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1.
Nucleic Acids Res ; 49(15): 8900-8922, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34370034

RESUMO

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.


Assuntos
Regiões 5' não Traduzidas , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/virologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Arabidopsis/virologia , Proteínas de Arabidopsis/fisiologia , Proteínas do Capsídeo/metabolismo , Caulimovirus/genética , Caulimovirus/metabolismo , Núcleo Celular/metabolismo , Doenças das Plantas/virologia , RNA Viral/química , DNA Polimerase Dirigida por RNA/metabolismo
2.
PLoS One ; 16(7): e0253488, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34242244

RESUMO

The untranslated regions (UTRs) of mRNAs are involved in many posttranscriptional regulatory pathways. The rice OsMac1 mRNA has three splicing variants of the 5' UTR (UTRa, UTRb, and UTRc), which include a CU-rich region and three upstream open reading frames (uORFs). UTRc contains an additional 38-nt sequence, termed sp38, which acts as a strong translational enhancer of the downstream ORF; reporter analysis revealed translational efficiencies >15-fold higher with UTRc than with the other splice variants. Mutation analysis of UTRc demonstrated that an optimal sequence length of sp38, rather than its nucleotide sequence is essential for UTRc to promote efficient translation. In addition, the 5' 100 nucleotides of CU-rich region contribute to UTRc translational enhancement. Strikingly, three uORFs did not reveal their inhibitory potential within the full-length leader, whereas deletion of the 5' leader fragment preceding the leader region with uORFs nearly abolished translation. Computational prediction of UTRc structural motifs revealed stem-loop structures, termed SL1-SL4, and two regions, A and B, involved in putative intramolecular interactions. Our data suggest that SL4 binding to Region-A and base pairing between Region-B and the UTRc 3'end are critically required for translational enhancement. Since UTRc is not capable of internal initiation, we presume that the three-dimensional leader structures can allow translation of the leader downstream ORF, likely allowing the bypass of uORFs.

3.
Nucleic Acids Res ; 49(12): 6908-6924, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34133725

RESUMO

Reinitiation supporting protein, RISP, interacts with 60S (60S ribosomal subunit) and eIF3 (eukaryotic initiation factor 3) in plants. TOR (target-of-rapamycin) mediates RISP phosphorylation at residue Ser267, favoring its binding to eL24 (60S ribosomal protein L24). In a viral context, RISP, when phosphorylated, binds the CaMV transactivator/ viroplasmin, TAV, to assist in an exceptional mechanism of reinitiation after long ORF translation. Moreover, we show here that RISP interacts with eIF2 via eIF2ß and TOR downstream target 40S ribosomal protein eS6. A RISP phosphorylation knockout, RISP-S267A, binds preferentially eIF2ß, and both form a ternary complex with eIF3a in vitro. Accordingly, transient overexpression in plant protoplasts of RISP-S267A, but not a RISP phosphorylation mimic, RISP-S267D, favors translation initiation. In contrast, RISP-S267D preferentially binds eS6, and, when bound to the C-terminus of eS6, can capture 60S in a highly specific manner in vitro, suggesting that it mediates 60S loading during reinitiation. Indeed, eS6-deficient plants are highly resistant to CaMV due to their reduced reinitiation capacity. Strikingly, an eS6 phosphomimic, when stably expressed in eS6-deficient plants, can fully restore the reinitiation deficiency of these plants in cellular and viral contexts. These results suggest that RISP function in translation (re)initiation is regulated by phosphorylation at Ser267.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Iniciação Traducional da Cadeia Peptídica , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Caulimovirus , Fator de Iniciação 2B em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Fosforilação , Proteína S6 Ribossômica/genética , Proteína S6 Ribossômica/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo
4.
Sci Rep ; 9(1): 7042, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31065034

RESUMO

During pathogenesis, viruses hijack the host cellular machinery to access molecules and sub-cellular structures needed for infection. We have evidence that the multifunctional viral translation transactivator/viroplasmin (TAV) protein from Cauliflower mosaic virus (CaMV) can function as a suppressor of nonsense-mediated mRNA decay (NMD). TAV interacts specifically with a scaffold protein of the decapping complex VARICOSE (VCS) in the yeast two-hybrid system, and co-localizes with components of the decapping complex in planta. Notably, plants transgenic for TAV accumulate endogenous NMD-elicited mRNAs, while decay of AU-rich instability element (ARE)-signal containing mRNAs are not affected. Using an agroinfiltration-based transient assay we confirmed that TAV specifically stabilizes mRNA containing a premature termination codon (PTC) in a VCS-dependent manner. We have identified a TAV motif consisting of 12 of the 520 amino acids in the full-length sequence that is critical for both VCS binding and the NMD suppression effect. Our data suggest that TAV can intercept NMD by targeting the decapping machinery through the scaffold protein VARICOSE, indicating that 5'-3' mRNA decapping is a late step in NMD-related mRNA degradation in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Caulimovirus/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Degradação do RNAm Mediada por Códon sem Sentido , Proteínas Virais/metabolismo , Arabidopsis/genética , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Caulimovirus/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Folhas de Planta/virologia , Plantas Geneticamente Modificadas , Tabaco/genética , Tabaco/virologia , Técnicas do Sistema de Duplo-Híbrido
6.
Front Microbiol ; 9: 644, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29692761

RESUMO

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.

8.
Front Plant Sci ; 8: 1014, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28659957

RESUMO

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.

9.
EMBO J ; 36(7): 886-903, 2017 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-28246118

RESUMO

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.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Ligação Proteica
10.
Front Plant Sci ; 7: 1611, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27877176

RESUMO

Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5' untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5' terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase.

11.
New Phytol ; 211(3): 1020-34, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27120694

RESUMO

Virus interactions with plant silencing and innate immunity pathways can potentially alter the susceptibility of virus-infected plants to secondary infections with nonviral pathogens. We found that Arabidopsis plants infected with Cauliflower mosaic virus (CaMV) or transgenic for CaMV silencing suppressor P6 exhibit increased susceptibility to Pseudomonas syringae pv. tomato (Pst) and allow robust growth of the Pst mutant hrcC-, which cannot deploy effectors to suppress innate immunity. The impaired antibacterial defense correlated with the suppressed oxidative burst, reduced accumulation of the defense hormone salicylic acid (SA) and diminished SA-dependent autophagy. The viral protein domain required for suppression of these plant defense responses is dispensable for silencing suppression but essential for binding and activation of the plant target-of-rapamycin (TOR) kinase which, in its active state, blocks cellular autophagy and promotes CaMV translation. Our findings imply that CaMV P6 is a versatile viral effector suppressing both silencing and innate immunity. P6-mediated suppression of oxidative burst and SA-dependent autophagy may predispose CaMV-infected plants to bacterial infection.


Assuntos
Arabidopsis/imunologia , Arabidopsis/virologia , Autofagia/efeitos dos fármacos , Caulimovirus/fisiologia , Pseudomonas syringae/crescimento & desenvolvimento , Explosão Respiratória , Ácido Salicílico/farmacologia , Proteínas Virais/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/microbiologia , Proteínas de Arabidopsis/metabolismo , Caulimovirus/efeitos dos fármacos , Caulimovirus/patogenicidade , Inativação Gênica/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Doenças das Plantas/microbiologia , Doenças das Plantas/virologia , Domínios Proteicos , Pseudomonas syringae/efeitos dos fármacos , Explosão Respiratória/efeitos dos fármacos , Deleção de Sequência , Proteínas Virais/química
12.
PLoS One ; 10(7): e0132665, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26162084

RESUMO

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.


Assuntos
Processamento Alternativo/genética , Caulimovirus/genética , Transcriptoma/genética , Sequência de Aminoácidos , Sequência de Bases , Caulimovirus/isolamento & purificação , Caulimovirus/patogenicidade , Simulação por Computador , Sequência Conservada/genética , Dados de Sequência Molecular , Mutação/genética , Sítios de Splice de RNA/genética , Proteínas Recombinantes de Fusão/metabolismo
13.
EMBO J ; 32(8): 1087-102, 2013 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-23524850

RESUMO

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.


Assuntos
Proteínas de Arabidopsis/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Arabidopsis , Ácidos Indolacéticos/metabolismo , Fases de Leitura Aberta , Fosforilação , Polirribossomos/metabolismo , Processamento de Proteína Pós-Traducional
14.
PLoS Pathog ; 8(3): e1002568, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22396650

RESUMO

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.


Assuntos
Oryza/virologia , Picornaviridae/genética , Doenças das Plantas/virologia , Ribossomos/genética , Tungrovirus/genética , DNA Viral , Genes de Plantas , Interações Hospedeiro-Patógeno , Fases de Leitura Aberta/genética , RNA Viral , Ribossomos/metabolismo , Transcrição Genética
15.
EMBO J ; 30(7): 1343-56, 2011 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-21343906

RESUMO

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.


Assuntos
Interações Hospedeiro-Patógeno , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Proteínas Virais/metabolismo , Arabidopsis , Proteínas de Arabidopsis , Fator de Iniciação 3 em Eucariotos/metabolismo , Imunoprecipitação , Fosfatidilinositol 3-Quinases , Ligação Proteica , Mapeamento de Interação de Proteínas , Ribossomos/metabolismo , Técnicas do Sistema de Duplo-Híbrido
16.
EMBO J ; 28(20): 3171-84, 2009 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-19745810

RESUMO

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.


Assuntos
Proteínas de Arabidopsis/metabolismo , Caulimovirus/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Regulação da Expressão Gênica de Plantas , Biossíntese de Proteínas/fisiologia , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas Virais/metabolismo , Proteínas de Arabidopsis/genética , Caulimovirus/genética , Caulimovirus/fisiologia , Modelos Biológicos , Reação em Cadeia da Polimerase , Polirribossomos/metabolismo , Ligação Proteica/genética , Ligação Proteica/fisiologia , Biossíntese de Proteínas/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/genética
17.
Nucleic Acids Res ; 37(17): 5838-47, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19638424

RESUMO

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.


Assuntos
Regiões 5' não Traduzidas , Iniciação Traducional da Cadeia Peptídica , RNA Viral/química , Spumavirus/genética , Animais , Linhagem Celular , Produtos do Gene gag/biossíntese , Produtos do Gene gag/genética , Fases de Leitura Aberta , Ribossomos/metabolismo
18.
RNA ; 12(5): 841-50, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16556934

RESUMO

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.


Assuntos
Badnavirus/genética , DNA Viral/genética , Oryza/virologia , Vírus de Plantas/genética , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Sequência de Bases , Caulimovirus/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Sequência Conservada , DNA Viral/química , Genes de Plantas , Genes Reporter , Técnicas In Vitro , Modelos Genéticos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Extratos Vegetais , Mutação Puntual , Biossíntese de Proteínas , Protoplastos/metabolismo , Ribossomos/genética , Transcrição Genética , Triticum
19.
Virus Res ; 119(1): 52-62, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16325949

RESUMO

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.


Assuntos
Iniciação Traducional da Cadeia Peptídica , Vírus de Plantas/genética , Ribossomos/metabolismo , Códon , Vírus de Plantas/metabolismo , Biossíntese de Proteínas , RNA Viral/genética , RNA Viral/metabolismo , Ribossomos/genética
20.
EMBO J ; 23(6): 1381-91, 2004 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-14988734

RESUMO

The cauliflower mosaic virus reinitiation factor TAV interacts with host translation initiation factor 3 (eIF3) and the 60S ribosomal subunit to accomplish translation of polycistronic mRNAs. Interaction between TAV and eIF3g is critical for the reinitiation process. Here, we show that eIF4B can preclude formation of the TAV/eIF3 complex via competition with TAV for eIF3g binding; indeed, the eIF4B- and TAV-binding sites on eIF3g overlap. Our data indicate that eIF4B interferes with TAV/eIF3/40S ribosome complex formation during the first initiation event. Consequently, overexpression of TAV in plant protoplasts affects only second initiation events. Transient overexpression of eIF4B in plant protoplasts specifically inhibits TAV-mediated reinitiation of a second ORF. These data suggest that TAV enters the host translation machinery at the eIF4B removal step to stabilize eIF3 on the translating ribosome, thereby allowing translation of polycistronic viral RNA.


Assuntos
Caulimovirus/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Fatores de Iniciação de Peptídeos/biossíntese , Fatores de Iniciação de Peptídeos/genética , Ribossomos/metabolismo , Proteínas Virais/biossíntese , Proteínas Virais/genética , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Dados de Sequência Molecular , Ligação Proteica , Protoplastos/metabolismo , Protoplastos/virologia , Alinhamento de Sequência , Tabaco/genética , Tabaco/metabolismo , Tabaco/virologia
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