A Zinc Finger Motif in the P1 N Terminus, Highly Conserved in a Subset of Potyviruses, Is Associated with the Host Range and Fitness of Telosma Mosaic Virus.

P1 is the first protein translated from the genomes of most viruses in the family Potyviridae, and it contains a C-terminal serine-protease domain that cis-cleaves the junction between P1 and HCPro in most cases. Intriguingly, P1 is the most divergent among all mature viral factors, and its roles during viral infection are still far from understood. In this study, we found that telosma mosaic virus (TelMV, genus Potyvirus) in passion fruit, unlike TelMV isolates present in other hosts, has two stretches at the P1 N terminus, named N1 and N2, with N1 harboring a Zn finger motif. Further analysis revealed that at least 14 different potyviruses, mostly belonging to the bean common mosaic virus subgroup, encode a domain equivalent to N1. Using the newly developed TelMV infectious cDNA clones from passion fruit, we demonstrated that N1, but not N2, is crucial for viral infection in both Nicotiana benthamiana and passion fruit. The regulatory effects of N1 domain on P1 cis cleavage, as well as the accumulation and RNA silencing suppression (RSS) activity of its cognate HCPro, were comprehensively investigated. We found that N1 deletion decreases HCPro abundance at the posttranslational level, likely by impairing P1 cis cleavage, thus reducing HCPro-mediated RSS activity. Remarkably, disruption of the Zn finger motif in N1 did not impair P1 cis cleavage and HCPro accumulation but severely debilitated TelMV fitness. Therefore, our results suggest that the Zn finger motif in P1s plays a critical role in viral infection that is independent of P1 protease activity and self-release, as well as HCPro accumulation and silencing suppression. IMPORTANCE Viruses belonging to the family Potyviridae represent the largest group of plant-infecting RNA viruses, including a variety of agriculturally and economically important viral pathogens. Like all picorna-like viruses, potyvirids employ polyprotein processing as the gene expression strategy. P1, the first protein translated from most potyvirid genomes, is the most variable viral factor and has attracted great scientific interest. Here, we defined a Zn finger motif-encompassing domain (N1) at the N terminus of P1 among diverse potyviruses phylogenetically related to bean common mosaic virus. Using TelMV as a model virus, we demonstrated that the N1 domain is key for viral infection, as it is involved both in regulating the abundance of its cognate HCPro and in an as-yet-undefined key function unrelated to protease processing and RNA silencing suppression. These results advance our knowledge of the hypervariable potyvirid P1s and highlight the importance for infection of a previously unstudied Zn finger domain at the P1 N terminus.

Modification of Papaya Ringspot Virus HC-Pro to Generate Effective Attenuated Mutants for Overcoming the Problem of Strain-Specific Cross Protection.

Cross protection application of HA5-1, an attenuated mutant of papaya ringspot virus (PRSV) HA strain from Hawaii, was withdrawn from Taiwan due to the narrow geographic strain specificity of HA5-1. Here, to overcome this problem, we created attenuated mutants of PRSV YK, a dominant severe strain from Taiwan, by mutating helper component protease (HC-Pro) at F7, R181, F206, and D397 residues critical for potyviral pathogenicity. PRSV YK HC-Pro R181I, F206L, and D397N single-mutant viruses induced mild symptoms, but their adverse effects on growth of papaya plants disqualified them as useful protective viruses. However, F7I single-mutant and F7I + F206L double-mutant viruses displayed mild symptoms followed by recovery, and they showed a zigzag pattern of accumulation in papaya plants, indicating their potential to trigger RNA silencing and retain partial antagonistic suppression of host defense. Although F7I + R181I and F7I + D397N double-mutant viruses caused symptomless infection, they accumulated barely above mock level and, thus, were not qualified as proper protective viruses. RNA silencing suppression (RSS) analysis by agroinfiltration in Nicotiana benthamiana plants revealed that the HC-Pro F7I and F7I + F206L mutant proteins were weaker in RSS ability than the wild-type protein. Under greenhouse conditions, F7I and F7I + F206L mutant viruses were genetically stable but not aphid transmissible. Compared with the HA5-1 mutant's low degree (10%) of protection to papaya plants, the F7I and F7I + F206L mutants provided complete (100%) protection to papaya and horn melon plants against strain YK. Thus, F7I and F7I + F206L mutants solve the problem of strain-specific protection and have great potential for control of PRSV in Taiwan.

A Newly Identified Virus in the Family Potyviridae Encodes Two Leader Cysteine Proteases in Tandem That Evolved Contrasting RNA Silencing Suppression Functions.

Potyviridae is the largest family of plant-infecting RNA viruses and includes many agriculturally and economically important viral pathogens. The viruses in the family, known as potyvirids, possess single-stranded, positive-sense RNA genomes with polyprotein processing as a gene expression strategy. The N-terminal regions of potyvirid polyproteins vary greatly in sequence. Previously, we identified a novel virus species within the family, Areca palm necrotic spindle-spot virus (ANSSV), which was predicted to encode two cysteine proteases, HCPro1 and HCPro2, in tandem at the N-terminal region. Here, we present evidence showing self-cleavage activity of these two proteins and define their cis-cleavage sites. We demonstrate that HCPro2 is a viral suppressor of RNA silencing (VSR), and both the variable N-terminal and conserved C-terminal (protease domain) moieties have antisilencing activity. Intriguingly, the N-terminal region of HCPro1 also has RNA silencing suppression activity, which is, however, suppressed by its C-terminal protease domain, leading to the functional divergence of HCPro1 and HCPro2 in RNA silencing suppression. Moreover, the deletion of HCPro1 or HCPro2 in a newly created infectious clone abolishes viral infection, and the deletion mutants cannot be rescued by addition of corresponding counterparts of a potyvirus. Altogether, these data suggest that the two closely related leader proteases of ANSSV have evolved differential and essential functions to concertedly maintain viral viability.IMPORTANCE The Potyviridae represent the largest group of known plant RNA viruses and account for more than half of the viral crop damage worldwide. The leader proteases of viruses within the family vary greatly in size and arrangement and play key roles during the infection. Here, we experimentally demonstrate the presence of a distinct pattern of leader proteases, HCPro1 and HCPro2 in tandem, in a newly identified member within the family. Moreover, HCPro1 and HCPro2, which are closely related and typically characterized with a short size, have evolved contrasting RNA silencing suppression activity and seem to function in a coordinated manner to maintain viral infectivity. Altogether, the new knowledge fills a missing piece in the evolutionary relationship history of potyvirids and improves our understanding of the diversification of potyvirid genomes.

A Single Amino Acid Substitution in the Intervening Region of 129K Protein of Cucumber Green Mottle Mosaic Virus Resulted in Attenuated Symptoms.

Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, is a major threat to economically important cucurbit crops worldwide. An attenuated strain (SH33b) derived from a severe strain (SH) of CGMMV caused a reduction in the viral RNA accumulation and the attenuation of symptoms, and it has been successfully used to protect muskmelon plants against severe strains in Japan. In this study, we compared GFP-induced silencing suppression by the 129K protein and the methyltransferase domain plus intervening region (MTIR) of the 129K protein between the SH and SH33b strains, respectively. As a result, silencing suppression activity (SSA) in the GFP-silenced plants was inhibited efficiently by the MTIR and 129K protein of SH strain, and it coincided with drastically reduced accumulation of GFP-specific small interfering RNAs (siRNAs) but not by that of SH33b strain. Furthermore, analyses of siRNA binding capability (SBC) by the MTIR of 129K protein and 129K protein using electrophoretic mobility shift assay revealed that SBC was found with the MTIR and 129K protein of SH but not with that of SH33b, suggesting that a single amino acid mutation (E to G) in the MTIR is responsible for impaired SSA and SBC of SH33b. These data suggest that a single amino acid substitution in the intervening region of 129K protein of CGMMV resulted in attenuated symptoms by affecting RNA silencing suppression.

Cucurbit chlorotic yellows virus p22 is a suppressor of local RNA silencing.

RNA silencing is a major antiviral mechanism in plants, which is counteracted by virus-encoded proteins with silencing suppression activity. ORFs encoding putative silencing suppressor proteins that share no structural or sequence homology have been identified in the genomes of four criniviruses. In this study, we investigated the RNA silencing suppression activity of several proteins encoded by the RNA1 (RdRp, p22) and RNA2 (CP, CPm and p26) of cucurbit chlorotic yellows virus (CCYV) using co-agroinfiltration assays on Nicotiana benthamiana plants. Our results indicate that p22 is a suppressor of local RNA silencing that does not interfere with cell-to-cell movement of the RNA silencing signal or with systemic silencing. Furthermore, comparisons of the suppression activity of CCYV p22 with that of two other well-known crinivirus suppressors (CYSDV p25 and ToCV p22) revealed that CCYV p22 is a weaker suppressor of local RNA silencing than the other two proteins. Finally, a comparative sequence analysis of the p22 genes of seven Greek CCYV isolates was performed, revealing a high level of conservation. Taken together, our research advances our knowledge about plant-virus interactions of criniviruses, an emergent group of pathogens that threatens global agriculture.

Identification and RNA binding characterization of plant virus RNA silencing suppressor proteins.

Suppression is a common mechanism employed by viruses to evade the antiviral effects of the host's RNA silencing pathway. The activity of suppression has commonly been localized to gene products in the virus, but the variety of mechanisms used in suppression by these viral proteins spans nearly the complete biochemical pathway of RNA silencing in the host. This review describes the agrofiltration assay and a slightly modified version of the agro-infiltration assay called co-infiltration, which are common methods used to observe RNA silencing and identify viral silencing suppressor proteins in plants, respectively. In addition, this review will provide an overview of two methods, electrophoretic mobility shift assay and fluorescence polarization, used to assess the binding of a suppressor protein to siRNA which has been shown to be a general mechanism to suppress RNA silencing by plant viruses.

Corrigendum: Development and Evaluation of Stable Sugarcane Mosaic Virus Mild Mutants for Cross-Protection Against Infection by Severe Strain.

[This corrects the article DOI: 10.3389/fpls.2021.788963.].

Development and Evaluation of Stable Sugarcane Mosaic Virus Mild Mutants for Cross-Protection Against Infection by Severe Strain.

Sugarcane mosaic virus (SCMV; genus Potyvirus) induces maize dwarf mosaic disease that has caused serious yield losses of maize in China. Cross-protection is one of the efficient strategies to fight against severe virus strains. Although many mild strains have been identified, the spontaneous mutation is one of the challenging problems affecting their application in cross-protection. In this study, we found that the substitution of cysteine (C) at positions 57 or 60 in the zinc finger-like motif of HC-Pro with alanine (A; C57A or C60A) significantly reduced its RNA silencing suppression activity and SCMV virulence. To reduce the risk of mild strains mutating to virulent ones by reverse or complementary mutations, we obtained attenuated SCMV mutants with double-mutations in the zinc finger-like and FRNK motifs of HC-Pro and evaluated their potential application in cross-protection. The results showed that the maize plants infected with FKNK/C60A double-mutant showed symptomless until 95 days post-inoculation and FKNK/C60A cross-protected plants displayed high resistance to severe SCMV strain. This study provides theoretical and material bases for the control of SCMV through cross-protection.

An Importin-ß-like Protein from Nicotiana benthamiana Interacts with the RNA Silencing Suppressor P1b of the Cucumber Vein Yellowing Virus, Modulating Its Activity.

During a plant viral infection, host-pathogen interactions are critical for successful replication and propagation of the virus through the plant. RNA silencing suppressors (RSSs) are key players of this interplay, and they often interact with different host proteins, developing multiple functions. In the Potyviridae family, viruses produce two main RSSs, HCPro and type B P1 proteins. We focused our efforts on the less known P1b of cucumber vein yellowing virus (CVYV), a type B P1 protein, to try to identify possible factors that could play a relevant role during viral infection. We used a chimeric expression system based on plum pox virus (PPV) encoding a tagged CVYV P1b in place of the canonical HCPro. We used that tag to purify P1b in Nicotiana-benthamiana-infected plants and identified by mass spectrometry an importin-ß-like protein similar to importin 7 of Arabidopsis thaliana. We further confirmed the interaction by bimolecular fluorescence complementation assays and defined its nuclear localization in the cell. Further analyses showed a possible role of this N. benthamiana homolog of Importin 7 as a modulator of the RNA silencing suppression activity of P1b.

The P1 Protein of Watermelon mosaic virus Compromises the Activity as RNA Silencing Suppressor of the P25 Protein of Cucurbit yellow stunting disorder virus.

Mixed viral infections in plants involving a potyvirus and other unrelated virus often result in synergistic effects, with significant increases in accumulation of the non-potyvirus partner, as in the case of melon plants infected by the potyvirus Watermelon mosaic virus (WMV) and the crinivirus Cucurbit yellow stunting disorder virus (CYSDV). To further explore the synergistic interaction between these two viruses, the activity of RNA silencing suppressors (RSSs) was addressed in transiently co-expressed combinations of heterologous viral products in Nicotiana benthamiana leaves. While the strong RSS activity of WMV Helper Component Proteinase (HCPro) was unaltered, including no evident additive effects observed when co-expressed with the weaker CYSDV P25, an unexpected negative effect of WMV P1 was found on the RSS activity of P25. Analysis of protein expression during the assays showed that the amount of P25 was not reduced when co-expressed with P1. The detrimental action of P1 on the activity of P25 was dose-dependent, and the subcellular localization of fluorescently labeled variants of P1 and P25 when transiently co-expressed showed coincidences both in nucleus and cytoplasm. Also, immunoprecipitation experiments showed interaction of tagged versions of the two proteins. This novel interaction, not previously described in other combinations of potyviruses and criniviruses, might play a role in modulating the complexities of the response to multiple viral infections in susceptible plants.

A Spontaneous Complementary Mutation Restores the RNA Silencing Suppression Activity of HC-Pro and the Virulence of Sugarcane Mosaic Virus.

Cross-protection is a promising measure to control plant viral diseases. Reverse genetics had been recently adopted to generate attenuated mutants that have potential in cross-protection. But studies on the variability of the progeny viruses of the attenuated mutants are scarce. Sugarcane mosaic virus (SCMV; genus Potyvirus, family Potyviridae) is the prevalent virus inducing maize dwarf mosaic disease in China. Here, we showed that the substitution of arginine with isoleucine in the FRNK motif at position 184 of helper component-proteinase (HC-Pro) abolished its RNA silencing suppression (RSS) activity, drastically reduced the virulence and accumulation level of SCMV, and impaired the synergism between SCMV and maize chlorotic mottle virus. The attenuated mutant could protect maize plants from a severe infection of SCMV. However, a spontaneous mutation of glycine at position 440 to arginine in HC-Pro rescued the virulence and synergism with maize chlorotic mottle virus of SCMV and the RSS activity of HC-Pro. Similar results were obtained with tobacco vein banding mosaic virus and watermelon mosaic virus. These results provide novel evidence for the complementary mutation of potyviruses in maintaining the HC-Pro RSS activity and potyviral virulence and remind us of evaluating the potential risk of attenuated mutants thoroughly before applying for the control of plant viral diseases via cross-protection.

Insights into the Functions of eIF4E-Biding Motif of VPg in Potato Virus A Infection.

The interaction between the viral protein genome-linked (VPg) and eukaryotic initiation factor 4E (eIF4E) or eIF(iso)4E of the host plays a crucial role in potyvirus infection. The VPg of potato virus A (PVA) contains the Tyr-X-X-X-X-Leu-phi (YXXXLΦ) binding motif for eIF(iso)4E. In order to investigate its role in PVA infection, we substituted the conserved tyrosine and leucine residues of the motif with alanine residues in the infectious cDNA of PVA (PVAVPgmut). PVAVPgmut RNA replicated in infiltrated leaves, but RNA accumulation remained low. Systemic infection occurred only if a reversion to wild type PVA occurred. VPg was able to stabilize PVA RNA and enhance the expression of Renilla luciferase (3'RLUC) from the 3' end of the PVA genome. VPgmut could not support either PVA RNA stabilization or enhanced 3'RLUC expression. The RNA silencing suppressor helper-component proteinase (HCPro) is responsible for the formation of PVA-induced RNA granules (PGs) during infection. While VPgmut increased the number of PG-like foci, the percentage of PVA RNA co-localization with PGs was reduced from 86% to 20%. A testable hypothesis for future studies based on these results is that the binding of eIF(iso)4E to PVA VPg via the YXXXLΦ motif is required for PVA RNA stabilization, as well as the transfer to the RNA silencing suppression pathway and, further, to polysomes for viral protein synthesis.

Truncation of a P1 leader proteinase facilitates potyvirus replication in a non-permissive host.

The Potyviridae family is a major group of plant viruses that includes c. 200 species, most of which have narrow host ranges. The potyvirid P1 leader proteinase self-cleaves from the remainder of the viral polyprotein and shows large sequence variability linked to host adaptation. P1 proteins can be classified as Type A or Type B on the basis, amongst other things, of their dependence or not on a host factor to develop their protease activity. In this work, we studied Type A proteases from the Potyviridae family, characterizing their host factor requirements. Our in vitro cleavage analyses of potyvirid P1 proteases showed that the N-terminal domain is relevant for host factor interaction and suggested that the C-terminal domain is also involved. In the absence of plant factors, the N-terminal end of Plum pox virus P1 antagonizes protease self-processing. We performed extended deletion mutagenesis analysis to define the N-terminal antagonistic domain of P1. In viral infections, removal of the P1 protease antagonistic domain led to a gain-of-function phenotype, strongly increasing local infection in a non-permissive host. Altogether, our results shed new insights into the adaptation and evolution of potyvirids.

The Luteovirus P4 Movement Protein Is a Suppressor of Systemic RNA Silencing.

The plant viral family Luteoviridae is divided into three genera: Luteovirus, Polerovirus and Enamovirus. Without assistance from another virus, members of the family are confined to the cells of the host plant's vascular system. The first open reading frame (ORF) of poleroviruses and enamoviruses encodes P0 proteins which act as silencing suppressor proteins (VSRs) against the plant's viral defense-mediating RNA silencing machinery. Luteoviruses, such as barley yellow dwarf virus-PAV (BYDV-PAV), however, have no P0 to carry out the VSR role, so we investigated whether other proteins or RNAs encoded by BYDV-PAV confer protection against the plant's silencing machinery. Deep-sequencing of small RNAs from plants infected with BYDV-PAV revealed that the virus is subjected to RNA silencing in the phloem tissues and there was no evidence of protection afforded by a possible decoy effect of the highly abundant subgenomic RNA3. However, analysis of VSR activity among the BYDV-PAV ORFs revealed systemic silencing suppression by the P4 movement protein, and a similar, but weaker, activity by P6. The closely related BYDV-PAS P4, but not the polerovirus potato leafroll virus P4, also displayed systemic VSR activity. Both luteovirus and the polerovirus P4 proteins also showed transient, weak local silencing suppression. This suggests that systemic silencing suppression is the principal mechanism by which the luteoviruses BYDV-PAV and BYDV-PAS minimize the effects of the plant's anti-viral defense.

Functional analysis of a weak viral RNA silencing suppressor using two GFP variants as silencing inducers.

RNA silencing in plants can be triggered by the introduction of an exogenous gene. Green fluorescent protein (GFP) has been widely used as a visual reporter to study RNA silencing and viral-mediated suppression of RNA silencing in the model plant Nicotiana benthamiana. In transgenic N. benthamiana plants expressing an endoplasmic reticulum targeted GFP variant (16c) known as mGFP5, RNA silencing can be induced by ectopic over-expression of mGFP5. However, other GFP variants can also be used to induce GFP silencing in these plants. We compared the efficiency to induce local and systemic silencing of two commonly used GFP variants: enhanced GFP (eGFP) and mGFP5. Using lettuce necrotic yellows virus (LNYV) P protein to suppress GFP silencing, we demonstrate that eGFP gene, which is 76% identical at the nucleotide level to the endogenously expressed mGFP5 in 16c plants, triggers silencing more slowly and concurrently prolongs detectable silencing suppressor activity of the weak LNYV P suppressor, compared to the homologous mGFP5 gene. The use of eGFP as RNA silencing inducer in wild type or 16c plants appears to be a useful tool in identifying and analysing weak viral RNA silencing suppressor proteins whose activity might otherwise have been masked when challenged by a stronger RNA silencing response. We also show that reducing the dosage of strong dsRNA silencing inducers in conjunction with their homologous GFP targets facilitates the discovery and analysis of "weaker" RNA silencing suppressor activities.

Intracellular Localization, Interactions and Functions of Capsicum Chlorosis Virus Proteins.

Tospoviruses are among the most devastating viruses of horticultural and field crops. Capsicum chlorosis virus (CaCV) has emerged as an important pathogen of capsicum and tomato in Australia and South-east Asia. Present knowledge about CaCV protein functions in host cells is lacking. We determined intracellular localization and interactions of CaCV proteins by live plant cell imaging to gain insight into the associations of viral proteins during infection. Proteins were transiently expressed as fusions to autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana and capsicum. All viral proteins localized at least partially in the cell periphery suggestive of cytoplasmic replication and assembly of CaCV. Nucleocapsid (N) and non-structural movement (NSm) proteins localized exclusively in the cell periphery, while non-structural suppressor of silencing (NSs) protein and Gc and Gn glycoproteins accumulated in both the cell periphery and the nucleus. Nuclear localization of CaCV Gn and NSs is unique among tospoviruses. We validated nuclear localization of NSs by immunofluorescence in protoplasts. Bimolecular fluorescence complementation showed self-interactions of CaCV N, NSs and NSm, and heterotypic interactions of N with NSs and Gn. All interactions occurred in the cytoplasm, except NSs self-interaction was exclusively nuclear. Interactions of a tospoviral NSs protein with itself and with N had not been reported previously. Functionally, CaCV NSs showed strong local and systemic RNA silencing suppressor activity and appears to delay short-distance spread of silencing signal. Cell-to-cell movement activity of NSm was demonstrated by trans-complementation of a movement-defective tobamovirus replicon. CaCV NSm localized at plasmodesmata and its transient expression led to the formation of tubular structures that protruded from protoplasts. The D155 residue in the 30K-like movement protein-specific LxD/N50-70G motif of NSm was critical for plasmodesmata localization and movement activity. Compared to other tospoviruses, CaCV proteins have both conserved and unique properties in terms of in planta localization, interactions and protein functions which will effect viral multiplication and movement in host plants.

A novel sweet potato potyvirus open reading frame (ORF) is expressed via polymerase slippage and suppresses RNA silencing.

The single-stranded, positive-sense RNA genome of viruses in the genus Potyvirus encodes a large polyprotein that is cleaved to yield 10 mature proteins. The first three cleavage products are P1, HCpro and P3. An additional short open reading frame (ORF), called pipo, overlaps the P3 region of the polyprotein ORF. Four related potyviruses infecting sweet potato (Ipomoea batatas) are predicted to contain a third ORF, called pispo, which overlaps the 3' third of the P1 region. Recently, pipo has been shown to be expressed via polymerase slippage at a conserved GA6 sequence. Here, we show that pispo is also expressed via polymerase slippage at a GA6 sequence, with higher slippage efficiency (∼5%) than at the pipo site (∼1%). Transient expression of recombinant P1 or the 'transframe' product, P1N-PISPO, in Nicotiana benthamiana suppressed local RNA silencing (RNAi), but only P1N-PISPO inhibited short-distance movement of the silencing signal. These results reveal that polymerase slippage in potyviruses is not limited to pipo expression, but can be co-opted for the evolution and expression of further novel gene products.

Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins.

The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far.

A Viral Noncoding RNA Complements a Weakened Viral RNA Silencing Suppressor and Promotes Efficient Systemic Host Infection.

Systemic movement of beet necrotic yellow vein virus (BNYVV) in Beta macrocarpa depends on viral RNA3, whereas in Nicotiana benthamiana this RNA is dispensable. RNA3 contains a coremin motif of 20 nucleotides essential for the stabilization of noncoding RNA3 (ncRNA3) and for long-distance movement in Beta species. Coremin mutants that are unable to accumulate ncRNA3 also do not achieve systemic movement in Beta species. A mutant virus carrying a mutation in the p14 viral suppressor of RNA silencing (VSR), unable to move long distances, can be complemented with the ncRNA3 in the lesion phenotype, viral RNA accumulation, and systemic spread. Analyses of the BNYVV VSR mechanism of action led to the identification of the RNA-dependent RNA polymerase 6 (RDR6) pathway as a target of the virus VSR and the assignment of a VSR function to the ncRNA3.

Cytorhabdovirus P protein suppresses RISC-mediated cleavage and RNA silencing amplification in planta.

Plant viruses have evolved to undermine the RNA silencing pathway by expressing suppressor protein(s) that interfere with one or more key components of this antiviral defense. Here we show that the recently identified RNA silencing suppressor (RSS) of lettuce necrotic yellows virus (LNYV), phosphoprotein P, binds to RNA silencing machinery proteins AGO1, AGO2, AGO4, RDR6 and SGS3 in protein-protein interaction assays when transiently expressed. In planta, we demonstrate that LNYV P inhibits miRNA-guided AGO1 cleavage and translational repression, and RDR6/SGS3-dependent amplification of silencing. Analysis of LNYV P deletion mutants identified a C-terminal protein domain essential for both local RNA silencing suppression and interaction with AGO1, AGO2, AGO4, RDR6 and SGS3. In contrast to other viral RSS known to disrupt AGO activity, LNYV P sequence does not contain any recognizable GW/WG or F-box motifs. This suggests that LNYV P may represent a new class of AGO binding proteins.