Véronique Ziegler-Graff

The Polerovirus F box protein P0 targets ARGONAUTE1 to suppress RNA silencing.

Plants employ post-transcriptional gene silencing (PTGS) as an antiviral defense response. In this mechanism, viral-derived small RNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation of the corresponding viral RNAs. ARGONAUTE1 (AGO1) is a key component of RISC: it carries the RNA slicer activity. As a counter-defense, viruses have evolved various proteins that suppress PTGS. Recently, we showed that the Polerovirus P0 protein carries an F box motif required to form an SCF-like complex, which is also essential for P0s silencing suppressor function. Here, we investigate the molecular mechanism by which P0 impairs PTGS. First we show that P0s expression does not affect the biogenesis of primary siRNAs in an inverted repeat-PTGS assay, but it does affect their activity. Moreover, P0s expression in transformed Arabidopsis plants leads to various developmental abnormalities reminiscent of mutants affected in miRNA pathways, which is accompanied by enhanced levels of several miRNA-target transcripts, suggesting that P0 acts at the level of RISC. Interestingly, ectopic expression of P0 triggered AGO1 protein decay in planta. Finally, we provide evidence that P0 physically interacts with AGO1. Based on these results, we propose that P0 hijacks the host SCF machinery to modulate gene silencing by destabilizing AGO1.

P0 of Beet Western Yellows Virus Is a Suppressor of Posttranscriptional Gene Silencing

ABSTRACT Higher plants employ a homology-dependent RNA-degradation system known as posttranscriptional gene silencing (PTGS) as a defense against virus infection. Several plant viruses are known to encode proteins that can suppress PTGS. Here we show that P0 of beet western yellows virus (BWYV) displays strong silencing suppressor activity in a transient expression assay based upon its ability to inhibit PTGS of green fluorescent protein (GFP) when expressed in agro-infiltrated leaves of Nicotiana benthamiana containing a GFP transgene. PTGS suppressor activity was also observed for the P0s of two other poleroviruses, cucurbit aphid-borne yellows virus and potato leafroll virus. P0 is encoded by the 5′-proximal gene in BWYV RNA but does not accumulate to detectable levels when expressed from the genome-length RNA during infection. The low accumulation of P0 and the resulting low PTGS suppressor activity are in part a consequence of the suboptimal translation initiation context of the P0 start codon in viral RNA, although other factors, probably related to the viral replication process, also play a role. A mutation to optimize the P0 translation initiation efficiency in BWYV RNA was not stable during virus multiplication in planta. Instead, the P0 initiation codon in the progeny was frequently replaced by a less efficient initiation codon such as ACG, GTG, or ATA, indicating that there is selection against overexpression of P0 from the viral genome.

Aphid transmission of beet western yellows luteovirus requires the minor capsid read-through protein P74.

Beet western yellows luteovirus is obligately transmitted by the aphid Myzus persicae in a circulative, non‐propagative fashion. Virus movement across the epithelial cells of the digestive tube into the hemocoel and from the hemocoel into the accessory salivary glands is believed to occur by receptor‐mediated endocytosis and exocytosis. Virions contain two types of protein; the major 22 kDa capsid protein and the minor read‐through protein, P74, which is composed of the major capsid protein fused by translational read‐through to a long C‐terminal extension called the read‐through domain. Beet western yellows virus carrying various mutations in the read‐through domain was tested for its ability to be transmitted to test plants by aphids fed on agro‐infected plants and semi‐purified or purified virus preparations. The results establish that the read‐through domain carries determinants that are essential for aphid transmission. The findings also reveal that the read‐through domain is important for accumulation of the virus in agro‐infected plants.

Degradation of the antiviral component ARGONAUTE1 by the autophagy pathway.

Posttranscriptional gene silencing (PTGS) mediated by siRNAs is an evolutionarily conserved antiviral defense mechanism in higher plants and invertebrates. In this mechanism, viral-derived siRNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation of the corresponding viral RNAs. In Arabidopsis, a key component of RISC is ARGONAUTE1 (AGO1), which not only binds to siRNAs but also carries the RNA slicer activity. At present little is known about posttranslational mechanisms regulating AGO1 turnover. Here we report that the viral suppressor of RNA silencing protein P0 triggers AGO1 degradation by the autophagy pathway. Using a P0-inducible transgenic line, we observed that AGO1 degradation is blocked by inhibition of autophagy. The engineering of a functional AGO1 fluorescent reporter protein further indicated that AGO1 colocalizes with autophagy-related (ATG) protein 8a (ATG8a) positive bodies when degradation is impaired. Moreover, this pathway also degrades AGO1 in a nonviral context, especially when the production of miRNAs is impaired. Our results demonstrate that a selective process such as ubiquitylation can lead to the degradation of a key regulatory protein such as AGO1 by a degradation process generally believed to be unspecific. We anticipate that this mechanism will not only lead to degradation of AGO1 but also of its associated proteins and eventually small RNAs.

Synthesis of full-length transcripts of beet western yellows virus RNA: Messenger properties and biological activity in protoplasts

Full-length cDNA of beet western yellows virus genomic RNA has been cloned behind the bacteriophage T7 RNA polymerase promoter of the transcription vector BS(-). The in vitro run-off transcription product obtained in the presence of T7 RNA polymerase and m7GpppG cap has the same messenger properties as natural viral RNA in in vitro translation systems. The full-length transcript was also able to infect Chenopodium quinoa protoplasts inoculated by electroporation. Infection could be followed by the appearance of viral coat protein in the inoculated protoplasts and the de novo synthesis of viral RNA. Site-directed mutagenesis experiments revealed that expression of beet western yellows virus open reading frame 1 and the C-terminal portion of open reading frame 6 were not required for infection of protoplasts. Additional experiments with these mutants and mutants in the other viral open reading frames should provide information concerning the requirements for beet western yellows virus replication and, ultimately, the role of virus genes in other important steps in the virus infection cycle, such as aphid transmission.

Viral and cellular factors involved in phloem transport of plant viruses

Phloem transport of plant viruses is an essential step in the setting-up of a complete infection of a host plant. After an initial replication step in the first cells, viruses spread from cell-to-cell through mesophyll cells, until they reach the vasculature where they rapidly move to distant sites in order to establish the infection of the whole plant. This last step is referred to as systemic transport, or long-distance movement, and involves virus crossings through several cellular barriers: bundle sheath, vascular parenchyma, and companion cells for virus loading into sieve elements (SE). Viruses are then passively transported within the source-to-sink flow of photoassimilates and are unloaded from SE into sink tissues. However, the molecular mechanisms governing virus long-distance movement are far from being understood. While most viruses seem to move systemically as virus particles, some viruses are transported in SE as viral ribonucleoprotein complexes (RNP). The nature of the cellular and viral factors constituting these RNPs is still poorly known. The topic of this review will mainly focus on the host and viral factors that facilitate or restrict virus long-distance movement.

Effects of Point Mutations in the Readthrough Domain of the Beet Western Yellows Virus Minor Capsid Protein on Virus Accumulation In Planta and on Transmission by Aphids

ABSTRACT Point mutations were introduced into or near five conserved sequence motifs of the readthrough domain of the beet western yellows virus minor capsid protein P74. The mutant virus was tested for its ability to accumulate efficiently in agroinfected plants and to be transmitted by its aphid vector, Myzus persicae. The stability of the mutants in the agroinfected and aphid-infected plants was followed by sequence analysis of the progeny virus. Only the mutation Y201D was found to strongly inhibit virus accumulation in planta following agroinfection, but high accumulation levels were restored by reversion or pseudoreversion at this site. Four of the five mutants were poorly aphid transmissible, but in three cases successful transmission was restored by pseudoreversion or second-site mutations. The same second-site mutations in the nonconserved motif PVT(32-34) were shown to compensate for two distinct primary mutations (R24A and E59A/D60A), one on each side of the PVT sequence. In the latter case, a second-site mutation in the PVT motif restored the ability of the virus to move from the hemocoel through the accessory salivary gland following microinjection of mutant virus into the aphid hemocoel but did not permit virus movement across the epithelium separating the intestine from the hemocoel. Successful movement of the mutant virus across both barriers was accompanied by conversion of A59 to E or T, indicating that distinct features of the readthrough domain in this region operate at different stages of the transmission process.

The Polerovirus Minor Capsid Protein Determines Vector Specificity and Intestinal Tropism in the Aphid

ABSTRACT Aphid transmission of poleroviruses is highly specific, but the viral determinants governing this specificity are unknown. We used a gene exchange strategy between two poleroviruses with different vectors, Beet western yellows virus (BWYV) and Cucurbit aphid-borne yellows virus (CABYV), to analyze the role of the major and minor capsid proteins in vector specificity. Virus recombinants obtained by exchanging the sequence of the readthrough domain (RTD) between the two viruses replicated in plant protoplasts and in whole plants. The hybrid readthrough protein of chimeric viruses was incorporated into virions. Aphid transmission experiments using infected plants or purified virions revealed that vector specificity is driven by the nature of the RTD. BWYV and CABYV have specific intestinal sites in the vectors for endocytosis: the midgut for BWYV and both midgut and hindgut for CABYV. Localization of hybrid virions in aphids by transmission electron microscopy revealed that gut tropism is also determined by the viral origin of the RTD.

Effects of Point Mutations in the Major Capsid Protein of Beet Western Yellows Virus on Capsid Formation, Virus Accumulation, and Aphid Transmission

ABSTRACT Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected ∼90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a “reverse” substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid.

Studies on the role of the minor capsid protein in transport of Beet western yellows virus through Myzus persicae

Beet western yellows virus (BWYV), family Luteoviridae, is an icosahedral plant virus which is strictly transmitted by aphids in a persistent and circulative manner. Virions cross two cellular barriers in the aphid by receptor-based mechanisms involving endocytosis and exocytosis. Particles are first transported across intestinal cells into the haemolymph and then across accessory salivary gland cells for delivery to the plant via saliva. We identified the midgut part of the digestive tract as the site of intestinal passage by BWYV virions. To analyse the role in transmission of the minor capsid component, the readthrough (RT) protein, the fate of a BWYV RT-deficient non-transmissible mutant was followed by transmission electron microscopy in the vector Myzus persicae. This mutant was observed in the gut lumen but was never found inside midgut cells. However, virion aggregates were detected in the basal lamina of midgut cells when BWYV antiserum was microinjected into the haemolymph. The presence of virions in the haemolymph was confirmed by a sensitive molecular technique for detecting viral RNA. Thus, transport of the mutant virions through intestinal cells occurred but at a low frequency. Even when microinjected into the haemolymph, the RT protein mutant was never detected near or in the accessory salivary gland cells. We conclude that the RT protein is not strictly required for the transport of virus particles through midgut cells, but is necessary for the maintenance of virions in the haemolymph and their passage through accessory salivary gland cells.