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1.
J Virol ; 95(18): e0016921, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34160262

RESUMO

Long noncoding RNAs (lncRNAs) of virus origin accumulate in cells infected by many positive-strand (+) RNA viruses to bolster viral infectivity. Their biogenesis mostly utilizes exoribonucleases of host cells that degrade viral genomic or subgenomic RNAs in the 5'-to-3' direction until being stalled by well-defined RNA structures. Here, we report a viral lncRNA that is produced by a novel replication-dependent mechanism. This lncRNA corresponds to the last 283 nucleotides of the turnip crinkle virus (TCV) genome and hence is designated tiny TCV subgenomic RNA (ttsgR). ttsgR accumulated to high levels in TCV-infected Nicotiana benthamiana cells when the TCV-encoded RNA-dependent RNA polymerase (RdRp), also known as p88, was overexpressed. Both (+) and (-) strand forms of ttsgR were produced in a manner dependent on the RdRp functionality. Strikingly, templates as short as ttsgR itself were sufficient to program ttsgR amplification, as long as the TCV-encoded replication proteins p28 and p88 were provided in trans. Consistent with its replicational origin, ttsgR accumulation required a 5' terminal carmovirus consensus sequence (CCS), a sequence motif shared by genomic and subgenomic RNAs of many viruses phylogenetically related to TCV. More importantly, introducing a new CCS motif elsewhere in the TCV genome was alone sufficient to cause the emergence of another lncRNA. Finally, abolishing ttsgR by mutating its 5' CCS gave rise to a TCV mutant that failed to compete with wild-type TCV in Arabidopsis. Collectively, our results unveil a replication-dependent mechanism for the biogenesis of viral lncRNAs, thus suggesting that multiple mechanisms, individually or in combination, may be responsible for viral lncRNA production. IMPORTANCE Many positive-strand (+) RNA viruses produce long noncoding RNAs (lncRNAs) during the process of cellular infections and mobilize these lncRNAs to counteract antiviral defenses, as well as coordinate the translation of viral proteins. Most viral lncRNAs arise from 5'-to-3' degradation of longer viral RNAs being stalled at stable secondary structures. Here, we report a viral lncRNA that is produced by the replication machinery of turnip crinkle virus (TCV). This lncRNA, designated ttsgR, shares the terminal characteristics with TCV genomic and subgenomic RNAs and overaccumulates in the presence of moderately overexpressed TCV RNA-dependent RNA polymerase (RdRp). Furthermore, templates that are of similar sizes as ttsgR are readily replicated by TCV replication proteins (p28 and RdRp) provided from nonviral sources. In summary, this study establishes an approach for uncovering low abundance viral lncRNAs, and characterizes a replicating TCV lncRNA. Similar investigations on human-pathogenic (+) RNA viruses could yield novel therapeutic targets.


Assuntos
Carmovirus/genética , Genoma Viral , RNA Longo não Codificante/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Arabidopsis/virologia , RNA Longo não Codificante/química , RNA Viral/química , RNA Polimerase Dependente de RNA/genética , Tabaco/virologia , Proteínas Virais/genética
2.
Biochem Mol Biol Educ ; 49(4): 605-618, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33914410

RESUMO

With the growing importance of the field of RNA biology, undergraduates need to perform RNA-related research. Systematic evolution of ligands by exponential enrichment (SELEX) has become an important method in RNA biology. The principles of SELEX were applied to a semester-long course-based undergraduate research experience (CURE) in which two rounds of in vivo functional selection of regions of a viral RNA were performed. As the labwork had an unknown outcome, students indicated that they were excited by the work and became invested in the experience. By completing two rounds of SELEX, the students repeated molecular methods (e.g., RNA extraction, RT-PCR, agarose gel electrophoresis, DNA purification, cloning, and sequence analysis) and reported that repetition reinforced their learning and helped them build confidence in their lab abilities. Students also appreciated that they did not learn a "technique-per-week" without context, but rather they understood why certain methods were used for certain molecular tasks. Results from a 19-question multiple-choice assessment indicated increased comprehension of theory underlying methods performed. Details regarding experimental methods and timeline, and assessment and attitudinal results from three student cohorts, are described herein.


Assuntos
Pesquisa Biomédica/educação , Carmovirus/genética , Biologia Molecular/educação , RNA Viral/análise , Técnica de Seleção de Aptâmeros/métodos , Estudantes/estatística & dados numéricos , Brassica napus/virologia , Humanos , Doenças das Plantas/genética , Doenças das Plantas/virologia , Folhas de Planta/virologia , RNA Viral/genética , Projetos de Pesquisa
3.
Virology ; 551: 64-74, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33038689

RESUMO

Previous study has shown that Hibiscus sulfite oxidase (SO) interacts with Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) and triggers sulfur enhanced defense (SED). In this study, we show the interaction of Arabidopsis SO (AtSO) and Turnip crinkle virus (TCV) CP in Arabidopsis thaliana plants. We identified the binding sites of TCV CP (W274) and AtSO (D223) using bioinformatics and confirmed it experimentally. Mutation of binding site W274 to A274 in TCV CP resulted in failure of TCV infection. TCV accumulation in SO over-expression (SO_OE) plants was lower than that in wild-type (WT) and SO knock-out (SO_KO) plants at 7 dpi but reached a level similar to that of WT and SO_KO plants at 10 dpi. AtSO competed with Argonaute 1 (AGO1) for TCV CP binding in vitro. AtSO may serve as an anti-viral factor through sequestering TCV CP for binding with AGO1 and confers virus resistance.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Proteínas do Capsídeo/metabolismo , Carmovirus/metabolismo , Doenças das Plantas/virologia , Sulfito Oxidase/metabolismo , Arabidopsis/enzimologia , Arabidopsis/virologia , Proteínas Argonauta/metabolismo , Interações entre Hospedeiro e Microrganismos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos
4.
Plant Physiol ; 184(3): 1482-1498, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32859754

RESUMO

Patatin-related phospholipase As (pPLAs) are major hydrolases acting on acyl-lipids and play important roles in various plant developmental processes. pPLAIII group members, which lack a canonical catalytic Ser motif, have been less studied than other pPLAs. We report here the characterization of pPLAIIIα in Arabidopsis (Arabidopsis thaliana) based on the biochemical and physiological characterization of pPLAIIIα knockouts, complementants, and overexpressors, as well as heterologous expression of the protein. In vitro activity assays on the purified recombinant protein showed that despite lack of canonical phospholipase motifs, pPLAIIIα had a phospholipase A activity on a wide variety of phospholipids. Overexpression of pPLAIIIα in Arabidopsis resulted in a decrease in many lipid molecular species, but the composition in major lipid classes was not affected. Fluorescence tagging indicated that pPLAIIIα localizes to the plasma membrane. Although Arabidopsis pplaIIIα knockout mutants showed some phenotypes comparable to other pPLAIIIs, such as reduced trichome length and increased hypocotyl length, control of seed size and germination were identified as distinctive pPLAIIIα-mediated functions. Expression of some PLD genes was strongly reduced in the pplaIIIα mutants. Overexpression of pPLAIIIα caused increased resistance to turnip crinkle virus, which associated with a 2-fold higher salicylic acid/jasmonic acid ratio and an increased expression of the defense gene pathogenesis-related protein1. These results therefore show that pPLAIIIα has functions that overlap with those of other pPLAIIIs but also distinctive functions, such as the control of seed germination. This study also provides new insights into the pathways downstream of pPLAIIIα.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Carmovirus/patogenicidade , Resistência à Doença/genética , Germinação/genética , Fosfolipases/metabolismo , Fosfolipídeos/metabolismo , Arabidopsis/virologia , Resistência à Doença/fisiologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Germinação/fisiologia , Mutação , Fosfolipases/genética , Fosfolipídeos/genética , Plantas Geneticamente Modificadas/metabolismo
5.
Viruses ; 12(3)2020 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-32235750

RESUMO

RNA secondary structures play diverse roles in positive-sense (+) RNA virus infections, but those located with the replication protein coding sequence can be difficult to investigate. Structures that regulate the translation of replication proteins pose particular challenges, as their potential involvement in post-translational steps cannot be easily discerned independent of their roles in regulating translation. In the current study, we attempted to overcome these difficulties by providing viral replication proteins in trans. Specifically, we modified the plant-infecting turnip crinkle virus (TCV) into variants that are unable to translate one (p88) or both (p28 and p88) replication proteins, and complemented their replication with the corresponding replication protein(s) produced from separate, non-replicating constructs. This approach permitted us to re-examine the p28/p88 coding region for potential RNA elements needed for TCV replication. We found that, while more than a third of the p88 coding sequence could be deleted without substantially affecting viral RNA levels, two relatively small regions, known as RSE and IRE, were essential for robust accumulation of TCV genomic RNA, but not subgenomic RNAs. In particular, the RSE element, found previously to be required for regulating the translational read-through of p28 stop codon to produce p88, contained sub-elements needed for efficient replication of the TCV genome. Application of this new approach in other viruses could reveal novel RNA secondary structures vital for viral multiplication.


Assuntos
Carmovirus/fisiologia , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Biossíntese de Proteínas , RNA Viral/química , RNA Viral/genética , Replicação Viral , Genoma Viral
6.
Sci Rep ; 10(1): 4758, 2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32179855

RESUMO

Due to their minimal genomes, plant viruses are forced to hijack specific cellular pathways to ensure host colonization, a condition that most frequently involves physical interaction between viral and host proteins. Among putative viral interactors are the movement proteins, responsible for plasmodesma gating and genome binding during viral transport. Two of them, DGBp1 and DGBp2, are required for alpha-, beta- and gammacarmovirus cell-to-cell movement, but the number of DGBp-host interactors identified at present is limited. By using two different approaches, yeast two-hybrid and bimolecular fluorescence complementation assays, we found three Arabidopsis factors, eIF3g1, RPP3A and WRKY36, interacting with DGBp1s from each genus mentioned above. eIF3g1 and RPP3A are mainly involved in protein translation initiation and elongation phases, respectively, while WRKY36 belongs to WRKY transcription factor family, important regulators of many defence responses. These host proteins are not expected to be associated with viral movement, but knocking out WRKY36 or silencing either RPP3A or eIF3g1 negatively affected Arabidopsis infection by Turnip crinkle virus. A highly conserved FNF motif at DGBp1 C-terminus was required for protein-protein interaction and cell-to-cell movement, suggesting an important biological role.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Interações Hospedeiro-Patógeno/genética , Proteínas do Movimento Viral em Plantas/genética , Proteínas do Movimento Viral em Plantas/fisiologia , Vírus de Plantas/fisiologia , Domínios e Motivos de Interação entre Proteínas , Motivos de Aminoácidos , Arabidopsis/virologia , Carmovirus/genética , Carmovirus/fisiologia , Vírus de Plantas/genética
7.
Mol Plant Microbe Interact ; 33(2): 364-375, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31880982

RESUMO

We recently reported that the p28 auxiliary replication protein encoded by turnip crinkle virus (TCV) is also responsible for eliciting superinfection exclusion (SIE) against superinfecting TCV. However, it remains unresolved whether the replication function of p28 could be separated from its ability to elicit SIE. Here, we report the identification of two single amino acid mutations that decouple these two functions. Using an Agrobacterium infiltration-based delivery system, we transiently expressed a series of p28 deletion and point mutants, and tested their ability to elicit SIE against a cointroduced TCV replicon. We found that substituting alanine (A) for valine (V) and phenylalanine (F) at p28 positions 181 and 182, respectively, modestly compromised SIE in transiently expressed p28 derivatives. Upon incorporation into TCV replicons, V181A and F182A decoupled TCV replication and SIE diametrically. Although V181A impaired SIE without detectably compromising replication, F182A abolished TCV replication but had no effect on SIE once the replication of the defective replicon was restored through complementation. Both mutations diminished accumulation of p28 protein, suggesting that p28 must reach a concentration threshold in order to elicit a strong SIE. Importantly, the severe reduction of F182A protein levels correlated with a dramatic loss in the number of intracellular p28 foci formed by p28-p28 interactions. Together, these findings not only decouple the replication and SIE functions of p28 but also unveil a concentration dependence for p28 coalescence and SIE elicitation. These data further highlight the role of p28 multimerization in driving the exclusion of secondary TCV infections.


Assuntos
Carmovirus , Replicação Viral , Carmovirus/genética , Carmovirus/fisiologia , Deleção de Sequência , Replicação Viral/genética
8.
J Virol ; 94(1)2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31597780

RESUMO

Recombination is one of the driving forces of viral evolution. RNA recombination events among similar RNA viruses are frequent, although RNA recombination could also take place among unrelated viruses. In this paper, we have established efficient interviral recombination systems based on yeast and plants. We show that diverse RNA viruses, including the plant viruses tomato bushy stunt virus, carnation Italian ringspot virus, and turnip crinkle virus-associated RNA; the insect plus-strand RNA [(+)RNA] viruses Flock House virus and Nodamura virus; and the double-stranded L-A virus of yeast, are involved in interviral recombination events. Most interviral recombinants are minus-strand recombinant RNAs, and the junction sites are not randomly distributed, but there are certain hot spot regions. Formation of interviral recombinants in yeast and plants is accelerated by depletion of the cellular SERCA-like Pmr1 ATPase-driven Ca2+/Mn2+ pump, regulating intracellular Ca2+ and Mn2+ influx into the Golgi apparatus from the cytosol. The interviral recombinants are generated by a template-switching mechanism during RNA replication by the viral replicase. Replication studies revealed that a group of interviral recombinants is replication competent in cell-free extracts, in yeast, and in the plant Nicotiana benthamiana We propose that there are major differences among the viral replicases to generate and maintain interviral recombinants. Altogether, the obtained data promote the model that host factors greatly contribute to the formation of recombinants among related and unrelated viruses. This is the first time that a host factor's role in affecting interviral recombination is established.IMPORTANCE Viruses with RNA genomes are abundant, and their genomic sequences show astonishing variation. Genetic recombination in RNA viruses is a major force behind their rapid evolution, enhanced pathogenesis, and adaptation to their hosts. We utilized a previously identified intracellular Ca2+/Mn2+ pump-deficient yeast to search for interviral recombinants. Noninfectious viral replication systems were used to avoid generating unwanted infectious interviral recombinants. Altogether, interviral RNA recombinants were observed between plant and insect viruses, and between a fungal double-stranded RNA (dsRNA) virus and an insect virus, in the yeast host. In addition, interviral recombinants between two plant virus replicon RNAs were identified in N. benthamiana plants, in which the intracellular Ca2+/Mn2+ pump was depleted. These findings underline the crucial role of the host in promoting RNA recombination among unrelated viruses.


Assuntos
ATPases Transportadoras de Cálcio/genética , Carmovirus/genética , Chaperonas Moleculares/genética , Nodaviridae/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Tombusvirus/genética , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/deficiência , Carmovirus/metabolismo , Cátions Bivalentes , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Sistema Livre de Células/virologia , Transporte de Íons , Manganês/metabolismo , Nodaviridae/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/virologia , Tabaco/genética , Tabaco/metabolismo , Tabaco/virologia , Tombusvirus/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
9.
Plant Physiol ; 180(3): 1418-1435, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31043494

RESUMO

RNA-based silencing functions as an important antiviral immunity mechanism in plants. Plant viruses evolved to encode viral suppressors of RNA silencing (VSRs) that interfere with the function of key components in the silencing pathway. As effectors in the RNA silencing pathway, ARGONAUTE (AGO) proteins are targeted by some VSRs, such as that encoded by Turnip crinkle virus (TCV). A VSR-deficient TCV mutant was used to identify AGO proteins with antiviral activities during infection. A quantitative phenotyping protocol using an image-based color trait analysis pipeline on the PlantCV platform, with temporal red, green, and blue imaging and a computational segmentation algorithm, was used to measure plant disease after TCV inoculation. This process captured and analyzed growth and leaf color of Arabidopsis (Arabidopsis thaliana) plants in response to virus infection over time. By combining this quantitative phenotypic data with molecular assays to detect local and systemic virus accumulation, AGO2, AGO3, and AGO7 were shown to play antiviral roles during TCV infection. In leaves, AGO2 and AGO7 functioned as prominent nonadditive, anti-TCV effectors, whereas AGO3 played a minor role. Other AGOs were required to protect inflorescence tissues against TCV. Overall, these results indicate that distinct AGO proteins have specialized, modular roles in antiviral defense across different tissues, and demonstrate the effectiveness of image-based phenotyping to quantify disease progression.


Assuntos
Proteínas de Arabidopsis/imunologia , Arabidopsis/imunologia , Proteínas Argonauta/imunologia , Carmovirus/imunologia , Processamento de Imagem Assistida por Computador/métodos , Arabidopsis/genética , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Carmovirus/genética , Carmovirus/fisiologia , Resistência à Doença/genética , Resistência à Doença/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Mutação , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/virologia , Ligação Proteica , Interferência de RNA/imunologia
10.
Virology ; 526: 165-172, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30391806

RESUMO

We recently reported that p28, one of the two turnip crinkle virus (TCV) replication proteins, trans-complemented a defective TCV lacking p28, yet repressed the replication of another TCV replicon encoding wild-type p28 (Zhang et al., 2017). Here we show that p88, the TCV-encoded RNA-dependent RNA polymerase, likewise trans-complemented a p88-defective TCV replicon, but repressed one encoding wild-type p88. Surprisingly, lowering p88 protein levels enhanced trans-complementation, but weakened repression. Repression by p88 was not simply due to protein over-expression, as deletion mutants missing 127 or 224 N-terminal amino acids accumulated to higher levels but were poor repressors. Finally, both trans-complementation and repression by p88 were accompanied by preferential accumulation of subgenomic RNA2, and a novel class of small TCV RNAs. Our results suggest that repression of TCV replication by p88 may manifest a viral mechanism that regulates the ratio of genomic and subgenomic RNAs based on p88 abundance.


Assuntos
Carmovirus/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/genética , Expressão Gênica , Doenças das Plantas/virologia , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/deficiência , RNA Polimerase Dependente de RNA/genética , Replicon/genética , Deleção de Sequência , Proteínas Virais/genética
11.
PLoS Pathog ; 14(11): e1007459, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30452463

RESUMO

Nonsense-mediated decay (NMD) is a host RNA control pathway that removes aberrant transcripts with long 3' untranslated regions (UTRs) due to premature termination codons (PTCs) that arise through mutation or defective splicing. To maximize coding potential, RNA viruses often contain internally located stop codons that should also be prime targets for NMD. Using an agroinfiltration-based NMD assay in Nicotiana benthamiana, we identified two segments conferring NMD-resistance in the carmovirus Turnip crinkle virus (TCV) genome. The ribosome readthrough structure just downstream of the TCV p28 termination codon stabilized an NMD-sensitive reporter as did a frameshifting element from umbravirus Pea enation mosaic virus. In addition, a 51-nt unstructured region (USR) at the beginning of the TCV 3' UTR increased NMD-resistance 3-fold when inserted into an unrelated NMD-sensitive 3' UTR. Several additional carmovirus 3' UTRs also conferred varying levels of NMD resistance depending on the construct despite no sequence similarity in the analogous region. Instead, these regions displayed a marked lack of RNA structure immediately following the NMD-targeted stop codon. NMD-resistance was only slightly reduced by conversion of 19 pyrimidines in the USR to purines, but resistance was abolished when a 2-nt mutation was introduced downstream of the USR that substantially increased the secondary structure in the USR through formation of a stable hairpin. The same 2-nt mutation also enhanced the NMD susceptibility of a subgenomic RNA expressed independently of the genomic RNA. The conserved lack of RNA structure among most carmoviruses at the 5' end of their 3' UTR could serve to enhance subgenomic RNA stability, which would increase expression of the encoded capsid protein that also functions as the RNA silencing suppressor. These results demonstrate that the TCV genome has features that are inherently NMD-resistant and these strategies could be widespread among RNA viruses and NMD-resistant host mRNAs with long 3' UTRs.


Assuntos
Carmovirus/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , Regiões 3' não Traduzidas/genética , Carmovirus/patogenicidade , Códon sem Sentido/genética , Códon de Terminação/genética , Biossíntese de Proteínas , Interferência de RNA/fisiologia , Estabilidade de RNA/genética , Vírus de RNA/genética , RNA Viral/genética , Ribossomos , Tabaco/genética
12.
Methods Mol Biol ; 1776: 3-17, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29869231

RESUMO

When the coat protein reading frame of turnip crinkle virus (TCV) is transiently expressed in leaves, virus-like particles (VLPs) are readily formed. However, after introducing genetic modifications to the full-length coat protein sequence, such as the introduction of an epitope-specific sequence within the coat protein sequence or the in-frame carboxyl terminal fusion of GFP, the formation of such modified VLPs is poor. However, by coexpression of one of these modified forms with wild-type TCV coat protein by the coinfiltration of appropriate Agrobacterium suspensions, VLP generation is enhanced through the formation of "mosaics," that is, individual VLPs consisting of both modified and wild-type subunits (also known as phenotypically mixed VLPs). Here we describe methods for the introduction of genetic modifications into the TCV coat protein sequence, the production of mosaic TCV VLPs and their characterization.


Assuntos
Arabidopsis/virologia , Proteínas do Capsídeo/genética , Carmovirus/genética , Vírus do Mosaico/genética , Proteínas Virais/genética , Capsídeo , Folhas de Planta/virologia
13.
Virology ; 520: 137-152, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29864677

RESUMO

Noncoding RNAs use their sequence and/or structure to mediate function(s). The 5' portion (166 nt) of the 356-nt noncoding satellite RNA C (satC) of Turnip crinkle virus (TCV) was previously modeled to contain a central region with two stem-loops (H6 and H7) and a large connecting hairpin (H2). We now report that in vivo functional selection (SELEX) experiments assessing sequence/structure requirements in H2, H6, and H7 reveal that H6 loop sequence motifs were recovered at nonrandom rates and only some residues are proposed to base-pair with accessible complementary sequences within the 5' central region. In vitro SHAPE of SELEX winners indicates that the central region is heavily base-paired, such that along with the lower stem and H2 region, one extensive hairpin exists composing the entire 5' region. As these SELEX winners are highly fit, these characteristics facilitate satRNA amplification in association with TCV in plants.


Assuntos
Carmovirus/genética , Evolução Molecular , Plantas/virologia , RNA Satélite/genética , RNA Viral/genética , Conformação de Ácido Nucleico , RNA Satélite/química , RNA Viral/química , Técnica de Seleção de Aptâmeros , Replicação Viral
14.
PLoS Pathog ; 14(3): e1006894, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29513740

RESUMO

The E3 ubiquitin ligase COP1 (Constitutive Photomorphogenesis 1) is a well known component of the light-mediated plant development that acts as a repressor of photomorphogenesis. Here we show that COP1 positively regulates defense against turnip crinkle virus (TCV) and avrRPM1 bacteria by contributing to stability of resistance (R) protein HRT and RPM1, respectively. HRT and RPM1 levels and thereby pathogen resistance is significantly reduced in the cop1 mutant background. Notably, the levels of at least two double-stranded RNA binding (DRB) proteins DRB1 and DRB4 are reduced in the cop1 mutant background suggesting that COP1 affects HRT stability via its effect on the DRB proteins. Indeed, a mutation in either drb1 or drb4 resulted in degradation of HRT. In contrast to COP1, a multi-subunit E3 ligase encoded by anaphase-promoting complex (APC) 10 negatively regulates DRB4 and TCV resistance but had no effect on DRB1 levels. We propose that COP1-mediated positive regulation of HRT is dependent on a balance between COP1 and negative regulators that target DRB1 and DRB4.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Carmovirus/imunologia , Resistência à Doença/imunologia , Doenças das Plantas/imunologia , Proteínas de Ligação a RNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Arabidopsis/metabolismo , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Luz , Morfogênese , Mutação , Desenvolvimento Vegetal , Doenças das Plantas/virologia , Tabaco/imunologia , Tabaco/virologia , Ubiquitina-Proteína Ligases/genética
15.
Methods Mol Biol ; 1746: 187-195, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29492896

RESUMO

Next-generation sequencing has opened the door to the reconstruction of viral populations and examination of the composition of mutant spectra in infected cells, tissues, and host organisms. In this chapter we present details on the use of the Shannon entropy method to estimate the site-specific nucleotide relative variability of turnip crinkle virus, a positive (+) stranded RNA plant virus, in a large dataset of short RNAs of Cicer arietinum L., a natural reservoir of the virus. We propose this method as a viral metagenomics tool to provide a more detailed description of the viral quasispecies in infected plant tissue. Viral replicative fitness relates to an optimal composition of variants that provide the molecular basis of virus behavior in the complex environment of natural infections. A complete description of viral quasispecies may have implications in determining fitness landscapes for host-virus coexistence and help to design specific diagnostic protocols and antiviral strategies.


Assuntos
Carmovirus/genética , Cicer/virologia , Entropia , Taxa de Mutação , Nucleotídeos/genética , RNA Interferente Pequeno/genética , RNA Viral/análise , Sequenciamento de Nucleotídeos em Larga Escala , RNA Viral/genética , Replicação Viral
16.
RNA Biol ; 14(11): 1466-1472, 2017 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-28548627

RESUMO

How plus-strand [+]RNA virus genomes transition from translation templates to replication templates is a matter of much speculation. We have previously proposed that, for Turnip crinkle virus, binding of the encoded RNA-dependent RNA polymerase (RdRp) to the 3'UTR of the [+]RNA template promotes a regional wide-spread conformational switch to an alternative structure that disassembles the cap-independent translation enhancer (CITE) in the 3'UTR. The active 3'CITE folds into a tRNA-like T-shaped structure (TSS) that binds to 80S ribosomes and 60S subunits in the P-site. In this Point-of-View, we discuss the history of our research on the TSS and our recent report combining coarse level single molecule force spectroscopy (optical tweezers) with fine-grain computer simulations of this experimental process and biochemical approaches to obtain a detailed understanding of how RdRp binding in the TSS vicinity might lead to an extensive rearrangement of the RNA structure.


Assuntos
Carmovirus/genética , Elementos Facilitadores Genéticos , Regulação Viral da Expressão Gênica , RNA Viral/química , RNA Polimerase Dependente de RNA/química , Ribossomos/metabolismo , Regiões 3' não Traduzidas , Pareamento de Bases , Sequência de Bases , Carmovirus/metabolismo , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Pinças Ópticas , Biossíntese de Proteínas , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Ribossomos/genética , Imagem Individual de Molécula
17.
Plant Physiol ; 174(2): 1067-1081, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28455401

RESUMO

RNA silencing is an innate antiviral mechanism conserved in organisms across kingdoms. Such a cellular defense involves DICER or DICER-LIKEs (DCLs) that process plant virus RNAs into viral small interfering RNAs (vsiRNAs). Plants encode four DCLs that play diverse roles in cell-autonomous intracellular virus-induced RNA silencing (known as VIGS) against viral invasion. VIGS can spread between cells. However, the genetic basis and involvement of vsiRNAs in non-cell-autonomous intercellular VIGS remains poorly understood. Using GFP as a reporter gene together with a suite of DCL RNAi transgenic lines, here we show that despite the well-established activities of DCLs in intracellular VIGS and vsiRNA biogenesis, DCL4 acts to inhibit intercellular VIGS whereas DCL2 is required (likely along with DCL2-processed/dependent vsiRNAs and their precursor RNAs) for efficient intercellular VIGS trafficking from epidermal to adjacent cells. DCL4 imposed an epistatic effect on DCL2 to impede cell-to-cell spread of VIGS. Our results reveal previously unknown functions for DCL2 and DCL4 that may form a dual defensive frontline for intra- and intercellular silencing to double-protect cells from virus infection in Nicotiana benthamiana.


Assuntos
Carmovirus/metabolismo , Proteínas de Plantas/metabolismo , Interferência de RNA , Tabaco/genética , Tabaco/virologia , Proteínas de Fluorescência Verde/metabolismo , Epiderme Vegetal/citologia , Proteínas do Movimento Viral em Plantas/metabolismo , RNA Interferente Pequeno/metabolismo , Transgenes
18.
PLoS Pathog ; 13(3): e1006253, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28267773

RESUMO

Diverse animal and plant viruses block the re-infection of host cells by the same or highly similar viruses through superinfection exclusion (SIE), a widely observed, yet poorly understood phenomenon. Here we demonstrate that SIE of turnip crinkle virus (TCV) is exclusively determined by p28, one of the two replication proteins encoded by this virus. p28 expressed from a TCV replicon exerts strong SIE to a different TCV replicon. Transiently expressed p28, delivered simultaneously with, or ahead of, a TCV replicon, largely recapitulates this repressive activity. Interestingly, p28-mediated SIE is dramatically enhanced by C-terminally fused epitope tags or fluorescent proteins, but weakened by N-terminal modifications, and it inversely correlates with the ability of p28 to complement the replication of a p28-defective TCV replicon. Strikingly, p28 in SIE-positive cells forms large, mobile punctate inclusions that trans-aggregate a non-coalescing, SIE-defective, yet replication-competent p28 mutant. These results support a model postulating that TCV SIE is caused by the formation of multimeric p28 complexes capable of intercepting fresh p28 monomers translated from superinfector genomes, thereby abolishing superinfector replication. This model could prove to be applicable to other RNA viruses, and offer novel targets for antiviral therapy.


Assuntos
Carmovirus/fisiologia , Superinfecção/microbiologia , Replicação Viral/fisiologia , Immunoblotting , Microscopia Confocal , Doenças das Plantas/virologia , Tabaco/virologia
19.
Virus Res ; 232: 96-105, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28215614

RESUMO

Nucleotide sequence of a distinct soybean yellow mottle mosaic virusisolate from Vignaradiata (mungbean isolate, SYMMV-Mb) from India was determined and compared with othermembers of the family Tombusviridae. The complete monopartite single-stranded RNA genome of SYMMV-Mb consisted of 3974nt with six putative open reading frames and includes 5' and 3' untranslated regions of 35 and 254nt, respectively. SYMMV-Mb genome shared 75% nt sequence identity at complete genome level and 67-92% identity at all ORFs level with SYMMV Korean and USA isolates (soybean isolates) followed by CPMoV, whereas it shared very low identity with other tombusviridae members (5-41%). A full-length infectious cDNA clone of the SYMMV-Mb placed under the control of the T7 RNA polymerase and the CaMV35S promoters was generated and French bean plants on mechanical inoculation with in vitro RNA transcripts, p35SSYMMV-O4 plasmid and agroinoculation with p35SSYMMV-O4 showed symptoms typical of SYMMV-Mb infection. The infection was confirmed by DAC-ELISA, ISEM, RT-PCR and mechanical transmission to new plant species. Further testing of different plant species with agroinoculation of p35SSYMMV-O4 showed delay in symptoms but indistinguishable from mechanical sap inoculation and the infection was confirmed by DAC-ELISA, RT-PCR and mechanical transmission to new plants. The system developed here will be useful for further studies on pathogenecity, viral gene functions, plant-virus-vector interactions of SYMMV-Mb and to utilize it as a gene expression and silencing vector.


Assuntos
Carmovirus/genética , Genoma Viral , Filogenia , RNA Viral/genética , Soja/virologia , Tombusvirus/genética , Carmovirus/classificação , Carmovirus/patogenicidade , Clonagem Molecular , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Expressão Gênica , Genótipo , Especificidade de Hospedeiro , Índia , Fases de Leitura Aberta , Doenças das Plantas/virologia , Plasmídeos/química , Plasmídeos/metabolismo , RNA Viral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tombusvirus/classificação , Tombusvirus/patogenicidade , Proteínas Virais/genética , Proteínas Virais/metabolismo
20.
Elife ; 62017 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-28186489

RESUMO

Turnip crinkle virus contains a T-shaped, ribosome-binding, translation enhancer (TSS) in its 3'UTR that serves as a hub for interactions throughout the region. The viral RNA-dependent RNA polymerase (RdRp) causes the TSS/surrounding region to undergo a conformational shift postulated to inhibit translation. Using optical tweezers (OT) and steered molecular dynamic simulations (SMD), we found that the unusual stability of pseudoknotted element H4a/Ψ3 required five upstream adenylates, and H4a/Ψ3 was necessary for cooperative association of two other hairpins (H5/H4b) in Mg2+. SMD recapitulated the TSS unfolding order in the absence of Mg2+, showed dependence of the resistance to pulling on the 3D orientation and gave structural insights into the measured contour lengths of the TSS structure elements. Adenylate mutations eliminated one-site RdRp binding to the 3'UTR, suggesting that RdRp binding to the adenylates disrupts H4a/Ψ3, leading to loss of H5/H4b interaction and promoting a conformational switch interrupting translation and promoting replication.


Assuntos
Carmovirus/genética , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Viral/química , RNA Viral/genética , RNA Polimerase Dependente de RNA/metabolismo , Ribossomos/metabolismo , Análise Mutacional de DNA , Simulação de Dinâmica Molecular
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