Molecular dissection of the prototype foamy virus (PFV) RNA 5′-UTR identifies essential elements of a ribosomal shunt

The prototype foamy virus (PFV) is a nonpathogenic retrovirus that shows promise as a vector for gene transfer. The PFV (pre)genomic RNA starts with a long complex leader that can be folded into an elongated hairpin, suggesting an alternative strategy to cap-dependent linear scanning for translation initiation of the downstream GAG open reading frame (ORF). We found that the PFV leader carries several short ORFs (sORFs), with the three 5′-proximal sORFs located upstream of a structural element. Scanning-inhibitory hairpin insertion analysis suggested a ribosomal shunt mechanism, whereby ribosomes start scanning at the leader 5′-end and initiate at the downstream ORF via bypass of the central leader regions, which are inhibitory for scanning. We show that the efficiency of shunting depends strongly on the stability of the structural element located downstream of either sORFs A/A′ or sORF B, and on the translation event at the corresponding 5′-proximal sORF. The PFV shunting strategy mirrors that of Cauliflower mosaic virus in plants; however, in mammals shunting can operate in the presence of a less stable structural element, although it is greatly improved by increasing the number of base pairings. At least one shunt configuration was found in primate FV (pre)genomic RNAs

Short ORF-Dependent Ribosome Shunting Operates in an RNA Picorna-Like Virus and a DNA Pararetrovirus that Cause Rice Tungro Disease

Rice tungro disease is caused by synergistic interaction of an RNA picorna-like virus Rice tungro spherical virus (RTSV) and a DNA pararetrovirus Rice tungro bacilliform virus (RTBV). It is spread by insects owing to an RTSV-encoded transmission factor. RTBV has evolved a ribosome shunt mechanism to initiate translation of its pregenomic RNA having a long and highly structured leader. We found that a long leader of RTSV genomic RNA remarkably resembles the RTBV leader: both contain several short ORFs (sORFs) and potentially fold into a large stem-loop structure with the first sORF terminating in front of the stem basal helix. Using translation assays in rice protoplasts and wheat germ extracts, we show that, like in RTBV, both initiation and proper termination of the first sORF translation in front of the stem are required for shunt-mediated translation of a reporter ORF placed downstream of the RTSV leader. The base pairing that forms the basal helix is required for shunting, but its sequence can be varied. Shunt efficiency in RTSV is lower than in RTBV. But in addition to shunting the RTSV leader sequence allows relatively efficient linear ribosome migration, which also contributes to translation initiation downstream of the leader. We conclude that RTSV and RTBV have developed a similar, sORF-dependent shunt mechanism possibly to adapt to the host translation system and/or coordinate their life cycles. Given that sORF-dependent shunting also operates in a pararetrovirus Cauliflower mosaic virus and likely in other pararetroviruses that possess a conserved shunt configuration in their leaders it is tempting to propose that RTSV may have acquired shunt cis-elements from RTBV during their co-existence

Réinitiation de la traduction dépendante de TAV au cours de l infection des plantes par le virus de la mosaïque du chou-fleur (Caractérisation fonctionnelle d'une nouvelle protéine cellulaire RISP)

Le virus de la mosaïque du chou fleur (CaMV) a développé une stratégie de réinitiation de la traduction pour synthétiser l ensemble des protéines virales à partir de leurs ARN polycistroniques.Cette stratégie encore peu comprise consiste à exploiter la machinerie traductionnelle grâce à une protéine virale, TAV (transactivator/ viroplasmin). Au cours de ma thèse, nous avons isolé un nouveau co-facteur de TAV qui favorise la réinitiation de la traduction nommé RISP, Reinitiation Supporting Factor. RISP interagit avec le domaine essentiel de TAV pour la réinitiation de la traduction ainsi qu avec la sous unité ribosomique 60S.L expression transitoire de RISP dans des protoplastes augmentent fortement l activité de transactivation traductionnelle. Nous avons démontré la formation du complexe TAV/RISP/60S in vitro et suggérons l importance de ce complexe pour le recrutement efficace de 60S par la sous unité 40S lors de la traduction afin de favoriser la réinitiation de la traduction.The cauliflower mosaic virus has developed an original translation reinitiation strategy to synthesize viral proteins from polycistronic mRNA. This strategy is very complex and not very well-known but involves the viral protein TAV (transactivator/ viroplasmin) wich activates alternative cellular pathways to allow translation reinitiation of major open reading frames on the polycistronic viral RNA. In my Phd, we have shown that TAV reinitiation function depends on a novel cellular protein RISP, reinitiation supporting protein. RISP physically interacts with TAV via TAV's essential transactivation domain, and may bind the 60S ribosomal subunit. Transient expression of RISP in plant protoplasts strongly increases TAV transactivation activity. We show TAV/RISP/60S complex formation in vitro, recruitment of RISP by TAV to polysomes, and suggest the involvement of the TAV/RISP/60S complex in efficient 60S recruitment to polysomes thus increasing the probability of the reinitiation event.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Auxin Signaling in Regulation of Plant Translation Reinitiation

The mRNA translation machinery directs protein production, and thus cell growth, according to prevailing cellular and environmental conditions. The target of rapamycin (TOR) signaling pathway—a major growth-related pathway—plays a pivotal role in optimizing protein synthesis in mammals, while its deregulation triggers uncontrolled cell proliferation and the development of severe diseases. In plants, several signaling pathways sensitive to environmental changes, hormones, and pathogens have been implicated in post-transcriptional control, and thus far phytohormones have attracted most attention as TOR upstream regulators in plants. Recent data have suggested that the coordinated actions of the phytohormone auxin, Rho-like small GTPases (ROPs) from plants, and TOR signaling contribute to translation regulation of mRNAs that harbor upstream open reading frames (uORFs) within their 5′-untranslated regions (5′-UTRs). This review will summarize recent advances in translational regulation of a specific set of uORF-containing mRNAs that encode regulatory proteins—transcription factors, protein kinases and other cellular controllers—and how their control can impact plant growth and development

Ribosome Shunting, Polycistronic Translation, and Evasion of Antiviral Defenses in Plant Pararetroviruses and Beyond

Viruses have compact genomes and usually translate more than one protein from polycistronic RNAs using leaky scanning, frameshifting, stop codon suppression or reinitiation mechanisms. Viral (pre-)genomic RNAs often contain long 5′-leader sequences with short upstream open reading frames (uORFs) and secondary structure elements, which control both translation initiation and replication. In plants, viral RNA and DNA are targeted by RNA interference (RNAi) generating small RNAs that silence viral gene expression, while viral proteins are recognized by innate immunity and autophagy that restrict viral infection. In this review we focus on plant pararetroviruses of the family Caulimoviridae and describe the mechanisms of uORF- and secondary structure-driven ribosome shunting, leaky scanning and reinitiation after translation of short and long uORFs. We discuss conservation of these mechanisms in different genera of Caulimoviridae, including host genome-integrated endogenous viral elements, as well as in other viral families, and highlight a multipurpose use of the highly-structured leader sequence of plant pararetroviruses in regulation of translation, splicing, packaging, and reverse transcription of pregenomic RNA (pgRNA), and in evasion of RNAi. Furthermore, we illustrate how targeting of several host factors by a pararetroviral effector protein can lead to transactivation of viral polycistronic translation and concomitant suppression of antiviral defenses. Thus, activation of the plant protein kinase target of rapamycin (TOR) by the Cauliflower mosaic virus transactivator/viroplasmin (TAV) promotes reinitiation of translation after long ORFs on viral pgRNA and blocks antiviral autophagy and innate immunity responses, while interaction of TAV with the plant RNAi machinery interferes with antiviral silencing

Target of Rapamycin kinase: central regulatory hub for plant growth and metabolism

International audienceThe adaptation of plants to their environment requires tight regulation of metabolism and growth processes through central and highly connected signalling pathways. The signalling cascade involving the evolutionarily conserved Target of Rapamycin (TOR) represents just such a central regulatory hub, and research on this protein kinase in plants has progressed significantly during the past decade. TOR is now firmly established as a central player in plant responses to the availability of nutrients such as sugars, stresses including those from pathogens, and hormones. Moreover plant-specific targets and regulators have recently been identified. The reviews in this special issue explore the various facets of regulation exerted by this fascinating kinase as well as its potential for crop improvement

Translation reinitiation and leaky scanning in plant viruses

While translation of mRNAs in eukaryotic cells in general follows strict rules, viruses infecting these cells break those rules in various ways. Viruses are under high selection pressure to compete with the host, to economize genome size, and to accommodate signals for replication, virus assembly, etc., on their RNAs as well as using them for translation. The cornucopia of extraordinary translation strategies, such as leaky scanning, internal initiation of translation, ribosome shunt, and virus-controlled reinitiation of translation, evolved by viruses continues to surprise and inform our understanding of general translation mechanisms. While internal initiation is treated in another section of this issue, we concentrate on leaky scanning, shunt and reinitiation, with emphasis on plant pararetroviruses

Mechanism of ribosome shunting in Rice tungro bacilliform pararetrovirus

In plant pararetroviruses, pregenomic RNA serves both as a template for replication through reverse transcription and a polysictronic mRNA. This RNA has a complex leader sequence preceding the first large ORF. The leader contains multiple short ORFs and strong secondary structure, both inhibiting ribosome scanning. Translation on this RNA is initiated by shunting, in which scanning ribosomes bypass a large portion of the leader with the inhibitory secondary structure and short ORFs. In Cauliflower mosaic virus (CaMV), the ribosome shunting mechanism involves translation of the 5′-proximal short ORF terminating in front of the secondary structure that appears to force ribosomes to take off and resume scanning at a landing site downstream of the structure. Using two plant protoplast systems and shunt-competent wheat-germ extracts, we demonstrate that in Rice tungro bacilliform virus (RTBV) shunting also depends on the first short ORF followed by strong secondary structure. Swapping of the conserved shunt elements between CaMV and RTBV revealed the importance of nucleotide composition of the landing sequence for efficient shunting. The results suggest that the mechanism of ribosome shunting is evolutionary conserved in plant pararetroviruses