RESUMO
Complete nucleotide sequences of the type isolate of Grapevine leafroll-associated virus 4 (GLRaV-4) and of an isolate of GLRaV-6 from cv 'Estellat' (GLRaV-6Est) were generated and compared mutually and with related viruses. The genome organization of both viruses resembled that of members of Subgroup I in the genus Ampelovirus (fam. Closteroviridae). The availability of these sequences, along with previously existing data on related GLRaVs, allowed critical review of the taxonomy and nomenclature of these viruses. In phylogenetic analyses, GLRaV-4 and -6Est consistently grouped with GLRaV-5, -9, and -Pr forming a poorly resolved sub-cluster ("GLRaV-4 group") within the genus Ampelovirus. In-depth study showed that genetic distances between these viruses do not exceed the intra-species diversity observed in other closteroviruses. In Western blots, partially purified preparations of GLRaVs -4, -5, -6 and -9 reacted only with homologous monoclonal antibodies, but were all recognized by polyclonal antisera to GLRaV-5 and GLRaV-9. Serological relatedness among these viruses was further confirmed in DAS-ELISA. In immuno-electron microscopy, GLRaV-6 particles appeared uniformly decorated with homologous monoclonal antibodies, whereas GLRaV-2, used as a control, showed "bipolar" morphology of the virion. Results of this study challenge taxonomy and nomenclature of several GLRaVs suggesting that they are divergent isolates of Grapevine leafroll-associated virus 4 and not, as has been assumed, distinct species (definitive and/or putative) in the genus Ampelovirus.
Assuntos
Closteroviridae/classificação , Ordem dos Genes , Genoma Viral , Filogenia , RNA Viral/genética , Sequência de Aminoácidos , Closteroviridae/genética , Closteroviridae/imunologia , Análise por Conglomerados , Ensaio de Imunoadsorção Enzimática , Genes Virais , Genótipo , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , SorotipagemRESUMO
To direct the genome synthesis, RNA viruses without a DNA stage in the replication cycle use RNA-dependent RNA polymerase (RdRp). All RdRps have conserved right hand-like shape that includes characteristic A-->B-->C sequence motifs forming the active site. Recently, the structural permutation of the RdRp active site (C-->A-->B) has been described in few double-stranded RNA birnaviruses and a subset of positive-stranded RNA tetraviruses distantly related to Picorna-like viruses. Here we describe a permuted RdRp in the newly identified plant alpha-like virus with 6.5 kb-long polyadenylated genome, dubbed Grapevine virus Q (GVQ). The multi-domain layout and sequence similarities place GVQ into the genus Marafivirus of the family Tymoviridae. In contrast to other tymovirids, GVQ has 21 amino acid residues corresponding to the motif C relocated upstream of the motif A in the putative RdRp. This unique sequence characteristic was extensively verified and identified in several GVQ isolates infecting wild and cultivated Vitis and Rubus spp.
Assuntos
Domínio Catalítico , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Tymoviridae/enzimologia , Tymoviridae/genética , Proteínas Virais/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Análise por Conglomerados , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Tymoviridae/classificação , Vitis/virologiaRESUMO
The nucleotide sequence of RNA-2 of Grapevine Anatolian ringspot virus (GARSV) and Grapevine deformation virus (GDefV), two recently described nepoviruses, has been determined. These RNAs are 3753 nt (GDefV) and 4607 nt (GARSV) in size and contain a single open reading frame encoding a polyprotein of 122 kDa (GDefV) and 150 kDa (GARSV). Full-length nucleotide sequence comparison disclosed 71-73% homology between GDefV RNA-2 and that of Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV), and 62-64% homology between GARSV RNA-2 and that of Grapevine chrome mosaic virus (GCMV) and Tomato black ring virus (TBRV). As previously observed in other nepoviruses, the 5' non-coding regions of both RNAs are capable of forming stem-loop structures. Phylogenetic analysis of the three proteins encoded by RNA-2 (i.e. protein 2A, movement protein and coat protein) confirmed that GDefV and GARSV are distinct viruses which can be assigned as definitive species in subgroup A and subgroup B of the genus Nepovirus, respectively.
Assuntos
Sequência de Bases/genética , Nepovirus/genética , RNA Viral/genética , Análise de Sequência de RNA , Regiões 5' não Traduzidas/genética , Proteínas do Capsídeo/genética , Dados de Sequência Molecular , Peso Molecular , Nepovirus/classificação , Conformação de Ácido Nucleico , Fases de Leitura Aberta/genética , Filogenia , Proteínas do Movimento Viral em Plantas , Poliproteínas/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Proteínas Virais/genéticaRESUMO
The 3' end of the genome of three Grapevine fleck virus-like viruses, i.e. Grapevine redglobe virus (GRGV), Grapevine asteroid mosaic-associated virus (GAMaV), and an unidentified virus from a Greek grapevine (accession GR8-19) was amplified from reverse transcribed total nucleic acid extracts from infected grapevine tissues and sequenced. The analysed genome portions differed in size and organization. The 3' ends of GAMaV (1852 nt) and of GR8-19 (1791 nt) resembled that of marafiviruses, as both encoded a single putative polyprotein containing the conserved "marafibox" sequence and lacked the stop codon between the replicase and coat protein genes. By contrast, the replicase and coat protein genes present in the terminal 2006 nt of GRGV genome were clearly separated and there was a 3'-proximal open reading frame encoding a putative proline rich protein with molecular mass of c. 17 kDa. The genome of all three viruses was polyadenylated. The organization of the 3' terminal genomic region and phylogenetic analysis of viral replicases and coat proteins suggest that GAMaV and the Greek virus GR8-19 belong in the genus Marafivirus, and GRGV in the genus Maculavirus, family Tymoviridae. Virus GR8-19 had molecular traits differing enough from GAMaV and other marafiviruses to be regarded as a new putative species in the genus Marafivirus, for which the name of Grapevine rupestris vein feathering virus is proposed.
Assuntos
Regiões 3' não Traduzidas/genética , Genoma Viral , Vírus de RNA/classificação , Análise de Sequência de DNA , Vitis/virologia , Dados de Sequência Molecular , Filogenia , Doenças das Plantas/virologia , Vírus de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The complete nucleotide sequence of Grapevine fleck virus (GFkV) genomic RNA was determined. The genome is 7564 nt in size, excluding the 3'-terminal poly(A) tail, is characterized by an extremely high cytosine content (ca. 50%), and contains four putative open reading frames and untranslated regions of 291 and 35 nt at the 5' and 3' ends, respectively. ORF 1 potentially encodes a 215.4 kDa polypeptide (p215), which has the conserved motifs of replication-associated proteins of positive-strand RNA viruses. ORF 2 encodes a 24.3 kDa polypeptide (p24) identified as the coat protein. ORFs 3 and 4 are located at the extreme 3' end of the viral genome and encode proline-rich proteins of 31.4 kDa (p31) and 15.9 kDa (p16), respectively, of unknown function. Phylogenetic analysis of the viral replicase and coat protein genes showed that GFkV is related to members of the Tymovirus and Marafivirus genera. Two subgenomic RNAs were present in the GFkV preparations as ascertained by molecular hybridization. The genome organization of GFkV resembles to some extent that of tymoviruses and marafiviruses. However, differences in the biological and epidemiological behaviour, cytopathology and molecular properties (i.e. size of genomic RNA and coat protein, and number of ORFs) support the notion that GFkV is a separate virus belonging in a new genus.
