A Viral Suppressor Modulates the Plant Immune Response Early in Infection by Regulating MicroRNA Activity.

Many viral suppressors (VSRs) counteract antiviral RNA silencing, a central component of the plant's immune response by sequestration of virus-derived antiviral small interfering RNAs (siRNAs). Here, we addressed how VSRs affect the activities of cellular microRNAs (miRNAs) during a viral infection by characterizing the interactions of two unrelated VSRs, the Tombusvirus p19 and the Cucumovirus 2b, with miRNA 162 (miR162), miR168, and miR403. These miRNAs regulate the expression of the important silencing factors Dicer-like protein 1 (DCL1) and Argonaute proteins 1 and 2 (AGO1 and AGO2), respectively. Interestingly, while the two VSRs showed similar binding profiles, the miRNAs were bound with significantly different affinities, for example, with the affinity of miR162 greatly exceeding that of miR168. In vitro silencing experiments revealed that p19 and 2b affect miRNA-mediated silencing of the DCL1, AGO1, and AGO2 mRNAs in strict accordance with the VSR's miRNA-binding profiles. In Tombusvirus-infected plants, the miRNA-binding behavior of p19 closely corresponded to that in vitro Most importantly, in contrast to controls with a Δp19 virus, infections with wild-type (wt) virus led to changes of the levels of the miRNA-targeted mRNAs, and these changes correlated with the miRNA-binding preferences of p19. This was observed exclusively in the early stage of infection when viral genomes are proposed to be susceptible to silencing and viral siRNA (vsiRNA) concentrations are low. Accordingly, our study suggests that differential binding of miRNAs by VSRs is a widespread viral mechanism to coordinately modulate cellular gene expression and the antiviral immune response during infection initiation.IMPORTANCE Plant viruses manipulate their hosts in various ways. Viral suppressor proteins (VSRs) interfere with the plant's immune response by sequestering small, antivirally acting vsiRNAs, which are processed from viral RNAs during the plant's RNA-silencing response. Here, we examined the effects of VSRs on cellular microRNAs (miRNAs), which show a high degree of similarity with vsiRNAs. Binding experiments with two unrelated VSRs and three important regulatory miRNAs revealed that the proteins exhibit similar miRNA-binding profiles but bind different miRNAs at considerably different affinities. Most interestingly, experiments in plants showed that in the early infection phase, the Tombusvirus VSR p19 modulates the activity of these miRNAs on their target mRNAs very differently and that this differential regulation strictly correlates with the binding affinities of p19 for the respective miRNAs. Our data suggest that VSRs may specifically control plant gene expression and the early immune response by differential sequestration of miRNAs.

Exploitation of a surrogate host, Saccharomyces cerevisiae, to identify cellular targets and develop novel antiviral approaches.

Plant RNA viruses are widespread pathogens that need to interact intricately with their hosts to co-opt numerous cellular factors to facilitate their replication. Currently, there are only a limited number of plant resistance genes against a limited number of viruses. To develop novel antiviral approaches, the interaction network between the given virus and the host cell could be targeted. Yeast (Saccharomyces cerevisiae) has been developed as a surrogate host for tomato bushy stunt virus (TBSV), allowing systematic genome-wide screens to identify both susceptibility and restriction factors for TBSV. Importantly, pro-viral or antiviral functions of several of the characterized yeast proteins have been validated in plant hosts. This paper describes how yeast susceptibility and restriction factors of TBSV could be used as antiviral approaches. The gained knowledge on host factors could lead to novel, inducible, broad-range, and durable antiviral tools against plant viruses.

Genetic Modifications of Icosahedral Plant Virus-based Nanoparticles for Vaccine and Immunotherapy Applications.

Vaccine development is one of the greatest achievements of modern medicine. Vaccines made of live-attenuated pathogens can revert to virulent live strains, which causes safety concerns. On the other hand, the use of purified antigenic components as subunit vaccines is safer, but less effective, as these components induce lower levels of protective immunity. Multiple copy presentation of an antigenic determinant in a well-ordered and well-defined orientation on a nanosized particle can mimic the natural host-pathogen surface interaction to provide antigen stability and immunogenicity similar to that of conventional vaccines with improved safety. The icosahedral symmetry of plant viral capsid based nanoparticles is highly ordered and their multivalent structured protein nanostructures facilitate genetic modifications that result in the display of heterologous epitopes or antigens attached to coat proteins. These recombinant plant virus-based nanoparticles (PVNs) provide platforms for the induction of humoral and cellular immune responses to genetically fused antigens from pathogenic viruses, bacteria, tumors, and toxins in man and animals. Here, we comprehensively review the developments of several recombinant PVNs as prophylactic and/or therapeutic vaccines for the prevention or treatment of several microbial diseases, pathologies, and toxin poisoning.

A biodistribution study of two differently shaped plant virus nanoparticles reveals new peculiar traits.

Self-assembling plant virus nanoparticles (pVNPs) have started to be explored as nanometre-sized objects for biomedical applications, such as vaccine or drug delivery and imaging. Plant VNPs may be ideal tools in terms of biocompatibility and biodegradability endowed with a wide diversity of symmetries and dimensions, easy chemical/biological engineering, and rapid production in plants. Recently, we defined that icosahedral Tomato bushy stunt virus (TBSV) and filamentous Potato virus X (PVX) are neither toxic nor teratogenic. We report here the results of an interdisciplinary study aimed to define for the first time the biodistribution of unlabelled, unpegylated, underivatized TBSV and PVX by proved detecting antibodies. These data add new insights on the in vivo behaviour of these nano-objects and demonstrate that the pVNPs under scrutiny are each intrinsically endowed with peculiar properties foreshadowing different applications in molecular medicine.

Positive Selection or Free to Vary? Assessing the Functional Significance of Sequence Change Using Molecular Dynamics.

Evolutionary arms races between pathogens and their hosts may be manifested as selection for rapid evolutionary change of key genes, and are sometimes detectable through sequence-level analyses. In the case of protein-coding genes, such analyses frequently predict that specific codons are under positive selection. However, detecting positive selection can be non-trivial, and false positive predictions are a common concern in such analyses. It is therefore helpful to place such predictions within a structural and functional context. Here, we focus on the p19 protein from tombusviruses. P19 is a homodimer that sequesters siRNAs, thereby preventing the host RNAi machinery from shutting down viral infection. Sequence analysis of the p19 gene is complicated by the fact that it is constrained at the sequence level by overprinting of a viral movement protein gene. Using homology modeling, in silico mutation and molecular dynamics simulations, we assess how non-synonymous changes to two residues involved in forming the dimer interface-one invariant, and one predicted to be under positive selection-impact molecular function. Interestingly, we find that both observed variation and potential variation (where a non-synonymous change to p19 would be synonymous for the overprinted movement protein) does not significantly impact protein structure or RNA binding. Consequently, while several methods identify residues at the dimer interface as being under positive selection, MD results suggest they are functionally indistinguishable from a site that is free to vary. Our analyses serve as a caveat to using sequence-level analyses in isolation to detect and assess positive selection, and emphasize the importance of also accounting for how non-synonymous changes impact structure and function.

RNA Silencing May Play a Role in but Is Not the Only Determinant of the Multiplicity of Infection.

UNLABELLED: The multiplicity of infection (MOI), i.e., the number of viral genomes that infect a cell, is an important parameter in virus evolution, which for each virus and environment may have an optimum value that maximizes virus fitness. Thus, the MOI might be controlled by virus functions, an underexplored hypothesis in eukaryote-infecting viruses. To analyze if the MOI is controlled by virus functions, we estimated the MOI in plants coinfected by two genetic variants of Tomato bushy stunt virus (TBSV); by TBSV and a TBSV-derived defective interfering RNA (DI-RNA); or by TBSV and a second tombusvirus, Cymbidium ringspot virus (CymRSV). The MOI was significantly larger in TBSV-CymRSV coinfections (~4.0) than in TBSV-TBSV or TBSV-DI-RNA coinfections (~1.7 to 2.2). Coinfections by CymRSV or TBSV with chimeras in which an open reading frame (ORF) of one virus species was replaced by that of the other identified a role of viral proteins in determining the MOI, which ranged from 1.6 to 3.9 depending on the coinfecting genotypes. However, no virus-encoded protein or genomic region was the sole MOI determinant. Coinfections by CymRSV and TBSV mutants in which the expression of the gene-silencing suppressor protein p19 was abolished also showed a possible role of gene silencing in MOI determination. Taken together, these results demonstrate that the MOI is a quantitative trait showing continuous variation and that as such it has a complex determination involving different virus-encoded functions. IMPORTANCE: The number of viral genomes infecting a cell, or the multiplicity of infection (MOI), is an important parameter in virus evolution affecting recombination rates, selection intensity on viral genes, evolution of multipartite genomes, or hyperparasitism by satellites or defective interfering particles. For each virus and environment, the MOI may have an optimum value that maximizes virus fitness, but little is known about MOI control in eukaryote-infecting viruses. We show here that in plants coinfected by two genotypes of Tomato bushy stunt virus (TBSV), the MOI was lower than in plants coinfected by TBSV and Cymbidium ringspot virus (CymRSV). Coinfections by CymRSV or TBSV with TBSV-CymRSV chimeras showed a role of viral proteins in MOI determination. Coinfections by CymRSV and TBSV mutants not expressing the gene-silencing suppressor protein also showed a role of gene silencing in MOI determination. The results demonstrate that the MOI is a quantitative trait with a complex determination involving different viral functions.

A survey of resistance to Tomato bushy stunt virus in the genus Nicotiana reveals that the hypersensitive response is triggered by one of three different viral proteins.

In this study, we screened 22 Nicotiana spp. for resistance to the tombusviruses Tomato bushy stunt virus (TBSV), Cucumber necrosis virus, and Cymbidium ringspot virus. Eighteen species were resistant, and resistance was manifested in at least two different categories. In all, 13 species responded with a hypersensitive response (HR)-type resistance, whereas another five were resistant but either had no visible response or responded with chlorotic lesions rather than necrotic lesions. Three different TBSV proteins were found to trigger HR in Nicotiana spp. in an agroinfiltration assay. The most common avirulence (avr) determinant was the TBSV coat protein P41, a protein that had not been previously recognized as an avr determinant. A mutational analysis confirmed that the coat protein rather than the viral RNA sequence was responsible for triggering HR, and it triggered HR in six species in the Alatae section. The TBSV P22 movement protein triggered HR in two species in section Undulatae (Nicotiana glutinosa and N. edwardsonii) and one species in section Alatae (N. forgetiana). The TBSV P19 RNA silencing suppressor protein triggered HR in sections Sylvestres (N. sylvestris), Nicotiana (N. tabacum), and Alatae (N. bonariensis). In general, Nicotiana spp. were capable of recognizing only one tombusvirus avirulence determinant, with the exceptions of N. bonariensis and N. forgetiana, which were each able to recognize P41, as well as P19 and P22, respectively. Agroinfiltration failed to detect the TBSV avr determinants responsible for triggering HR in N. arentsii, N. undulata, and N. rustica. This study illustrates the breadth and variety of resistance responses to tombusviruses that exists in the Nicotiana genus.

Genetic analysis and mapping of resistance to lettuce dieback: a soilborne disease caused by tombusviruses.

A diverse collection of modern, heirloom and specialty cultivars, plant introduction (PI) accessions, and breeding lines of lettuce were screened for susceptibility to lettuce dieback, which is a disease caused by soilborne viruses of the family Tombusviridae. Susceptibility was evaluated by visual symptom assessment in fields that had been previously shown to be infested with Lettuce necrotic stunt virus. Of the 241 genotypes tested in multiple field experiments, 76 remained symptom-free in infested fields and were therefore classified as resistant to dieback. Overall, resistant genotypes were as prevalent among modern cultivars as in heirloom cultivars or primitive germplasm. Within modern germplasm, however, all crisphead (iceberg) cultivars were resistant, while all romaine cultivars were susceptible. Using enzyme-linked immunosorbent assay, tombusviruses were detected in leaves of some plants of resistant genotypes that were grown in infested fields, suggesting that symptom-free plants are not immune to viral infection. The inheritance of resistance was studied for 'Salinas', a modern iceberg cultivar, and PI 491224, the progenitor of recently released romaine germplasm with resistance to lettuce dieback. Resistance was conferred by a dominant allele at a single locus in both genotypes. The tombusvirus resistance locus from 'Salinas', Tvr1, was mapped in an intraspecific Lactuca sativa population to a location that corresponds to linkage group 2 on the consensus map of Lactuca. The largest cluster of resistance genes in lettuce, the Dm1/Dm3 cluster, is found on this linkage group; however, the precise position of Tvr1 relative to this cluster has not yet been determined. To our knowledge, Tvr1 is the first tombusvirus resistance gene identified for any plant host.

Immunodetection of the 33 K/92 K polymerase proteins in cymbidium ringspot virus-infected and in transgenic plant tissue extracts.

An antiserum was raised against the 33 K protein encoded by the 5' proximal gene of cymbidium ringspot tombusvirus RNA. This antiserum reacts specifically with the 33 K and 92 K proteins, which constitute the viral replicase, in CyRSV-infected Nicotiana benthamiana plants and in transgenic plants transformed with the full-length replicase gene. In inoculated leaves of infected plants, synthesis of the 33 K/92 K proteins stops ten days after inoculation, whereas in newly produced systemically infected leaves there was continuous production of these proteins. In transgenic plants, both proteins were detected showing that readthrough of the termination codon of the 33 K protein does not depend on the presence of the replicating virus. The subcellular localization of the 33 K/92 K proteins is similar in infected and transgenic plants. No correlation was found between the level of expression of integrated virus gene and level of resistance to the challenging virus.

Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack.

Expression of viral genes in transgenic plants is a very effective tool for attenuating plant viral infection. Nevertheless, the lack of generality and risk issues related to the expression of viral genes in plants might limit the exploitation of this strategy. Expression in plants of antibodies against essential viral proteins could provide an alternative approach to engineer viral resistance. Recently, expression of complete or engineered antibodies has been successfully achieved in plants. The engineered single-chain Fv antibody scFv (refs 10, 11) is particularly suitable for expression in plants because of its small size and the lack of assembly requirements. Here we present evidence that constitutive expression in transgenic plants of a scFv antibody, directed against the plant icosahedral tombusvirus artichoke mottled crinkle virus, causes reduction of infection incidence and delay in symptom development.