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1.
Arch Virol ; 166(9): 2573-2578, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34148142

RESUMO

A novel geminivirus was identified in France and Spain in asymptomatic plants of white clover (Trifolium repens) and shrub medick (Medicago arborea). Its genome has the hallmarks of a capulavirus, and its relationship to other capulaviruses was confirmed by phylogenetic analysis. White clover isolates formed a tight cluster in the phylogenetic tree, while shrub medick isolates formed two distinct, more divergent groups with sequence identity values close to the species cutoff. These three groups have likely participated in recombination events involving alfalfa leaf curl virus and French bean severe leaf curl virus. The name "trifolium virus 1" (TrV1) is proposed for this new Capulavirus. Three TrV1 genotypes (TrV1-A, TrV1-B, and TrV1-C) were clearly distinguished.


Assuntos
Filogenia , Trifolium/virologia , Vírus não Classificados/classificação , Vírus não Classificados/genética , Vírus não Classificados/isolamento & purificação , Sequência de Aminoácidos , Biodiversidade , Vírus de DNA/genética , Fabaceae/virologia , Geminiviridae/classificação , Geminiviridae/genética , Geminiviridae/isolamento & purificação , Genótipo , Fases de Leitura Aberta , Doenças das Plantas/virologia , Análise de Sequência de DNA
2.
Arch Virol ; 163(12): 3447-3449, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30159684

RESUMO

Using high-throughput sequencing, a novel waikavirus was identified in a mixed virus infection of red clover (Trifolium pratense L.). Its complete genomic sequence was determined and characterized. The virus, tentatively named red clover associated virus 1 (RCaV1), is phylogenetically related to members of the genus Waikavirus (family Secoviridae, order Picornavirales).


Assuntos
Genoma Viral , Doenças das Plantas/virologia , Vírus Satélites/genética , Vírus Satélites/isolamento & purificação , Trifolium/virologia , Waikavirus/genética , Waikavirus/isolamento & purificação , Sequência de Bases , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Vírus Satélites/classificação , Análise de Sequência de DNA , Waikavirus/classificação
3.
Arch Virol ; 163(12): 3439-3442, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30143875

RESUMO

This study reports the complete genomic sequence of a novel virus isolated from red clover. According to its genomic organization, its similarity to luteoviruses, and a greater than 10% difference in all genes, this virus isolate likely represents a new luteovirus species. As seen in nectarine stem pitting-associated virus (NSPaV) and NSPaV-South Korea (SK) luteoviruses, it differs from typical luteoviruses through the absence of ORF3a and ORF4 encoding movement proteins. Furthermore, its P5 protein (responsible for aphid transmission) is more similar to the P5 of enamoviruses than that of luteoviruses. The virus isolate has been named red clover-associated luteovirus (RCaV).


Assuntos
Luteovirus/classificação , Luteovirus/isolamento & purificação , Doenças das Plantas/virologia , Trifolium/virologia , Genoma Viral , Luteovirus/genética , Fases de Leitura Aberta , Filogenia , Análise de Sequência de DNA , Proteínas Virais/genética
4.
Arch Virol ; 163(8): 2213-2218, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29619600

RESUMO

During aetiological study of diseased red clover (Trifolium pratense L.) using high throughput sequencing, a novel virus with a 10 kb genome divided into two segments was discovered. The virus, tentatively named red clover associated varicosavirus (RCaVV), is phylogenetically related to classifiable members of the genus Varicosavirus (family Rhabdoviridae, order Mononegavirales). Analysis of mRNA levels from the individual RCaVV genes suggested possible differences in transcription regulation between rhabdoviruses with divided and undivided genomes.


Assuntos
Vírus de DNA/isolamento & purificação , Doenças das Plantas/virologia , Vírus de Plantas/isolamento & purificação , Trifolium/virologia , Vírus de DNA/classificação , Vírus de DNA/genética , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , Vírus de Plantas/classificação , Vírus de Plantas/genética , Análise de Sequência de DNA , Proteínas Virais/genética
5.
Arch Virol ; 163(5): 1387-1389, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29397455

RESUMO

During high throughput sequencing (HTS) of leaves from a symptomatic red clover plant, a new RNA virus, tentatively named red clover nepovirus A (RCNVA), was discovered. The complete genomic sequence was determined and characterized. Particularly noteworthy was that RCNVA shares high sequence identities in RNA1 with a group of phylogenetically related nepoviruses while homologies in the RNA2 segments are markedly lower. Based on the genomic organization and phylogenetic attributes, RCNVA should be classified as a novel virus of the genus Nepovirus (subfamily Comovirinae, family Secoviridae, order Picornavirales).


Assuntos
Genoma Viral , Nepovirus/genética , Trifolium/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Nepovirus/classificação , Nepovirus/isolamento & purificação , Filogenia , Doenças das Plantas/virologia , RNA Viral/genética , Proteínas Virais/genética
6.
Arch Virol ; 160(5): 1303-11, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25701210

RESUMO

The genomes of a large number of highly diverse novel circular DNA viruses from a wide range of sources have been characterised in recent years, including circular single-stranded DNA (ssDNA) viruses that share similarities with plant-infecting ssDNA viruses of the family Geminiviridae. Here, we describe six novel circular DNA viral genomes that encode replication-associated (Rep) proteins that are most closely related to those of either geminiviruses or gemycircularviruses (a new group of ssDNA viruses that are closely related to geminiviruses). Four possible viral genomes were recovered from Bromus hordeaceus sampled in New Zealand, and two were recovered from B. hordeaceus and Trifolium resupinatum sampled in France. Two of the viral genomes from New Zealand (one from the North Island and one from the South Island each) share >99 % sequence identity, and two genomes recovered from B. hordeaceus and T. resupinatum sampled in France share 74 % identity. All of the viral genomes that were recovered were found to have a major open reading frame on both their complementary and virion-sense strands, one of which likely encodes a Rep and the other a capsid protein. Although future infectivity studies are needed to identify the host range of these viruses, this is the first report of circular DNA viruses associated with grasses in New Zealand.


Assuntos
Bromus/virologia , Vírus de DNA/classificação , DNA Circular/genética , DNA Viral/genética , Vírus de Plantas/classificação , Trifolium/virologia , Sequência de Aminoácidos , Proteínas do Capsídeo/genética , Análise por Conglomerados , DNA Helicases/genética , Vírus de DNA/genética , Vírus de DNA/isolamento & purificação , DNA Viral/química , França , Geminiviridae , Dados de Sequência Molecular , Nova Zelândia , Fases de Leitura Aberta , Filogenia , Vírus de Plantas/genética , Vírus de Plantas/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência
7.
Viruses ; 5(10): 2512-30, 2013 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-24113719

RESUMO

Plant-infecting viruses of the genera Alpha- and Betacryptovirus within the family Partitiviridae cause no visible effects on their hosts and are only transmitted by cell division and through gametes. The bipartite dsRNA genome is encoding a RNA-dependent RNA polymerase (RdRp) and a coat protein (CP). Aside from sequence and structural analysis, the investigation of protein interactions is another step towards virus characterization. Therefore, ORFs of two type members White Clover Cryptic Virus 1 and 2 (WCCV-1 and WCCV-2), as well as the related viruses from Red Clover and Dill were introduced into a bimolecular fluorescence complementation assay. We showed CP-CP dimerization for all tested viruses with localization for alphacryptoviruses at the nuclear membrane and for betacryptoviruses close to cell walls within the cytoplasm. For CPs of WCCV-1 and WCCV-2, deletion mutants were created to determine internal interaction sites. Moreover, RdRp self-interaction was found for all viruses, whereas CP-RdRp interactions were only detectable for the alphacryptoviruses. An intra-genus test of CPs was successful in various virus combinations, whereas an inter-genus interaction of WCCV-1CP and WCCV-2CP was absent. This is the first report of in vivo protein interactions of members in the family Partitiviridae, indicating distinct features of the alpha- and betacryptoviruses.


Assuntos
Anethum graveolens/virologia , Vírus de Plantas/fisiologia , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Vírus de RNA/fisiologia , Trifolium/virologia , Proteínas Virais/metabolismo , Fluorescência , Dados de Sequência Molecular , Vírus de Plantas/genética , Vírus de RNA/genética , RNA Viral/genética , Análise de Sequência de DNA , Proteínas Virais/genética
8.
Arch Virol ; 158(9): 1943-52, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23584419

RESUMO

The family Partitiviridae includes plant (Alphacryptovirus and Betacryptovirus), fungal (Partitivirus) and protozoan (Cryspovirus) viruses with bisegmented dsRNA genomes and isometric virions. Cryptic viruses commonly occur in different plant species without causing any symptoms. So far, numerous sequences have been determined for viruses of the genus Alphacryptovirus, but no sequence is available for any assigned member of the genus Betacryptovirus. Following extraction, cloning and sequence analysis of double-stranded RNA in this study, we report the molecular properties of three assigned members of the genus Betacryptovirus, white clover cryptic virus 2, red clover cryptic virus 2 and hop trefoil cryptic virus 2, and two new putative betacryptoviruses found in crimson clover and dill. Betacryptoviruses share sequence motifs with members of the genus Partitivirus. In phylogenetic analyses, members of the genus Betacryptovirus formed a new sub-cluster within the clusters containing members of the genus Partitivirus. Our results provide evidence for a distinct evolutionary lineage of dsRNA viruses of plants and fungi.


Assuntos
Vírus de RNA/classificação , Vírus de RNA/genética , Regiões 5' não Traduzidas/genética , Anethum graveolens/virologia , Sequência de Bases , Sequência Consenso , Evolução Molecular , Fungos/genética , Fungos/virologia , Genoma Viral , Medicago/classificação , Medicago/microbiologia , Dados de Sequência Molecular , Filogenia , Doenças das Plantas/virologia , RNA de Cadeia Dupla/análise , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/isolamento & purificação , RNA Viral/genética , Análise de Sequência de DNA , Trifolium/virologia
9.
Virology ; 433(1): 131-41, 2012 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-22898643

RESUMO

Positive-strand RNA viruses require host intracellular membranes for replicating their genomic RNAs. In this study, we determined the domains and critical amino acids in p27 of Red clover necrotic mosaic virus (RCNMV) required for its association with and targeting of ER membranes in Nicotiana benthamiana plants using a C-terminally GFP-fused and biologically functional p27. Confocal microscopy and membrane-flotation assays using an Agrobacterium-mediated expression system showed that a stretch of 20 amino acids in the N-terminal region of p27 is essential for the association of p27 with membranes. We identified the amino acids in this domain required for the association of p27 with membranes using alanine-scanning mutagenesis. We also found that this domain contains amino acids not critical for the membrane association but required for the formation of viral RNA replication complexes and negative-strand RNA synthesis. Our results extend our understanding of the multifunctional role of p27 in RCNMV replication.


Assuntos
Retículo Endoplasmático/virologia , Nicotiana/virologia , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/metabolismo , Tombusviridae/fisiologia , Proteínas Virais/metabolismo , Agrobacterium tumefaciens/genética , Sequência de Aminoácidos , Retículo Endoplasmático/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Membranas Intracelulares/metabolismo , Membranas Intracelulares/virologia , Dados de Sequência Molecular , Mutação , Doenças das Plantas , Estrutura Terciária de Proteína , RNA Viral/genética , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Trifolium/virologia , Proteínas Virais/química , Proteínas Virais/genética , Replicação Viral
10.
Oecologia ; 170(2): 433-44, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22526939

RESUMO

Plant pathogens and insect herbivores are prone to share hosts under natural conditions. Consequently, pathogen-induced changes in the host plant can affect herbivory, and vice versa. Even though plant viruses are ubiquitous in the field, little is known about plant-mediated interactions between viruses and non-vectoring herbivores. We investigated the effects of virus infection on subsequent infestation by a non-vectoring herbivore in a natural genotype of Trifolium repens (white clover). We tested whether infection with White clover mosaic virus (WClMV) alters (1) the effects of fungus gnat feeding on plant growth, (2) the attractiveness of white clover for adult fungus gnat females, and (3) the volatile emission of white clover plants. We observed only marginal effects of WClMV infection on the interaction between fungus gnat larvae and white clover. However, adult fungus gnat females clearly preferred non-infected over WClMV-infected plants. Non-infected and virus-infected plants could easily be discriminated based on their volatile blends, suggesting that the preference of fungus gnats for non-infected plants may be mediated by virus-induced changes in volatile emissions. The compound ß-caryophyllene was exclusively detected in the headspace of virus-infected plants and may hence be particularly important for the preference of fungus gnat females. Our results demonstrate that WClMV infection can decrease the attractiveness of white clover plants for fungus gnat females. This suggests that virus infections may contribute to protecting their hosts by decreasing herbivore infestation rates. Consequently, it is conceivable that viruses play a more beneficial role in plant-herbivore interactions than generally thought.


Assuntos
Herbivoria , Vírus do Mosaico/patogenicidade , Trifolium/virologia , Animais , Dípteros , Doenças das Plantas , Plantas Comestíveis , Sesquiterpenos Policíclicos , Sesquiterpenos/metabolismo , Trifolium/química , Volatilização
11.
Transgenic Res ; 21(3): 619-32, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21947755

RESUMO

Viral diseases, such as Alfalfa mosaic virus (AMV), cause significant reductions in the productivity and vegetative persistence of white clover plants in the field. Transgenic white clover plants ectopically expressing the viral coat protein gene encoded by the sub-genomic RNA4 of AMV were generated. Lines carrying a single copy of the transgene were analysed at the molecular, biochemical and phenotypic level under glasshouse and field conditions. Field resistance to AMV infection, as well as mitotic and meiotic stability of the transgene, were confirmed by phenotypic evaluation of the transgenic plants at two sites within Australia. The T(0) and T(1) generations of transgenic plants showed immunity to infection by AMV under glasshouse and field conditions, while the T(4) generation in an agronomically elite 'Grasslands Sustain' genetic background, showed a very high level of resistance to AMV in the field. An extensive biochemical study of the T(4) generation of transgenic plants, aiming to evaluate the level and composition of natural toxicants and key nutritional parameters, showed that the composition of the transgenic plants was within the range of variation seen in non-transgenic populations.


Assuntos
Vírus do Mosaico da Alfafa/patogenicidade , Proteínas do Capsídeo/metabolismo , Embaralhamento de DNA/métodos , Trifolium/imunologia , Agrobacterium/genética , Agrobacterium/metabolismo , Vírus do Mosaico da Alfafa/imunologia , Austrália , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Resistência à Doença , Dosagem de Genes , Fluxo Gênico , Genes Virais , Instabilidade Genômica , Meiose , Mitose , Fenótipo , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/imunologia , Plantas Geneticamente Modificadas/virologia , Transgenes , Trifolium/genética , Trifolium/virologia
12.
Ann Bot ; 107(8): 1391-7, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21515605

RESUMO

BACKGROUND AND AIMS: Genotype by environment (G × E) interactions are important for the long-term persistence of plant species in heterogeneous environments. It has often been suggested that disease is a key factor for the maintenance of genotypic diversity in plant populations. However, empirical evidence for this contention is scarce. Here virus infection is proposed as a possible candidate for maintaining genotypic diversity in their host plants. METHODS: The effects of White clover mosaic virus (WClMV) on the performance and development of different Trifolium repens genotypes were analysed and the G × E interactions were examined with respect to genotype-specific plant responses to WClMV infection. Thus, the environment is defined as the presence or absence of the virus. KEY RESULTS: WClMV had a negative effect on plant performance as shown by a decrease in biomass and number of ramets. These effects of virus infection differ greatly among host genotypes, representing a strong G × E interaction. Moreover, the relative fitness and associated ranking of genotypes changed significantly between control and virus treatments. This shift in relative fitness among genotypes suggests the potential for WClMV to provoke differential selection on T. repens genotypes, which may lead to negative frequency-dependent selection in host populations. CONCLUSIONS: The apparent G × E interaction and evident repercussions for relative fitness reported in this study stress the importance of viruses for ecological and evolutionary processes and suggest an important role for viruses in shaping population dynamics and micro-evolutionary processes.


Assuntos
Variação Genética/genética , Potexvirus/fisiologia , Trifolium/genética , Trifolium/virologia , Análise de Variância , Evolução Biológica , Biomassa , Meio Ambiente , Aptidão Genética , Genótipo , Países Baixos , Doenças das Plantas/virologia , Potexvirus/patogenicidade , Seleção Genética , Trifolium/crescimento & desenvolvimento
13.
J Plant Physiol ; 166(14): 1557-67, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19660828

RESUMO

The lack of naturally occurring resistance to white clover mosaic virus (WCMV) has demanded exploration of a transgenic approach for the development of WCMV-resistant white clover plants. Transgenic white clover plants producing sense (co-suppression), antisense and hairpin RNA (hpRNA) transcripts corresponding to the WCMV replicase gene were produced and analysed at the molecular and phenotypic levels. Expression of hpRNA and antisense transgenes provided a high level resistance to WCMV, while the sense transgene provided partial resistance. The presence of small interfering RNA molecules (siRNAs) in the transgenic white clover plants prior to virus challenge indicated that WCMV resistance was due to pre-activated RNA silencing, and the presence of siRNAs acted as reliable biomarkers for prediction of the degree of virus resistance in these plants.


Assuntos
Inativação Gênica/fisiologia , Imunidade Inata/genética , Vírus do Mosaico/genética , Vírus do Mosaico/patogenicidade , Doenças das Plantas/virologia , Trifolium/virologia , Imunidade Inata/fisiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/virologia , RNA Polimerase Dependente de RNA/genética , Trifolium/genética , Proteínas Virais/genética
14.
Phytopathology ; 99(3): 251-7, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19203277

RESUMO

Attenuated isolate M11 of Bean yellow mosaic virus (BYMV), obtained after exposing BYMV-infected plants to low temperature, and its efficacy in cross-protecting against infection by BYMV isolates from gladiolus, broad bean (Vicia faba) and white clover (Trifolium repens) was assessed with western blotting and reverse transcription-polymerase chain reaction. The level of cross-protection varied depending on the challenge virus isolates. Cross-protection was complete against BYMV isolates from gladiolus, but incomplete against BYMV isolates from other hosts. M11 also partially cross-protected against an isolate of Clover yellow vein virus. A comparison of the nucleotide sequence of M11 and those of BYMV isolates from gladiolus and from other hosts showed higher homology among gladiolus isolates than the homology between gladiolus isolates and nongladiolus isolates. In the phylogenetic trees, constructed using the nucleotide sequences of an overall polyprotein of the genomes, five gladiolus isolates clustered together, completely separated from the three BYMV isolates from other hosts. A comparison of the amino acid sequences between M11 and its parental isolate IbG, and analysis of recombinant infectious clones between M11 and IbG revealed that an amino acid at position 314 was involved in the attenuation of BYMV.


Assuntos
Interações Hospedeiro-Patógeno , Iridaceae/virologia , Doenças das Plantas/imunologia , Potyvirus/fisiologia , Trifolium/virologia , Vicia faba/virologia , Sequência de Aminoácidos , Genoma Viral , Filogenia , Potyvirus/genética , RNA Viral/genética , Análise de Sequência de RNA , Proteínas Virais/química
15.
Virology ; 384(1): 169-78, 2009 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-19062064

RESUMO

The bipartite RNA genome of Red clover necrotic mosaic virus (RCNMV) is encapsidated into icosahedral virions that exist as two populations: i) virions that co-package both genomic RNAs and ii) virions packaging multiple copies of RNA-2. To elucidate the packaging mechanism, we sought to identify the RCNMV origin of assembly sequence (OAS). RCNMV RNA-1 cannot package in the absence of RNA-2 suggesting that it does not contain an independent packaging signal. A 209 nt RNA-2 element expressed from the Tomato bushy stunt virus CP subgenomic promoter is co-assembled with genomic RNA-1 into virions. Deletion mutagenesis delimited the previously characterized 34 nt trans-activator (TA) as the minimal RCNMV OAS. From this study we hypothesize that RNA-1 must be base-paired with RNA-2 at the TA to initiate co-packaging. The addition of viral assembly illustrates the critical importance of the multifunctional TA element as a key regulatory switch in the RCNMV life cycle.


Assuntos
Genoma Viral , RNA Viral/genética , Tombusviridae/genética , Trifolium/virologia , Pareamento de Bases , Proteínas do Capsídeo/genética , Fases de Leitura Aberta , Regiões Promotoras Genéticas , Tombusviridae/fisiologia , Tombusvirus/genética , Transativadores/genética , Transcrição Gênica , Proteínas Virais/genética , Vírion/genética , Vírion/fisiologia , Replicação Viral
16.
Proc Natl Acad Sci U S A ; 104(8): 2756-60, 2007 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-17299054

RESUMO

Predicting the magnitude of enemy release in host-pathogen systems after introduction of novel disease resistance genes has become a central problem in ecology. Here, we develop a general quantitative framework for predicting changes in realized niche size and intrinsic population growth rate after introgression of disease resistance genes into wild host populations. We then apply this framework to a model host-pathogen system targeted by genetically modified and conventionally bred disease-resistant host lines (Trifolium repens lines expressing resistance to Clover yellow vein potyvirus) and show that, under a range of ecologically realistic conditions, the introduction of novel pathogen resistance genes into host populations can pose a quantifiable risk to associated nontarget native plant communities. In the host-pathogen system studied, we predict that pathogen release could result in an increase in the intrinsic rate of population growth of up to 15% and the expansion of host populations into some marginal environments. This approach has general applicability to the ecological risk assessment of all novel disease-resistant plant genotypes that target coevolutionary host-pathogen systems for improvement of agricultural productivity.


Assuntos
Genótipo , Imunidade Inata/genética , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Potyvirus/fisiologia , Trifolium/virologia , Interações Hospedeiro-Parasita , Modelos Biológicos , Dinâmica Populacional
17.
J Virol ; 80(8): 3781-91, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16571795

RESUMO

The genome of Red clover necrotic mosaic virus (RCNMV) in the genus Dianthovirus is divided into two RNA molecules of RNA1 and RNA2, which have no cap structure at the 5' end and no poly(A) tail at the 3' end. The 3' untranslated region (3' UTR) of RCNMV RNA1 contains an essential RNA element (3'TE-DR1), which is required for cap-independent translation. In this study, we investigated a cap-independent translational mechanism of RNA2 using a firefly luciferase (Luc) gene expression assay system in cowpea protoplasts and a cell-free lysate (BYL) prepared from evacuolated tobacco BY2 protoplasts. We were unable to detect cis-acting RNA sequences in RNA2 that can replace the function of a cap structure, such as the 3'TE-DR1 of RNA1. However, the uncapped reporter RNA2, RNA2-Luc, in which the Luc open reading frame (ORF) was inserted between the 5' UTR and the movement protein ORF, was effectively translated in the presence of p27 and p88 in protoplasts in which RNA2-Luc was replicated. Time course experiments in protoplasts showed that the translational activity of RNA2-Luc did not reflect the amount of RNA2. Mutations in cis-acting RNA replication elements of RNA2 abolished the cap-independent translational activity of RNA2-Luc, suggesting that the translational activity of RNA2-Luc is coupled to RNA replication. Our results show that the translational mechanism differs between two segmented genomic RNAs of RCNMV. We present a model in which only RNA2 that is generated de novo through the viral RNA replication machinery functions as mRNA for translation.


Assuntos
Biossíntese de Proteínas , Capuzes de RNA/fisiologia , RNA Viral/biossíntese , Tombusviridae/genética , Trifolium/virologia , Sequência de Bases , Genoma Viral
18.
Virology ; 345(2): 532-9, 2006 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-16297955

RESUMO

Red clover necrotic mosaic virus (RCNMV) is a small icosahedral plant virus with a bipartite RNA genome. While the RCNMV genome consists of two RNAs, it has not been definitively established whether these RNAs are co-packaged into a single virion or packaged individually into separate virions. Biochemical evidence exists to support both hypotheses. To determine the genomic RNA complement within RCNMV, virions were subjected to heat treatments and UV crosslinking. A stable RNA-1:RNA-2 heterodimer was formed with both treatments establishing that RCNMV genomic RNAs are co-packaged into a single virion. Furthermore, RNA-2 homodimer and homotrimers were also observed indicating that some virions contain multiple copies of RNA-2 exclusively. These results indicate that RCNMV virions consist of two distinct populations: (i) virions containing both genomic RNAs; and (ii) virions with multiple copies of RNA-2. This type of hybrid packaging arrangement was unexpected and appears to be unique among the multipartite RNA viruses.


Assuntos
Genoma Viral , RNA Viral/metabolismo , Tombusviridae/metabolismo , Trifolium/virologia , Montagem de Vírus , Dimerização , Doenças das Plantas/virologia , Vírus de Plantas/genética , Vírus de Plantas/metabolismo , Nicotiana/virologia , Tombusviridae/genética , Vírion/metabolismo
19.
J Econ Entomol ; 98(6): 1816-23, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16539099

RESUMO

After characterization of the natural spread of necrosis-inducing Bean yellow mosaic potyvirus (family Potyviridae, genus Potyvirus, BYMV(N)), nonpersistently transmitted from clover, Trifolium repens L., to an adjacent field of snap bean, Phaseolus vulgaris L., in western Oregon, we established a study site enabling us to investigate the virus reservoir, to observe en masse transmission of BYMV(N) to bean plants, and to identify aphid species associated with virus spread. Colonies of Myzus persicae (Sulzer), Acyrthosiphon pisum (Harris), and Aphis fabae Scopoli associated with virus spread were established in an insectary and shown to vector this virus. Although Nearctaphis bakeri (Cowen) comprised 68% of aphid alatae taken from bean leaves during virus spread, we were unable to show that this species could vector the virus by using the same methods that were successful for the other species. Instead, we found that when two distinct N. bakeri colonies unexpectedly emerged from the roots of T. repens BYMV(N) source plants (WZwc #6 and #11) that were present in the laboratory (insectary), these aphids transmitted BYMVN at rates comparable with those of M. persicae and A. pisum. Transmission of BYMVN also occurred with two other N. bakeri colonies maintained for 4 mo on Trifolium pratense L. (NZwc Sch 3B and Sch 7C) BYMVN source plants. Each of these four BYMVN transmission successes also demonstrated an unprecedented once-only transmission of BYMV(N) by N. bakeri colonies. Our experience with western Oregon N. bakeri colonies was compared with descriptions of this native North American species after its 1960-1980s arrival in France, Germany, and Italy.


Assuntos
Afídeos/virologia , Fabaceae/virologia , Doenças das Plantas/virologia , Potyvirus , Trifolium/virologia , Animais , Especificidade da Espécie
20.
J Virol ; 79(2): 978-86, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15613326

RESUMO

The expression of the coat protein gene requires RNA-mediated trans-activation of subgenomic RNA synthesis in Red clover necrotic mosaic virus (RCNMV), the genome of which consists of two positive-strand RNAs, RNA1 and RNA2. The trans-acting RNA element required for subgenomic RNA synthesis from RNA1 has been mapped previously to the protein-coding region of RNA2, whereas RNA2 is not required for the replication of RNA1. In this study, we investigated the roles of the protein-coding region in RNA2 replication by analyzing the replication competence of RNA2 mutants containing deletions or nucleotide substitutions. Our results indicate that the same stem-loop structure (SL2) that functions as a trans-activator for RNA-mediated coat protein expression is critically required for the replication of RNA2 itself. Interestingly, however, disruption of the RNA-RNA interaction by nucleotide substitutions in the region of RNA1 corresponding to the SL2 loop of RNA2 does not affect RNA2 replication, indicating that the RNA-RNA interaction is not required for RNA2 replication. Further mutational analysis showed that, in addition to the stem-loop structure itself, nucleotide sequences in the stem and in the loop of SL2 are important for the replication of RNA2. These findings suggest that the structure and nucleotide sequence of SL2 in RNA2 play multiple roles in the virus life cycle.


Assuntos
RNA Viral/biossíntese , Tombusviridae/genética , Transativadores/fisiologia , Trifolium/virologia , Sequência de Bases , Proteínas do Capsídeo/fisiologia , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteínas do Movimento Viral em Plantas , RNA Viral/química , Proteínas Virais/fisiologia
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