribosomes and specifically binds to the 60S ribosomal subunits during infection. In vitro translation assays of reporter proteins suggested that TEV P1 stimulates protein translation, particularly when driven from the TEV internal ribosome entry site. These in vitro assays also suggested that TEV helper-component proteinase (HC-Pro) inhibits protein translation. Based on these findings, we propose that TEV P1 stimulates translation of viral proteins in infected cells. In this work, we researched the role during infection of tobacco etch virus P1 protease. P1 is the most mysterious protein of potyviruses, a relevant group of RNA viruses infecting plants. Our experiments showed that the viral P1 protein exclusively accumulates in infected cells at an early stage of infection and moves in and out of the nucleus of infected cells, particularly targeting the nucleolus. Our experiments also showed that P1 protein binds host ribosomes during infection. Based on these findings and other in vitro experiments we propose that P1 protein stimulates translation of viral proteins during infection.

Virus infection cycle events coupled to RNA replication.

Replication, the process by which the genetic material of a virus is copied to generate multiple progeny genomes, is the central part of the virus infection cycle. For an infection to be productive, it is essential that this process is coordinated with other aspects of the cycle, such as translation of the viral genome, encapsidation, and movement of the genome between cells. In the case of positive-strand RNA viruses, this represents a particular challenge, as the infecting genome must not only be replicated but also serve as an mRNA for the production of the replication-associated proteins. In recent years, it has become apparent that in positive-strand RNA plant viruses all the aspects of the infection cycle are intertwined. This article reviews the current state of knowledge regarding replication-associated events in such viruses.

Host factors used by positive-strand RNA plant viruses for genome replication

Replication of positive-strand RNA [(+)RNA] viruses proceeds through well-orchestrated actions of both viral and host factors. Remarkable features of eukaryotic (+)RNA virus replication include hijacking of host factors by viral components and remodeling of intracellular membranes to establish the viral replication factory, where viral RNA is synthesized. Here we review recent progress in our understanding of how (+)RNA plant viruses use host factors to create favorable environments for viral RNA replication.

3′ Cap-Independent Translation Enhancers of Plant Viruses

In the absence of a 5' cap, plant positive-strand RNA viruses have evolved a number of different elements in their 3' untranslated region (UTR) to attract initiation factors and/or ribosomes to their templates. These 3' cap-independent translational enhancers (3' CITEs) take different forms, such as I-shaped, Y-shaped, T-shaped, or pseudoknotted structures, or radiate multiple helices from a central hub. Common features of most 3' CITEs include the ability to bind a component of the translation initiation factor eIF4F complex and to engage in an RNA-RNA kissing-loop interaction with a hairpin loop located at the 5' end of the RNA. The two T-shaped structures can bind to ribosomes and ribosomal subunits, with one structure also able to engage in a simultaneous long-distance RNA-RNA interaction. Several of these 3' CITEs are interchangeable and there is evidence that natural recombination allows exchange of modular CITE units, which may overcome genetic resistance or extend the virus's host range.

Crystal Structure of the Superfamily 1 Helicase from Tomato Mosaic Virus

The genomes of the Tomato mosaic virus and many other plant and animal positive-strand RNA viruses of agronomic and medical importance encode superfamily 1 helicases. Although helicases play important roles in viral replication, the crystal structures of viral superfamily 1 helicases have not been determined. Here, we report the crystal structure of a fragment (S666 to Q1116) of the replication protein from Tomato mosaic virus. The structure reveals a novel N-terminal domain tightly associated with a helicase core. The helicase core contains two RecA-like α/β domains without any of the accessory domain insertions that are found in other superfamily 1 helicases. The N-terminal domain contains a flexible loop, a long α-helix, and an antiparallel six-stranded β-sheet. On the basis of the structure, we constructed deletion mutants of the S666-to-Q1116 fragment and performed split-ubiquitin-based interaction assays in Saccharomyces cerevisiae with TOM1 and ARL8, host proteins that are essential for tomato mosaic virus RNA replication. The results suggested that both TOM1 and ARL8 interact with the long α-helix in the N-terminal domain and that TOM1 also interacts with the helicase core. Prediction of secondary structures in other viral superfamily 1 helicases and comparison of those structures with the S666-to-Q1116 structure suggested that these helicases have a similar fold. Our results provide a structural basis of viral superfamily 1 helicases.

Role of tRNA-like structures in controlling plant virus replication

Transfer RNA-like structures (TLSs) that are sophisticated functional mimics of tRNAs are found at the 3′-termini of the genomes of a number of plant positive strand RNA viruses. Three natural aminoacylation identities are represented: valine, histidine, and tyrosine. Paralleling this variety in structure, the roles of TLSs vary widely between different viruses. For Turnip yellow mosaic virus, the TLS must be capable of valylation in order to support infectivity, major roles being the provision of translational enhancement and down-regulation of minus strand initiation. In contrast, valylation of the Peanut clump virus TLS is not essential. An intermediate situation seems to exist for Brome mosaic virus, whose RNAs 1 and 2, but not RNA 3, need to be capable of tyrosylation to support infectivity. Other known roles for certain TLSs include: (i) the recruitment of host CCA nucleotidyltransferase as a telomerase to maintain intact 3′ CCA termini, (ii) involvement in the encapsidation of viral RNAs, and (iii) presentation of minus strand promoter elements for replicase recognition. In the latter role, the promoter elements reside within the TLS but are not functionally dependent on tRNA mimicry. The phylogenetic distribution of TLSs indicates that their evolutionary history includes frequent horizontal exchange, as has been observed for protein-coding regions of plant positive strand RNA viruses.

Subgenomic mRNA transcription in an aureusvirus: Down-regulation of transcription and evolution of regulatory RNA elements

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.

Cloning of Large Positive‐Strand RNA Viruses

Full-length, biologically active cDNA clones of the positive-strand RNA plant viruses are indispensable for investigating the functions of viral genes and control elements as well as generating virus-derived gene expression and silencing vectors. Even though engineering of such clones for 4- to 10-kb viral RNAs has become routine, it remains a challenging task for 15- to 20-kb RNA genomes of the monopartite viruses in a family Closteroviridae. This unit describes strategic considerations and techniques used to generate an infectious cDNA clone of a closterovirus. The use of agroinfection to improve specific infectivity of the resulting clone is also explained.

Identification of a Ribonucleoprotein Intermediate of Tomato Mosaic Virus RNA Replication Complex Formation

The replication of eukaryotic positive-strand RNA virus genomes occurs in the membrane-bound RNA replication complexes. Previously, we found that the extract of evacuolated tobacco BY-2 protoplasts (BYL) is capable of supporting the translation and subsequent replication of the genomic RNAs of plant positive-strand RNA viruses, including Tomato mosaic virus (ToMV). Here, to dissect the process that precedes the formation of ToMV RNA replication complexes, we prepared membrane-depleted BYL (mdBYL), in which the membranes were removed by centrifugation. In mdBYL, ToMV RNA was translated to produce the 130-kDa and 180-kDa replication proteins, but the synthesis of any ToMV-related RNAs did not occur. When BYL membranes were added back to the ToMV RNA-translated mdBYL after the termination of translation with puromycin, ToMV RNA was replicated. Using a replication-competent ToMV derivative that encodes the FLAG-tagged 180-kDa replication protein, it was shown by affinity purification that a complex that contained the 130-kDa and 180-kDa proteins and ToMV genomic RNA was formed after translation in mdBYL. When the complex was mixed with BYL membranes, ToMV RNA was replicated, which suggests that this ribonucleoprotein complex is an intermediate of ToMV RNA replication complex formation. We have named this ribonucleoprotein complex the "pre-membrane-targeting complex." Our data suggest that the formation of the pre-membrane-targeting complex is coupled with the translation of ToMV RNA, while posttranslationally added exogenous 180-kDa protein and replication templates can contribute to replication and can be replicated, respectively. Based on these results, we discuss the mechanisms of ToMV RNA replication complex formation.