Nuclear export of plant pararetrovirus mRNAs involves the TREX complex, two viral proteins and the highly structured 5' leader region.

In eukaryotes, the major nuclear export pathway for mature mRNAs uses the dimeric receptor TAP/p15, which is recruited to mRNAs via the multisubunit TREX complex, comprising the THO core and different export adaptors. Viruses that replicate in the nucleus adopt different strategies to hijack cellular export factors and achieve cytoplasmic translation of their mRNAs. No export receptors are known in plants, but Arabidopsis TREX resembles the mammalian complex, with a conserved hexameric THO core associated with ALY and UIEF proteins, as well as UAP56 and MOS11. The latter protein is an orthologue of mammalian CIP29. The nuclear export mechanism for viral mRNAs has not been described in plants. To understand this process, we investigated the export of mRNAs of the pararetrovirus CaMV in Arabidopsis and demonstrated that it is inhibited in plants deficient in ALY, MOS11 and/or TEX1. Deficiency for these factors renders plants partially resistant to CaMV infection. Two CaMV proteins, the coat protein P4 and reverse transcriptase P5, are important for nuclear export. P4 and P5 interact and co-localise in the nucleus with the cellular export factor MOS11. The highly structured 5' leader region of 35S RNAs was identified as an export enhancing element that interacts with ALY1, ALY3 and MOS11 in vitro.

Strength in Diversity: Nuclear Export of Viral RNAs.

The nuclear export of cellular mRNAs is a complex process that requires the orchestrated participation of many proteins that are recruited during the early steps of mRNA synthesis and processing. This strategy allows the cell to guarantee the conformity of the messengers accessing the cytoplasm and the translation machinery. Most transcripts are exported by the exportin dimer Nuclear RNA export factor 1 (NXF1)-NTF2-related export protein 1 (NXT1) and the transcription-export complex 1 (TREX1). Some mRNAs that do not possess all the common messenger characteristics use either variants of the NXF1-NXT1 pathway or CRM1, a different exportin. Viruses whose mRNAs are synthesized in the nucleus (retroviruses, the vast majority of DNA viruses, and influenza viruses) exploit both these cellular export pathways. Viral mRNAs hijack the cellular export machinery via complex secondary structures recognized by cellular export factors and/or viral adapter proteins. This way, the viral transcripts succeed in escaping the host surveillance system and are efficiently exported for translation, allowing the infectious cycle to proceed. This review gives an overview of the cellular mRNA nuclear export mechanisms and presents detailed insights into the most important strategies that viruses use to export the different forms of their RNAs from the nucleus to the cytoplasm.

[Nuclear export of viral RNAs in metazoan]. / Export nucléaire des ARN viraux chez les métazoaires.

The nuclear export of mRNAs is a complex process, involving the participaton of numerous proteins, the recruitement of which starts during the early steps of mRNAs biosynthesis and maturation. This strategy allows the cell to export only mature and non-defective transcripts to the cytoplasm where they are directed to the translational machinery. The vast majority of mRNAs is exported by the dimeric transport receptor TAP-p15, which is mainly recruited by the large multiprotein complex TREX-1. Other mRNAs that do not display all typical features of a mature transcript use variants of the TAP-p15 export pathway or recruit the alternative export receptor CRM1. Most DNA viruses, retroviruses, and influenza viruses, the mRNAs of which are synthesized in the nucleus, also use TAP-p15 and/or CRM1 to export their mRNAs. The highjacking of the cellular export machinery by viral mRNAs usually involves the presence of constitutive structural elements that directly load cellular export factors and/or viral adaptor proteins. Associated with the host export machinery, viral mRNAs escape host surveillance, are efficiently exported in the cytoplasm in order to be translated, and thus make possible the progress toward the later events of the virus life cycles.

Transfection of Small Noncoding RNAs into Arabidopsis thaliana Protoplasts.

Polyethylene glycol transfection of plant protoplasts represents an efficient method to incorporate foreign DNA and study transient gene expression. Here, we describe an optimized protocol to deliver small noncoding RNAs into Arabidopsis thaliana protoplasts. An example of application is provided by demonstrating the incorporation of a 20 nt long small noncoding RNA deriving from the 5' extremity of an A. thaliana cytosolic alanine tRNA into freshly isolated protoplasts.

Formation of large viroplasms and virulence of Cauliflower mosaic virus in turnip plants depend on the N-terminal EKI sequence of viral protein TAV.

Cauliflower mosaic virus (CaMV) TAV protein (TransActivator/Viroplasmin) plays a pivotal role during the infection cycle since it activates translation reinitiation of viral polycistronic RNAs and suppresses RNA silencing. It is also the major component of cytoplasmic electron-dense inclusion bodies (EDIBs) called viroplasms that are particularly evident in cells infected by the virulent CaMV Cabb B-JI isolate. These EDIBs are considered as virion factories, vehicles for CaMV intracellular movement and reservoirs for CaMV transmission by aphids. In this study, focused on different TAV mutants in vivo, we demonstrate that three physically separated domains collectively participate to the formation of large EDIBs: the N-terminal EKI motif, a sequence of the MAV domain involved in translation reinitiation and a C-terminal region encompassing the zinc finger. Surprisingly, EKI mutant TAVm3, corresponding to a substitution of the EKI motif at amino acids 11-13 by three alanines (AAA), which completely abolished the formation of large viroplasms, was not lethal for CaMV but highly reduced its virulence without affecting the rate of systemic infection. Expression of TAVm3 in a viral context led to formation of small irregularly shaped inclusion bodies, mild symptoms and low levels of viral DNA and particles accumulation, despite the production of significant amounts of mature capsid proteins. Unexpectedly, for CaMV-TAVm3 the formation of viral P2-containing electron-light inclusion body (ELIB), which is essential for CaMV aphid transmission, was also altered, thus suggesting an indirect role of the EKI tripeptide in CaMV plant-to-plant propagation. This important functional contribution of the EKI motif in CaMV biology can explain the strict conservation of this motif in the TAV sequences of all CaMV isolates.

GTPase ROP2 binds and promotes activation of target of rapamycin, TOR, in response to auxin.

Target of rapamycin (TOR) promotes reinitiation at upstream ORFs (uORFs) in genes that play important roles in stem cell regulation and organogenesis in plants. Here, we report that the small GTPase ROP2, if activated by the phytohormone auxin, promotes activation of TOR, and thus translation reinitiation of uORF-containing mRNAs. Plants with high levels of active ROP2, including those expressing constitutively active ROP2 (CA-ROP2), contain high levels of active TOR ROP2 physically interacts with and, when GTP-bound, activates TOR in vitro TOR activation in response to auxin is abolished in ROP-deficient rop2 rop6 ROP4 RNAi plants. GFP-TOR can associate with endosome-like structures in ROP2-overexpressing plants, indicating that endosomes mediate ROP2 effects on TOR activation. CA-ROP2 is efficient in loading uORF-containing mRNAs onto polysomes and stimulates translation in protoplasts, and both processes are sensitive to TOR inhibitor AZD-8055. TOR inactivation abolishes ROP2 regulation of translation reinitiation, but not its effects on cytoskeleton or intracellular trafficking. These findings imply a mode of translation control whereby, as an upstream effector of TOR, ROP2 coordinates TOR function in translation reinitiation pathways in response to auxin.

Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern.

The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splicing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5' region and suggested that the main role of CaMV splicing is to downregulate expression of open reading frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA.

Le virus de la mosaïque du chou-fleur (CaMV), encore et toujours.

As a pararetrovirus and because of the non-canonical translation of its polycistronic pregenomic 35S RNA, Cauliflower mosaic virus (CaMV) is an original model system that has been extensively studied. Recent advances have improved our understanding of CaMV aphid transmission, cell-to-cell movement, protein expression and virus counter-defense strategy against host plant defense. Since P6/TAV is involved in many aspects of viral pathogenesis as well as in some replication steps, it is considered as the key player of CaMV infectious cycle. This paper reviews our current knowledge on CaMV multiplication and pathogenesis, with special emphasis on steps in which P6/TAV has a major role.

TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h.

Mammalian target-of-rapamycin (mTOR) triggers S6 kinase (S6K) activation to phosphorylate targets linked to translation in response to energy, nutrients, and hormones. Pathways of TOR activation in plants remain unknown. Here, we uncover the role of the phytohormone auxin in TOR signalling activation and reinitiation after upstream open reading frame (uORF) translation, which in plants is dependent on translation initiation factor eIF3h. We show that auxin triggers TOR activation followed by S6K1 phosphorylation at T449 and efficient loading of uORF-mRNAs onto polysomes in a manner sensitive to the TOR inhibitor Torin-1. Torin-1 mediates recruitment of inactive S6K1 to polysomes, while auxin triggers S6K1 dissociation and recruitment of activated TOR instead. A putative target of TOR/S6K1-eIF3h-is phosphorylated and detected in polysomes in response to auxin. In TOR-deficient plants, polysomes were prebound by inactive S6K1, and loading of uORF-mRNAs and eIF3h was impaired. Transient expression of eIF3h-S178D in plant protoplasts specifically upregulates uORF-mRNA translation. We propose that TOR functions in polysomes to maintain the active S6K1 (and thus eIF3h) phosphorylation status that is critical for translation reinitiation.

Viral factor TAV recruits TOR/S6K1 signalling to activate reinitiation after long ORF translation.

The protein kinase TOR (target-of-rapamycin) upregulates translation initiation in eukaryotes, but initiation restart after long ORF translation is restricted by largely unknown pathways. The plant viral reinitiation factor transactivator-viroplasmin (TAV) exceptionally promotes reinitiation through a mechanism involving retention on 80S and reuse of eIF3 and the host factor reinitiation-supporting protein (RISP) to regenerate reinitiation-competent ribosomal complexes. Here, we show that TAV function in reinitiation depends on physical association with TOR, with TAV-TOR binding being critical for both translation reinitiation and viral fitness. Consistently, TOR-deficient plants are resistant to viral infection. TAV triggers TOR hyperactivation and S6K1 phosphorylation in planta. When activated, TOR binds polyribosomes concomitantly with polysomal accumulation of eIF3 and RISP--a novel and specific target of TOR/S6K1--in a TAV-dependent manner, with RISP being phosphorylated. TAV mutants defective in TOR binding fail to recruit TOR, thereby abolishing RISP phosphorylation in polysomes and reinitiation. Thus, activation of reinitiation after long ORF translation is more complex than previously appreciated, with TOR/S6K1 upregulation being the key event in the formation of reinitiation-competent ribosomal complexes.

A new plant protein interacts with eIF3 and 60S to enhance virus-activated translation re-initiation.

The plant viral re-initiation factor transactivator viroplasmin (TAV) activates translation of polycistronic mRNA by a re-initiation mechanism involving translation initiation factor 3 (eIF3) and the 60S ribosomal subunit (60S). QJ;Here, we report a new plant factor-re-initiation supporting protein (RISP)-that enhances TAV function in re-initiation. RISP interacts physically with TAV in vitro and in vivo. Mutants defective in interaction are less active, or inactive, in transactivation and viral amplification. RISP alone can serve as a scaffold protein, which is able to interact with eIF3 subunits a/c and 60S, apparently through the C-terminus of ribosomal protein L24. RISP pre-bound to eIF3 binds 40S, suggesting that RISP enters the translational machinery at the 43S formation step. RISP, TAV and 60S co-localize in epidermal cells of infected plants, and eIF3-TAV-RISP-L24 complex formation can be shown in vitro. These results suggest that RISP and TAV bridge interactions between eIF3-bound 40S and L24 of 60S after translation termination to ensure 60S recruitment during repetitive initiation events on polycistronic mRNA; RISP can thus be considered as a new component of the cell translation machinery.

Nuclear import of CaMV P6 is required for infection and suppression of the RNA silencing factor DRB4.

Replication of Cauliflower mosaic virus (CaMV), a plant double-stranded DNA virus, requires the viral translational transactivator protein P6. Although P6 is known to form cytoplasmic inclusion bodies (viroplasms) so far considered essential for virus biology, a fraction of the protein is also present in the nucleus. Here, we report that monomeric P6 is imported into the nucleus through two importin-alpha-dependent nuclear localization signals, and show that this process is mandatory for CaMV infectivity and is independent of translational transactivation and viroplasm formation. One nuclear function of P6 is to suppress RNA silencing, a gene regulation mechanism with antiviral roles, commonly counteracted by dedicated viral suppressor proteins (viral silencing suppressors; VSRs). Transgenic P6 expression in Arabidopsis is genetically equivalent to inactivating the nuclear protein DRB4 that facilitates the activity of the major plant antiviral silencing factor DCL4. We further show that a fraction of P6 immunoprecipitates with DRB4 in CaMV-infected cells. This study identifies both genetic and physical interactions between a VSR to a host RNA silencing component, and highlights the importance of subcellular compartmentalization in VSR function.

Functional characterization of the Beet necrotic yellow vein virus RNA-5-encoded p26 protein: evidence for structural pathogenicity determinants.

A Beet necrotic yellow vein virus isolate containing a fifth RNA is present in the Pithiviers area of France. A full-length cDNA clone of RNA-5 was obtained and placed under the control of a T(7)-RNA-pol promoter that allowed the production of infectious transcripts. "Pithiviers" isolate-specific necrotic symptoms were obtained on Chenopodium quinoa when RNA-5-encoded p26 was expressed either from RNA-5 or from an RNA-3-derived replicon. By using haemagglutinin- and green fluorescent protein-tagged constructs, virally expressed p26-fusion proteins induced the same necrotic local lesions on host plants and were localized mainly in the nucleus of infected cells. Deletion mutagenesis permitted identification of two domains, responsible respectively for nuclear export and cytoplasmic retention of the p26 mutated proteins. By using a yeast two-hybrid system, Gal4DB-p26 protein self-activated transcription of the His3 reporter gene. The p26 transcription-activation domain was located within its first 55 aa and has been studied by alanine scanning. Resulting p26 mutants were tested for their capability to induce necrotic symptoms and to localize in the nuclear compartment.

Quantification of the 35S promoter in DNA extracts from genetically modified organisms using real-time polymerase chain reaction and specificity assessment on various genetically modified organisms, part I: operating procedure.

A highly sensitive quantitative real-time assay targeted on the 35S promoter of a commercial genetically modified organism (GMO) was characterized (sF/sR primers) and developed for an ABI Prism 7700 Sequence Detection System and TaqMan chemistry. The specificity assessment and performance criteria of sF/sR assay were compared to other P35S-targeted published assays. sF/sR primers amplified a 79 base pair DNA sequence located in a part of P35S that is highly conserved among many caulimovirus strains, i.e., this consensus part of CaMV P35S is likely to be present in many GM events. According to the experimental conditions, the absolute limit of detection for Bt176 corn was estimated between 0.2 and 2 copies of equivalent genome (CEG). The limit of quantification was reached below 0.1% Bt176 content. A Cauliflower Mosaic Virus control (CaMV) qualitative assay targeted on the ORF III of the viral genome was also used as a control (primers 3F/3R) to assess the presence of CaMV in plant-derived products. The specificity of this test was assessed on various CaMV strains, including the Figwort Mosaic Virus (FMV) and solanaceous CaMV strains. Considering the performance of sF/sR quantification test, the highly conserved sequence, and the small size of the amplicon, this assay was tested in a collaborative study in order to be proposed as an international standard.

The open reading frame VI product of Cauliflower mosaic virus is a nucleocytoplasmic protein: its N terminus mediates its nuclear export and formation of electron-dense viroplasms.

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replication and assembly occur. In this study, the mechanism involved in viroplasm formation was investigated by in vitro and in vivo experiments. Far protein gel blot assays using a collection of P6 deletion mutants demonstrated that the N-terminal alpha-helix of P6 mediates interaction between P6 molecules. Transient expression in tobacco (Nicotiana tabacum) BY-2 cells of full-length P6 and P6 mutants fused to enhanced green fluorescent protein revealed that viroplasms are formed at the periphery of the nucleus and that the N-terminal domain of P6 is an important determinant in this process. Finally, this study led to the unexpected finding that P6 is a nucleocytoplasmic shuttle protein and that its nuclear export is mediated by a Leu-rich sequence that is part of the alpha-helix domain implicated in viroplasm formation. The discovery that P6 can localize to the nucleus opens new prospects for understanding yet unknown roles of this viral protein in the course of the CaMV infection cycle.

P6 protein of Cauliflower mosaic virus, a translation reinitiator, interacts with ribosomal protein L13 from Arabidopsis thaliana.

The P6 protein of Cauliflower mosaic virus (CaMV) transactivates translation of the CaMV 35S polycistronic pregenomic RNA and its spliced versions, and thus allows synthesis of a complete set of viral proteins. Previous studies have shown that P6 interacts with plant L18 and L24 ribosomal proteins and initiation factor eIF3, and it has been proposed that these interactions are involved in the reinitiation of translation of polycistronic viral RNAs. This study characterizes a novel cellular partner of P6, the ribosomal protein L13 from Arabidopsis thaliana. Far-Western assays performed with several P6 deletion mutants have shown that L13 interacts with the miniTAV of P6, which represents the minimal domain for transactivation, suggesting that the P6-L13 interaction might also be involved in this process. L13 and L18 were found to bind to the same region within the miniTAV. Competition assays between L18 and L13 for binding to miniTAV suggest that interactions between P6 and these ribosomal proteins involve separate P6 molecules, and/or occur at different stages of translation or in the context of another function also mediated by P6.

Cauliflower mosaic virus: still in the news.

SUMMARY Taxonomic relationship: Cauliflower mosaic virus (CaMV) is the type member of the Caulimovirus genus in the Caulimoviridae family, which comprises five other genera. CaMV replicates its DNA genome by reverse transcription of a pregenomic RNA and thus belongs to the pararetrovirus supergroup, which includes the Hepadnaviridae family infecting vertebrates. Physical properties: Virions are non-enveloped isometric particles, 53 nm in diameter (Fig. 1). They are constituted by 420 capsid protein subunits organized following T= 7 icosahedral symmetry (Cheng, R.H., Olson, N.H. and Baker, T.S. (1992) Cauliflower mosaic virus: a 420 subunit (T= 7), multilayer structure. Virology, 16, 655-668). The genome consists of a double-stranded circular DNA of approximately 8000 bp that is embedded in the inner surface of the capsid. Viral proteins: The CaMV genome encodes six proteins, a cell-to-cell movement protein (P1), two aphid transmission factors (P2 and P3), the precursor of the capsid proteins (P4), a polyprotein precursor of proteinase, reverse transcriptase and ribonuclease H (P5) and an inclusion body protein/translation transactivator (P6). Hosts: The host range of CaMV is limited to plants of the Cruciferae family, i.e. Brassicae species and Arabidopsis thaliana, but some viral strains can also infect solanaceous plants. In nature, CaMV is transmitted by aphids in a non-circulative manner.