Orchid fleck dichorhavirus movement protein shows RNA silencing suppressor activity.

To counteract RNA interference-mediated antiviral defence, virus genomes evolved to express proteins that inhibit this plant defence mechanism. Using six independent biological approaches, we show that orchid fleck dichorhavirus citrus strain (OFV-citrus) movement protein (MP) may act as a viral suppressor of RNA silencing (VSR). By using the alfalfa mosaic virus (AMV) RNA 3 expression vector, it was observed that the MP triggered necrosis response in transgenic tobacco leaves and increased the viral RNA (vRNA) accumulation. The use of the potato virus X (PVX) expression system revealed that the cis expression of MP increased both the severity of the PVX infection and the accumulation of PVX RNAs, further supporting that MP could act as an RNA silencing suppressor (RSS). From the analysis of the RSS-defective turnip crinkle virus (TCV), we do not find local RSS activity for MP, suggesting a link between MP suppressor activity and the prevention of systemic silencing. In the analysis of local suppressive activity using the GFP-based agroinfiltration assay in Nicotiana benthamiana (16 c line), we do not identify local RSS activity for the five OFV RNA1-encoded proteins. However, when evaluating the small interfering RNA (siRNA) accumulation, we find that the expression of MP significantly reduces the accumulation of GFP-derived siRNA. Finally, we examine whether the MP can prevent systemic silencing in 16c plants. Our findings show that MP inhibits the long-distance spread of RNA silencing, but does not affect the short-distance spread. Together, our findings indicate that MP is part of OFV's counter-defence mechanism, acting mainly in the prevention of systemic long-distance silencing. This work presents the first report of a VSR for a member of the genus Dichorhavirus.

Identification and genomic characterization of a novel tobamovirus from prickly pear cactus.

In this work, we describe the complete sequence and genome organization of a novel tobamovirus detected in a prickly pear plant (Opuntia sp.) by high-throughput sequencing, tentatively named "opuntia virus 2". The full genome of opuntia virus 2 is 6,453 nucleotides in length and contains four open reading frames (ORFs) coding for the two subunits of the RNA polymerase, the movement protein, and the coat protein, respectively. Phylogenetic analysis using the complete nucleotide sequence revealed that the virus belongs to the genus Tobamovirus (family Virgaviridae), showing the highest nucleotide sequence identity (49.8%) with cactus mild mottle virus (CMMoV), being indicating that it belongs in the Cactaceae subgroup of tobamoviruses.

The capsid protein of citrus leprosis virus C shows a nuclear distribution and interacts with the nucleolar fibrillarin protein.

Brevipalpus-transmitted viruses (BTVs) have a significant negative economic impact on the citrus industry in Central and South America. Until now, only a few studies have explored the intracellular distribution and interaction of BTVs-encoded proteins with host factors, particularly for cileviruses, the main BTV responsible for the Citrus Leprosis (CL) disease. This study describes the nuclear localization of citrus leprosis virus C (CiLV-C) capsid protein (p29) and its interaction with the fibrillarin (Fib2) within the nucleolar compartment and cell cytoplasm. Our results, obtained by computer predictions and laser scanning confocal microscopy analyses, including colocalization and bimolecular fluorescence complementation (BiFC) approaches, revealed that a fraction of the p29 is localized in the nucleus and colocalizes with the Fib2 in both the nucleolus and cytosol. The nuclear localization of p29 correlated with a smaller nucleus size. Furthermore, co-immunoprecipitation (Co-IP) assays confirmed the interactions between p29 and Fib2. The implications of these findings for the functionalities of the cilevirus capsid protein are discussed.

Rescue of a Cilevirus from infectious cDNA clones.

Reverse genetics systems represent an important tool for studying the molecular and functional processes of viral infection. Citrus leprosis virus C (CiLV-C) (genus Cilevirus, family Kitaviridae) is the main pathogen responsible for the citrus leprosis (CL) disease in Latin America, one of the most economically important diseases of the citrus industry. Molecular studies of this pathosystem are limited due to the lack of infectious clones. Here, we report the construction and validation of a CiLV-C infectious cDNA clone based on an agroinfection system. The two viral RNA segments (RNA1 and RNA2) were assembled into two binary vectors (pJL89 and pLXAS). Agroinfiltrated Nicotiana benthamiana plants showed a response similar to that observed in the natural infection process with the formation of localized lesions restricted to the inoculated leaves. The virus recovered from the plant tissue infected with the infectious clones can be mechanically transmitted between N. benthamiana plants. Detection of CiLV-C subgenomic RNAs (sgRNAs) from agroinfiltrated and mechanically inoculated leaves further confirmed the infectivity of the clones. Finally, partial particle-purification preparations or sections of CiLV-C-infected tissue followed by transmission electron microscopy (TEM) analysis showed the formation of CiLV-C virions rescued by the infectious clone. The CiLV-C reverse genetic system now provides a powerful molecular tool to unravel the peculiarities of the CL pathosystem.

Spontaneous Mutation in the Movement Protein of Citrus Leprosis Virus C2, in a Heterologous Virus Infection Context, Increases Cell-to-Cell Transport and Generates Fitness Advantage.

Previous results using a movement defective alfalfa mosaic virus (AMV) vector revealed that citrus leprosis virus C (CiLV-C) movement protein (MP) generates a more efficient local movement, but not more systemic transport, than citrus leprosis virus C2 (CiLV-C2) MP, MPs belonging to two important viruses for the citrus industry. Here, competition experiment assays in transgenic tobacco plants (P12) between transcripts of AMV constructs expressing the cilevirus MPs, followed by several biological passages, showed the prevalence of the AMV construct carrying the CiLV-C2 MP. The analysis of AMV RNA 3 progeny recovered from P12 plant at the second viral passage revealed the presence of a mix of progeny encompassing the CiLV-C2 MP wild type (MPWT) and two variants carrying serines instead phenylalanines at positions 72 (MPS72F) or 259 (MPS259F), respectively. We evaluated the effects of each modified residue in virus replication, and cell-to-cell and long-distance movements. Results indicated that phenylalanine at position 259 favors viral cell-to-cell transport with an improvement in viral fitness, but has no effect on viral replication, whereas mutation at position 72 (MPS72F) has a penalty in the viral fitness. Our findings indicate that the prevalence of a viral population may be correlated with its greater efficiency in cell-to-cell and systemic movements.

Citrus Leprosis Virus C Encodes Three Proteins With Gene Silencing Suppression Activity.

Citrus leprosis virus C (CiLV-C) belongs to the genus Cilevirus, family Kitaviridae, and is considered the most devastating virus infecting citrus in Brazil, being the main viral pathogen responsible for citrus leprosis (CL), a severe disease that affects citrus orchards in Latin America. Here, proteins encoded by CiLV-C genomic RNA 1 and 2 were screened for potential RNA silencing suppressor (RSS) activity by five methods. Using the GFP-based reporter agroinfiltration assay, we have not found potential local suppressor activity for the five CiLV-C encoded proteins. However, when RSS activity was evaluated using the alfalfa mosaic virus (AMV) system, we found that the p29, p15, and p61 CiLV-C proteins triggered necrosis response and increased the AMV RNA 3 accumulation, suggesting a suppressive functionality. From the analysis of small interfering RNAs (siRNAs) accumulation, we observed that the ectopic expression of the p29, p15, and p61 reduced significantly the accumulation of GFP derived siRNAs. The use of the RSS defective turnip crinkle virus (TCV) system revealed that only the trans-expression of the p15 protein restored the cell-to-cell viral movement. Finally, the potato virus X (PVX) system revealed that the expression of p29, p15, and p61 increased the PVX RNA accumulation; in addition, the p29 and p15 enhanced the pathogenicity of PVX resulting in the death of tobacco plants. Furthermore, PVX-p61 infection resulted in a hypersensitive response (HR), suggesting that p61 could also activate a plant defense response mechanism. This is the first report describing the RSS activity for CiLV-C proteins and, moreover, for a member of the family Kitaviridae.

Dichorhaviruses Movement Protein and Nucleoprotein Form a Protein Complex That May Be Required for Virus Spread and Interacts in vivo With Viral Movement-Related Cilevirus Proteins.

Brevipalpus-transmitted viruses (BTVs) belong to the genera Dichorhavirus and Cilevirus and are the main causal agents of the citrus leprosis (CL) disease. In this report, we explored aspects related to the movement mechanism mediated by dichorhaviruses movement proteins (MPs) and the homologous and heterologous interactions among viral proteins related to the movement of citrus leprosis-associated viruses. The membrane-spanning property and topology analysis of the nucleocapsid (N) and MP proteins from two dichorhaviruses revealed that the MPs are proteins tightly associated with the cell membrane, exposing their N- and C-termini to the cytoplasm and the inner part of the nucleus, whereas the N proteins are not membrane-associated. Subcellular localization analysis revealed the presence of dichorhavirus MPs at the cell surface and in the nucleus, while the phosphoproteins (P) were located exclusively in the nucleus and the N proteins in both the cytoplasm and the nucleus. Co-expression analysis with the MP, P, and N proteins showed an interaction network formed between them. We highlight the MP capability to partially redistribute the previously reported N-P core complex, redirecting a portion of the N from the nucleus to the plasmodesmata at the cell periphery, which indicates not only that the MP might guide the intracellular trafficking of the viral infective complex but also that the N protein may be associated with the cell-to-cell movement mechanism of dichorhaviruses. The movement functionality of these MPs was analyzed by using three movement-defective infectious systems. Also, the MP capacity to generate tubular structures on the protoplast surface by ectopic expression was analyzed. Finally, we evaluated the in vivo protein-protein interaction networks between the dichorhavirus MP and/or N proteins with the heterologous cilevirus movement components, which suggest a broad spectrum of interactions, highlighting those among capsid proteins (CP), MPs, and Ns from citrus leprosis-associated viruses. These data may aid in understanding the mixed infection process naturally observed in the field caused by distinct BTVs.

Patellins 3 and 6, two members of the Plant Patellin family, interact with the movement protein of Alfalfa mosaic virus and interfere with viral movement.

Movement proteins (MPs) encoded by plant viruses interact with host proteins to facilitate or interfere with intra- and/or intercellular viral movement. Using yeast two-hybrid and bimolecular fluorescence complementation assays, we herein present in vivo evidence for the interaction between Alfalfa mosaic virus (AMV) MP and Arabidopsis Patellin 3 (atPATL3) and Patellin 6 (atPATL6), two proteins containing a Sec14 domain. Proteins with Sec14 domains are implicated in membrane trafficking, cytoskeleton dynamics, lipid metabolism and lipid-mediated regulatory functions. Interestingly, the overexpression of atPATL3 and/or atPATL6 interfered with the plasmodesmata targeting of AMV MP and correlated with reduced infection foci size. Consistently, the viral RNA levels increased in the single and double Arabidopsis knockout mutants for atPATL3 and atPATL6. Our results indicate that, in general, MP-PATL interactions interfere with the correct subcellular targeting of MP, thus rendering the intracellular transport of viral MP-containing complexes less efficient and diminishing cell-to-cell movement.

A plant virus movement protein regulates the Gcn2p kinase in budding yeast.

Virus life cycle heavily depends on their ability to command the host machinery in order to translate their genomes. Animal viruses have been shown to interfere with host translation machinery by expressing viral proteins that either maintain or inhibit eIF2α function by phosphorylation. However, this interference mechanism has not been described for any plant virus yet. Prunnus necrotic ringspot virus (PNRSV) is a serious pathogen of cultivated stone fruit trees. The movement protein (MP) of PNRSV is necessary for the cell-to-cell movement of the virus. By using a yeast-based approach we have found that over-expression of the PNRSV MP caused a severe growth defect in yeast cells. cDNA microarrays analysis carried out to characterise at the molecular level the growth interference phenotype reported the induction of genes related to amino acid deprivation suggesting that expression of MP activates the GCN pathway in yeast cells. Accordingly, PNRSV MP triggered activation of the Gcn2p kinase, as judged by increased eIF2α phosphorylation. Activation of Gcn2p by MP expression required a functional Tor1p kinase, since rapamycin treatment alleviated the yeast cell growth defect and blocked eIF2α phosphorylation triggered by MP expression. Overall, these findings uncover a previously uncharacterised function for PNRSV MP viral protein, and point out at Tor1p and Gcn2p kinases as candidate susceptibility factors for plant viral infections.

Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement.

The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutants and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed.