Characterization of Variant RNAs Encapsidated during Bromovirus Infection by High-Throughput Sequencing.

Previously, we described the RNA recombinants accumulating in tissues infected with the bromoviruses BMV (Brome mosaic virus) and CCMV (Cowpea chlorotic mottle virus). In this work, we characterize the recombinants encapsidated inside the purified virion particles of BMV and CCMV. By using a tool called the Viral Recombination Mapper (ViReMa) that detects recombination junctions, we analyzed a high number of high-throughput sequencing (HTS) short RNA sequence reads. Over 28% of BMV or CCMV RNA reads did not perfectly map to the viral genomes. ViReMa identified 1.40% and 1.83% of these unmapped reads as the RNA recombinants, respectively, in BMV and CCMV. Intra-segmental crosses were more frequent than the inter-segmental ones. Most intra-segmental junctions carried short insertions/deletions (indels) and caused frameshift mutations. The mutation hotspots clustered mainly within the open reading frames. Substitutions of various lengths were also identified, whereas a small fraction of crosses occurred between viral and their host RNAs. Our data reveal that the virions can package detectable amounts of multivariate recombinant RNAs, contributing to the flexible nature of the viral genomes.

Variability among the Isolates of Broad Bean Mottle Virus and Encapsidation of Host RNAs.

Broad bean mottle bromovirus infects legume plants and is transmissible by insects. Several broad bean mottle virus (BBMV) isolates have been identified, including one in England (isolate Ba) and five in the Mediterranean countries: Libya (LyV), Morocco (MV), Syria (SV), Sudan (TU) and Tunisia (TV). Previously, we analyzed the nucleotide sequence of the Ba RNA and here we report on and compare it with another five Mediterranean variants. The RNA segments in the latter ones were extensively homologous, with some SNPs, single nucleotide deletions and insertions, while the number of mutations was higher in isolate Ba. Both the 5' and 3' untranslated terminal regions (UTRs) among the corresponding RNAs are highly conserved, reflecting their functionality in virus replication. The AUG initiation codons are within suboptimal contexts, possibly to adjust/regulate translation. The proteins 1a, 2a, 3a and coat protein (CP) are almost identical among the five isolates, but in Ba they have more amino acid (aa) substitutions. Phylogenetic analysis revealed the isolates from Morocco and Syria clustering with the isolate from England, while the variants from Libya, Tunisia and Sudan created a different clade. The BBMV isolates encapsidate a high content of host (ribosomal and messenger) RNAs. Our studies present BBMV as a useful model for bromoviruses infecting legumes.

Modulation of Expression of PVYNTN RNA-Dependent RNA Polymerase (NIb) and Heat Shock Cognate Host Protein HSC70 in Susceptible and Hypersensitive Potato Cultivars.

Potato virus Y (PVY) belongs to the genus Potyvirus and is considered to be one of the most harmful and important plant pathogens. Its RNA-dependent RNA polymerase (RdRp) is known as nuclear inclusion protein b (NIb). The recent findings show that the genome of PVY replicates in the cytoplasm of the plant cell by binding the virus replication complex to the membranous structures of different organelles. In some potyviruses, NIb has been found to be localized in the nucleus and associated with the endoplasmic reticulum membranes. Moreover, NIb has been shown to interact with other host proteins that are particularly involved in promoting the virus infection cycle, such as the heat shock proteins (HSPs). HSP70 is the most conserved among the five major HSP families that are known to affect the plant-pathogen interactions. Some plant viruses can induce the production of HSP70 during the development of infection. To understand the molecular mechanisms underlying the interactive response to PVYNTN (necrotic tuber necrosis strain of PVY), the present study focused on StHSC70-8 and PVYNTN-NIb gene expression via localization of HSC70 and NIb proteins during compatible (susceptible) and incompatible (hypersensitive) potato-PVYNTN interactions. Our results demonstrate that NIb and HSC70 are involved in the response to PVYNTN infections and probably cooperate at some stages of the virus infection cycle. Enhanced deposition of HSC70 proteins during the infection cycle was associated with the dynamic induction of PVYNTN-NIb gene expression and NIb localization during susceptible infections. In hypersensitive response (HR), a significant increase in HSC70 expression was observed up to 3 days post-inoculation (dpi) in the nucleus and chloroplasts. Thereafter, between 3 and 21 dpi, the deposition of NIb decreased, which can be attributed to a reduction in the levels of both virus accumulation and PVYNTN-NIb gene expression. Therefore, we postulate that increase in the expression of both StHSC70-8 and PVYNTN-NIb induces the PVY infection during susceptible infections. In contrast, during HRs, HSC70 cooperates with PVYNTN only at the early stages of interaction and mediates the defense response signaling pathway at the later stages of infection.

Long Noncoding RNAs in Plant Viroids and Viruses: A Review.

Infectious long-noncoding (lnc) RNAs related to plants can be of both viral and non-viral origin. Viroids are infectious plant lncRNAs that are not related to viruses and carry the circular, single-stranded, non-coding RNAs that replicate with host enzymatic activities via a rolling circle mechanism. Viroids interact with host processes in complex ways, emerging as one of the most productive tools for studying the functions of lncRNAs. Defective (D) RNAs, another category of lnc RNAs, are found in a variety of plant RNA viruses, most of which are noncoding. These are derived from and are replicated by the helper virus. D RNA-virus interactions evolve into mutually beneficial combinations, enhancing virus fitness via competitive advantages of moderated symptoms. Yet the satellite RNAs are single-stranded and include either large linear protein-coding ss RNAs, small linear ss RNAs, or small circular ss RNAs (virusoids). The satellite RNAs lack sequence homology to the helper virus, but unlike viroids need a helper virus to replicate and encapsidate. They can attenuate symptoms via RNA silencing and enhancement of host defense, but some can be lethal as RNA silencing suppressor antagonists. Moreover, selected viruses produce lncRNAs by incomplete degradation of genomic RNAs. They do not replicate but may impact viral infection, gene regulation, and cellular functions. Finally, the host plant lncRNAs can also contribute during plant-virus interactions, inducing plant defense and the regulation of gene expression, often in conjunction with micro and/or circRNAs.

Modifications in Tissue and Cell Ultrastructure as Elements of Immunity-Like Reaction in Chenopodium quinoa against Prune Dwarf Virus (PDV).

Prune dwarf virus (PDV) is a plant RNA viral pathogen in many orchard trees worldwide. Our knowledge about resistance genes or resistant reactions of plant hosts to PDV is scant. To fill in part of this gap, an aim of this study was to investigate reactions to PDV infection in a model host, Chenopodium quinoa. Our investigations concentrated on morphological and ultrastructural changes after inoculation with PDV strain 0599. It turned out that PDV infection can cause deformations in host cells but also induce changes in the organelles, such as chloroplasts in inoculated leaves. Moreover, we also demonstrated specific reactions/changes, which could be associated with both types of vascular tissue capable of effectively blocking the systemic spread of PDV to upper leaves. Furthermore, the relative amount of virus, P1 protein deposition, and movement protein (MP) gene expression consequently decreased in PDV-inoculated leaves.

The Expression of Potato Expansin A3 (StEXPA3) and Extensin4 (StEXT4) Genes with Distribution of StEXPAs and HRGPs-Extensin Changes as an Effect of Cell Wall Rebuilding in Two Types of PVYNTN-Solanum tuberosum Interactions.

The plant cell wall acts not only as a physical barrier, but also as a complex and dynamic structure that actively changes under different biotic and abiotic stress conditions. The question is, how are the different cell wall compounds modified during different interactions with exogenous stimuli such as pathogens? Plants exposed to viral pathogens respond to unfavorable conditions on multiple levels. One challenge that plants face under viral stress is the number of processes required for differential cell wall remodeling. The key players in these conditions are the cell wall genes and proteins, which can be regulated in specific ways during the interactions and have direct influences on the rebuilding of the cell wall structure. The cell wall modifications occurring in plants during viral infection remain poorly described. Therefore, this study focuses on cell wall dynamics as an effect of incompatible interactions between the potato virus Y (PVYNTN) and resistant potatoes (hypersensitive plant), as well as compatible (susceptible plant) interactions. Our analysis describes, for the first time, the expression of the potato expansin A3 (StEXPA3) and potato extensin 4 (StEXT4) genes in PVYNTN-susceptible and -resistant potato plant interactions. The results indicated a statistically significant induction of the StEXPA3 gene during a susceptible response. By contrast, we demonstrated the predominantly gradual activation of the StEXT4 gene during the hypersensitive response to PVYNTN inoculation. Moreover, the in situ distributions of expansins (StEXPAs), which are essential cell wall-associated proteins, and the hydroxyproline-rich glycoprotein (HRGP) extensin were investigated in two types of interactions. Furthermore, cell wall loosening was accompanied by an increase in StEXPA deposition in a PVYNTN-susceptible potato, whereas the HRGP content dynamically increased during the hypersensitive response, when the cell wall was reinforced. Ultrastructural localization and quantification revealed that the HRGP extensin was preferably located in the apoplast, but deposition in the symplast was also observed in resistant plants. Interestingly, during the hypersensitive response, StEXPA proteins were mainly located in the symplast area, in contrast to the susceptible potato where StEXPA proteins were mainly observed in the cell wall. These findings revealed that changes in the intracellular distribution and abundance of StEXPAs and HRGPs can be differentially regulated, depending on different types of PVYNTN-potato plant interactions, and confirmed the involvement of apoplast and symplast activation as a defense response mechanism.

Spatiotemporal Changes in Xylan-1/Xyloglucan and Xyloglucan Xyloglucosyl Transferase (XTH-Xet5) as a Step-In of Ultrastructural Cell Wall Remodelling in Potato⁻Potato Virus Y (PVYNTN) Hypersensitive and Susceptible Reaction.

One type of monitoring system in a plant cell is the cell wall, which intensively changes its structure during interaction with pathogen-stress factors. The wall plays a role as a dynamic and controlled structure, although it is not fully understood how relevant these modifications are to the molecular mechanisms during plant⁻virus interactions. In this work we localise the non-cellulosic polysaccharides such as xyloglucan, xylan (xylan-1) and xyloglucosyl transferase (XTH-Xet5), the enzyme that participates in the metabolism of xyloglucan. This provided us with information about the in situ distribution of the components of the hemicellulotic cell wall matrix in hypersensitive and susceptible potato⁻PVYNTN interactions. The loosening of the cell wall was accompanied by an increase in xylan depositions during susceptible interactions, whereas, during the hypersensitive response, when the cell wall was reinforced, the xylan content decreased. Moreover, the PVY inoculation significantly redirected XTH-Xet5 depositions, regardless of types of interactions, compared to mock-inoculated tissues. Furthermore, the immunogold localisation clearly revealed the domination of Xet5 in the cell wall and in vesicles in the susceptible host. In contrast, in the resistant host increased levels of Xet5 were observed in cytoplasm, in the cell wall and in the trans-Golgi network. These findings show that the hypersensitive reaction activated XTH-Xet5 in the areas of xyloglucan endo-transglycosylase (XET) synthesis, which was then actively transported to cytoplasm, cell wall and to vacuoles. Our results provide novel insight into cell wall reorganisation during PVYNTN infection as a response to biotic stress factors. These novel findings help us to understand the mechanisms of defence responses that are incorporated into the cell wall signalling network.

Ultrastructural Analysis of Prune DwarfVirus Intercellular Transport and Pathogenesis.

Prune dwarf virus (PDV) is an important viral pathogen of plum, sweet cherry, peach, and many herbaceous test plants. Although PDV has been intensively investigated, mainly in the context of phylogenetic relationship of its genes and proteins, many gaps exist in our knowledge about the mechanism of intercellular transport of this virus. The aim of this work was to investigate alterations in cellular organelles and the cell-to-cell transport of PDV in Cucumis sativus cv. Polan at ultrastructural level. To analyze the role of viral proteins in local transport, double-immunogold assays were applied to localize PDV coat protein (CP) and movement protein (MP). We observe structural changes in chloroplasts, mitochondria, and cellular membranes. We prove that PDV is transported as viral particles via MP-generated tubular structures through plasmodesmata. Moreover, the computer-run 3D modeling reveals structural resemblances between MPs of PDV and of Alfalfa mosaic virus (AMV), implying similarities of transport mechanisms for both viruses.

Molecular Biology of Prune Dwarf Virus-A Lesser Known Member of the Bromoviridae but a Vital Component in the Dynamic Virus-Host Cell Interaction Network.

Prune dwarf virus (PDV) is one of the members of Bromoviridae family, genus Ilarvirus. Host components that participate in the regulation of viral replication or cell-to-cell movement via plasmodesmata are still unknown. In contrast, viral infections caused by some other Bromoviridae members are well characterized. Bromoviridae can be distinguished based on localization of their replication process in infected cells, cell-to-cell movement mechanisms, and plant-specific response reactions. Depending upon the genus, "genome activation" and viral replication are linked to various membranous structures ranging from endoplasmic reticulum, to tonoplast. In the case of PDV, there is still no evidence of natural resistance sources in the host plants susceptible to virus infection. Apparently, PDV has a great ability to overcome the natural defense responses in a wide spectrum of plant hosts. The first manifestations of PDV infection are specific cell membrane alterations, and the formation of replicase complexes that support PDV RNA replication inside the spherules. During each stage of its life cycle, the virus uses cell components to replicate and to spread in whole plants, within the largely suppressed cellular immunity environment. This work presents the above stages of the PDV life cycle in the context of current knowledge about other Bromoviridae members.

Co-infection with two strains of Brome mosaic bromovirus reveals common RNA recombination sites in different hosts.

We have previously reported intra-segmental crossovers in Brome mosaic virus (BMV) RNAs. In this work, we studied the homologous recombination of BMV RNA in three different hosts: barley (Hordeum vulgare), Chenopodium quinoa, and Nicotiana benthamiana that were co-infected with two strains of BMV: Russian (R) and Fescue (F). Our work aimed at (1) establishing the frequency of recombination, (2) mapping the recombination hot spots, and (3) addressing host effects. The F and R nucleotide sequences differ from each other at many translationally silent nucleotide substitutions. We exploited this natural variability to track the crossover sites. Sequencing of a large number of cDNA clones revealed multiple homologous crossovers in each BMV RNA segment, in both the whole plants and protoplasts. Some recombination hot spots mapped at similar locations in different hosts, suggesting a role for viral factors, but other sites depended on the host. Our results demonstrate the chimeric ('mosaic') nature of the BMV RNA genome.

Multiple functions of capsid proteins in (+) stranded RNA viruses during plant-virus interactions.

In addition to providing a protective shell for genomic RNA(s), the coat (capsid) proteins (CPs) of plus-stranded RNA viruses play a variety of other functions that condition the plant-virus relationship. In this review we outline the extensive research progress that has been made within the last decade on those CP characteristics that relate to virus infectivity, pathogenicity, symptom expression, interactions with host factors, virus movement, vector transmission, host range, as well as those used to study virus evolution. By discussing the examples among a variety of plant RNA viruses we show that in addition to general features and pathways, the involvement of CPs may assume very distinct tasks that depend on the particular virus life style. Research perspectives and potential applications are discussed at the end.

Genetic recombination in plant-infecting messenger-sense RNA viruses: overview and research perspectives.

RNA recombination is one of the driving forces of genetic variability in (+)-strand RNA viruses. Various types of RNA-RNA crossovers were described including crosses between the same or different viral RNAs or between viral and cellular RNAs. Likewise, a variety of molecular mechanisms are known to support RNA recombination, such as replicative events (based on internal or end-to-end replicase switchings) along with non-replicative joining among RNA fragments of viral and/or cellular origin. Such mechanisms as RNA decay or RNA interference are responsible for RNA fragmentation and trans-esterification reactions which are likely accountable for ligation of RNA fragments. Numerous host factors were found to affect the profiles of viral RNA recombinants and significant differences in recombination frequency were observed among various RNA viruses. Comparative analyses of viral sequences allowed for the development of evolutionary models in order to explain adaptive phenotypic changes and co-evolving sites. Many questions remain to be answered by forthcoming RNA recombination research. (1) How various factors modulate the ability of viral replicase to switch templates, (2) What is the intracellular location of RNA-RNA template switchings, (3) Mechanisms and factors responsible for non-replicative RNA recombination, (4) Mechanisms of integration of RNA viral sequences with cellular genomic DNA, and (5) What is the role of RNA splicing and ribozyme activity. From an evolutionary stand point, it is not known how RNA viruses parasitize new host species via recombination, nor is it obvious what the contribution of RNA recombination is among other RNA modification pathways. We do not understand why the frequency of RNA recombination varies so much among RNA viruses and the status of RNA recombination as a form of sex is not well documented.

Mutations in the coat protein-binding cis-acting RNA motifs debilitate RNA recombination of Brome mosaic virus.

We have previously described the efficient homologous recombination system between 5' subgenomic RNA3a (sgRNA3a) and genomic RNA3 of Brome mosaic virus (BMV) in barley protoplasts (Sztuba-Solinska et al., 2011a). Here, we demonstrated that sequence alterations in the coat protein (CP)-binding cis-acting RNA motifs, the Bbox region (in the intercistronic RNA3 sequence) and the RNA3 packaging element (PE, in the movement protein ORF), reduced crossover frequencies in protoplasts. Additionally, the modification of Bbox-like element in the 5' UTR region strongly debilitated crossovers. Along the lines of these observations, RNA3 mutants not expressing CP or expressing mutated CPs also reduced recombination. A series of reciprocal transfections demonstrated a functional crosstalk between the Bbox and PE elements. Altogether, our data imply the role of CP in sgRNA3a-directed recombination by either facilitating the interaction of the RNA substrates and/or by creating roadblocks for the viral replicase.

P-bodies and their functions during mRNA cell cycle: mini-review.

P-bodies (processing bodies) are observed in different organisms such as yeast, Caenorhabditis elegans and mammals. A typical eukaryotic cell contains several types of spatially formed granules, such as P-bodies, stress granules and a variety of ribonucleoprotein bodies. These microdomains play important role in mRNA processing, including RNA interference, repression of translation and mRNA decay. The P-bodies components as well as stress granules may play an important role in host defense against viral infection. The complete set of P-bodies protein elements is still poor known. They contain conserved protein core limited to different organisms or to stress status of the cell. P-bodies are related also to some neuronal mRNA granules as well as to maternal RNA granules or male germ cell granules. In this mini-review, we focus on the structure of P-bodies and their function in the mRNA utilization and processing because of the high mRNA's dynamics between different cellular compartments and its key role in modulation of gene expression.

Mutations in the antiviral RNAi defense pathway modify Brome mosaic virus RNA recombinant profiles.

RNA interference (RNAi) mechanism targets viral RNA for degradation. To test whether RNAi gene products contributed to viral RNA recombination, a series of Arabidopsis thaliana RNAi-defective mutants were infected with Brome mosaic virus (BMV) RNAs that have been engineered to support crossovers within the RNA3 segment. Single-cross RNA3-RNA1, RNA3-RNA2, and RNA3-RNA3 recombinants accumulated in both the wild-type (wt) and all knock-out lines at comparable frequencies. However, a reduced accumulation of novel 3' mosaic RNA3 recombinants was observed in ago1, dcl2, dcl4, and rdr6 lines but not in wt Col-0 or the dcl3 line. A BMV replicase mutant accumulated a low level of RNA3-RNA1 single-cross recombinants in Col-0 plants while, in a dcl2 dcl4 double mutant, the formation of both RNA3-RNA1 and mosaic recombinants was at a low level. A control infection in the cpr5-2 mutant, a more susceptible BMV Arabidopsis host, generated similar-to-Col-0 profiles of both single-cross and mosaic recombinants, indicating that recombinant profiles were, to some extent, independent of a viral replication rate. Also, the relative growth experiments revealed similar selection pressure for recombinants among the host lines. Thus, the altered recombinant RNA profiles have originated at the level of recombinant formation rather than because of altered selection. In conclusion, the viral replicase and the host RNAi gene products contribute in distinct ways to BMV RNA recombination. Our studies reveal that the antiviral RNAi mechanisms are utilized by plant RNA viruses to increase their variability, reminiscent of phenomena previously demonstrated in fungi.

RNA-RNA recombination in plant virus replication and evolution.

RNA-RNA recombination is one of the strongest forces shaping the genomes of plant RNA viruses. The detection of recombination is a challenging task that prompted the development of both in vitro and in vivo experimental systems. In the divided genome of Brome mosaic virus system, both inter- and intrasegmental crossovers are described. Other systems utilize satellite or defective interfering RNAs (DI-RNAs) of Turnip crinkle virus, Tomato bushy stunt virus, Cucumber necrosis virus, and Potato virus X. These assays identified the mechanistic details of the recombination process, revealing the role of RNA structure and proteins in the replicase-mediated copy-choice mechanism. In copy choice, the polymerase and the nascent RNA chain from which it is synthesized switch from one RNA template to another. RNA recombination was found to mediate the rearrangement of viral genes, the repair of deleterious mutations, and the acquisition of nonself sequences influencing the phylogenetics of viral taxa. The evidence for recombination, not only between related viruses but also among distantly related viruses, and even with host RNAs, suggests that plant viruses unabashedly test recombination with any genetic material at hand.

Subgenomic messenger RNAs: mastering regulation of (+)-strand RNA virus life cycle.

Many (+)-strand RNA viruses use subgenomic (SG) RNAs as messengers for protein expression, or to regulate their viral life cycle. Three different mechanisms have been described for the synthesis of SG RNAs. The first mechanism involves internal initiation on a (-)-strand RNA template and requires an internal SGP promoter. The second mechanism makes a prematurely terminated (-)-strand RNA which is used as template to make the SG RNA. The third mechanism uses discontinuous RNA synthesis while making the (-)-strand RNA templates. Most SG RNAs are translated into structural proteins or proteins related to pathogenesis: however other SG RNAs regulate the transition between translation and replication, function as riboregulators of replication or translation, or support RNA-RNA recombination. In this review we discuss these functions of SG RNAs and how they influence viral replication, translation and recombination.

Recombination of 5' subgenomic RNA3a with genomic RNA3 of Brome mosaic bromovirus in vitro and in vivo.

RNA-RNA recombination salvages viral RNAs and contributes to their genomic variability. A recombinationally-active subgenomic promoter (sgp) has been mapped in Brome mosaic bromovirus (BMV) RNA3 (Wierzchoslawski et al., 2004. J. Virol.78, 8552-8864) and mRNA-like 5' sgRNA3a was characterized (Wierzchoslawski et al., 2006. J. Virol. 80, 12357-12366). In this paper we describe sgRNA3a-mediated recombination in both in vitro and in vivo experiments. BMV replicase-directed co-copying of (-) RNA3 with wt sgRNA3a generated RNA3 recombinants in vitro, but it failed to when 3'-truncated sgRNA3a was substituted, demonstrating a role for the 3' polyA tail. Barley protoplast co-transfections revealed that (i) wt sgRNA3a recombines at the 3' and the internal sites; (ii) 3'-truncated sgRNA3as recombine more upstream; and (iii) 5'-truncated sgRNA3 recombine at a low rate. In planta co-inoculations confirmed the RNA3-sgRNA3a crossovers. In summary, the non-replicating sgRNA3a recombines with replicating RNA3, most likely via primer extension and/or internal template switching.

Characterization of a novel 5' subgenomic RNA3a derived from RNA3 of Brome mosaic bromovirus.

The synthesis of 3' subgenomic RNA4 (sgRNA4) by initiation from an internal sg promoter in the RNA3 segment was first described for Brome mosaic bromovirus (BMV), a model tripartite positive-sense RNA virus (W. A. Miller, T. W. Dreher, and T. C. Hall, Nature 313:68-70, 1985). In this work, we describe a novel 5' sgRNA of BMV (sgRNA3a) that we propose arises by premature internal termination and that encapsidates in BMV virions. Cloning and sequencing revealed that, unlike any other BMV RNA segment, sgRNA3a carries a 3' oligo(A) tail, in which respect it resembles cellular mRNAs. Indeed, both the accumulation of sgRNA3a in polysomes and the synthesis of movement protein 3a in in vitro systems suggest active functions of sgRNA3a during protein synthesis. Moreover, when copied in the BMV replicase in vitro reaction, the minus-strand RNA3 template generated the sgRNA3a product, likely by premature termination at the minus-strand oligo(U) tract. Deletion of the oligo(A) tract in BMV RNA3 inhibited synthesis of sgRNA3a during infection. We propose a model in which the synthesis of RNA3 is terminated prematurely near the sg promoter. The discovery of 5' sgRNA3a sheds new light on strategies viruses can use to separate replication from the translation functions of their genomic RNAs.