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At least 145 records · Page 8Linked to original sources

PubMed2006

Potato virus X RNA-mediated assembly of single-tailed ternary 'coat protein-RNA-movement protein' complexes.

Different models have been proposed for the nature of the potexvirus transport form that moves from cell to cell over the infected plant: (i) genomic RNA moves as native virions; or (ii) in vitro-assembled non-virion ribonucleoprotein (RNP) complexes consisting of viral RNA, coat protein (CP) and movement protein (MP), termed TGBp1, serve as the transport form in vivo. As the structure of these RNPs has not been elucidated, the products assembled in vitro from potato virus X (PVX) RNA, CP and TGBp1 were characterized. The complexes appeared as single-tailed particles (STPs) with a helical, head-like structure composed of CP subunits located at the 5'-proximal region of PVX RNA; the TGBp1 was bound to the terminal CP molecules of the head. Remarkably, no particular non-virion RNP complexes were observed. These data suggest that the CP-RNA interactions resulting in head formation prevailed over TGBp1-RNA binding upon STP assembly from RNA, CP and TGBp1. STPs could be assembled from the 5' end of PVX RNA and CP in the absence of TGBp1. The translational ability of STPs was characterized in a cell-free translation system. STPs lacking TGBp1 were entirely non-translatable; however, they were rendered translatable by binding of TGBp1 to the end of the head. It is suggested that the RNA-mediated assembly of STPs proceeds via two steps. Firstly, non-translatable CP-RNA STPs are produced, due to encapsidation of the 5'-terminal region. Secondly, the TGBp1 molecules bind to the end of a polar head, resulting in conversion of the STPs into a translatable form.

Biological Transport, Active↗

PubMed2006

Peptide display on Potato virus X: molecular features of the coat protein-fused peptide affecting cell-to-cell and phloem movement of chimeric virus particles.

The potexvirus Potato virus X (PVX) can be modified genetically to generate chimeric virus particles (CVPs) carrying heterologous peptides fused to coat protein (CP) subunits. A spontaneous PVX mutant expressing a truncated, but functional, form of the CP has been isolated. With the aim of exploiting this virus to display peptides useful for vaccine formulations, two novel viral expression vectors based on pPVX201 (bearing the wild-type PVX genome) were constructed encoding the truncated CP. Both vectors were able to produce infectious virus particles in planta and were used to insert a panel of sequences encoding peptides of biopharmaceutical interest as N-terminal fusions to the truncated cp gene. The analysis of infection progression induced by the different constructs enabled identification of two important structural features of the fused peptide, namely tryptophan content and isoelectric point, critically affecting the formation of PVX CVPs and virus movement through the plant. These results are discussed in view of the rising interest in engineered plant viruses for development of peptide-based epitope vaccines.

Capsid Proteins↗

PubMed2002

A surveillance system regulates selective entry of RNA into the shoot apex.

Phloem-mobile endogenous RNA is trafficked selectively into the shoot apex. In contrast, most viruses and long-distance post-transcriptional gene silencing (PTGS) signals are excluded from the shoot apex. These observations suggest the operation of an underlying regulatory mechanism. To examine this possibility, a potexvirus movement protein, known to modify cell-to-cell trafficking and PTGS, was expressed ectopically in transgenic plants. These plants were found to be compromised in their capacity to exclude both viral RNA and silencing signals from the shoot apex. The transgenic plants also displayed various degrees of abnormal leaf polarity depending on transgene expression level. Normal patterns of organ development were restored by either virus- or Agrobacterium tumefaciens-mediated induction of PTGS. This revealed the presence of an RNA signal surveillance system that acts to allow the selective entry of RNA into the shoot apex. We propose that this surveillance system regulates signaling and protects the shoot apex, in particular the cells that give rise to reproductive structures, from viral invasion.

Cloning, Molecular↗

PubMed1998

Identification and characterization of the Escherichia coli-expressed RNA-dependent RNA polymerase of bamboo mosaic virus.

Bamboo mosaic virus (BaMV), a member of the potexvirus group, infects primarily members of the Bambusoideae. The open reading frame 1 (ORF1) of BaMV encodes a 155-kDa polypeptide that was postulated to be involved in the replication and the formation of cap structure at the 5' end of the viral genome. To characterize the activities associated with the 155-kDa viral protein, it was expressed in Escherichia coli BL21(DE3) cells with thioredoxin, hexahistidine, and S. Tag fused consecutively at its amino terminus, and the fusion protein was purified by metal affinity chromatography. Several RNA fragments, prepared by in vitro transcription, were tested as substrates for the RNA-dependent RNA polymerase (RdRp) activity. Among them, the expressed fusion enzyme was able to generate a 32P-labeled RNA product when 3'-end RNA fragments of the positive strand or negative strand of BaMV were included in the assay mixture. Dot hybridization assay revealed that the reaction products are complementary to their RNA substrates. Taken together, the evidence suggests that the 155-kDa protein encoded by ORF1 of BaMV has an RdRp activity and should be involved in the replication of BaMV. Mutational analyses demonstrate the importance of the GDD motif in the polymerase activity, and deletion studies suggest that the polymerase activity resides in the carboxyl terminus of the 155-kDa viral protein.

Base Sequence↗

PubMed2004

Critical residues for GTP methylation and formation of the covalent m7GMP-enzyme intermediate in the capping enzyme domain of bamboo mosaic virus.

Open reading frame 1 of Bamboo mosaic virus (BaMV), a Potexvirus in the alphavirus-like superfamily, encodes a 155-kDa replicase responsible for the formation of the 5' cap structure and replication of the viral RNA genome. The N-terminal domain of the viral replicase functions as an mRNA capping enzyme, which exhibits both GTP methyltransferase and S-adenosylmethionine (AdoMet)-dependent guanylyltransferase activities. We mutated each of the four conserved amino acids among the capping enzymes of members within alphavirus-like superfamily and a dozen of other residues to gain insight into the structure-function relationship of the viral enzyme. The mutant enzymes were purified and subsequently characterized. H68A, the mutant enzyme bearing a substitution at the conserved histidine residue, has an approximately 10-fold increase in GTP methyltransferase activity but completely loses the ability to form the covalent m(7)GMP-enzyme intermediate. High-pressure liquid chromatography analysis confirmed the production of m(7)GTP by the GTP methyltransferase activity of H68A. Furthermore, the produced m(7)GTP sustained the formation of the m(7)GMP-enzyme intermediate for the wild-type enzyme in the presence of S-adenosylhomocysteine (AdoHcy), suggesting that the previously observed AdoMet-dependent guanylation of the enzyme using GTP results from reactions of GTP methylation and subsequently guanylation of the enzyme using m(7)GTP. Mutations occurred at the other three conserved residues (D122, R125, and Y213), and H66 resulted in abolition of activities for both GTP methylation and formation of the covalent m(7)GMP-enzyme intermediate. Mutations of amino acids such as K121, C234, D310, W312, R316, K344, W406, and K409 decreased both activities by various degrees, and the extents of mutational effects follow similar trends. The affinity to AdoMet of the various BaMV capping enzymes, except H68A, was found in good correlations with not only the magnitude of GTP methyltransferase activity but also the capability of forming the m(7)GMP-enzyme intermediate. Taken together with the AdoHcy dependence of guanylation of the enzyme using m(7)GTP, a basic working mechanism, with the contents of critical roles played by the binding of AdoMet/AdoHcy, of the BaMV capping enzyme is proposed and discussed.

Amino Acid Sequence↗

PubMed2026

Characterization of yam virus X isolates from Dioscorea trifida in Brazil.

OBJECTIVE: Yam virus X (YVX; Potexvirus ecsdioscoreae) is a positive-sense, flexuous RNA virus belonging to the family Alphaflexiviridae. It has been first reported from Guadeloupe, a French archipelago located in the Caribbean Sea. In this study, we investigated the virome in yam (Dioscorea spp.) plant material collected in the state of Bahia (Brazil) by high-throughput sequencing (HTS) on Illumina platform. The objective of the investigation was to explore the occurrence of YVX in yam from South America, and to study its genetic diversity compared to the only one YVX genome sequence available in the GenBank public database. RESULTS: An initial investigation by HTS of bulked RNA extracts (n=23, combined into 4 pools) revealed occurrence of YVX only in samples collected in the region of Valença. Subsequent screening by RT-PCR of the individual samples composing the pool uncovered infection with YVX only in Discorea trifida. Total RNA extracts from three infected plants were individually sequenced, resulting in the assembly of three complete genome sequences of YVX, showing ~84% nucleotide identity to the reference sequence from Guadeloupe. Our results contribute to expanding the pool of sequences available for YVX, supporting detection purposes and stimulating additional investigations for future studies on YVX diversity and evolution.

Brazil↗

PubMed2006

Long-distance RNA-RNA interactions between terminal elements and the same subset of internal elements on the potato virus X genome mediate minus- and plus-strand RNA synthesis.

Potexvirus genomes contain conserved terminal elements that are complementary to multiple internal octanucleotide elements. Both local sequences and structures at the 5' terminus and long-distance interactions between this region and internal elements are important for accumulation of potato virus X (PVX) plus-strand RNA in vivo. In this study, the role of the conserved hexanucleotide motif within SL3 of the 3' NTR and internal conserved octanucleotide elements in minus-strand RNA synthesis was analyzed using both a template-dependent, PVX RNA-dependent RNA polymerase (RdRp) extract and a protoplast replication system. Template analyses in vitro indicated that 3' terminal templates of 850 nucleotides (nt), but not 200 nt, supported efficient, minus-strand RNA synthesis. Mutational analyses of the longer templates indicated that optimal transcription requires the hexanucleotide motif in SL3 within the 3' NTR and the complementary CP octanucleotide element 747 nt upstream. Additional experiments to disrupt interactions between one or more internal conserved elements and the 3' hexanucleotide element showed that long-distance interactions were necessary for minus-strand RNA synthesis both in vitro and in vivo. Additionally, multiple internal octanucleotide elements could serve as pairing partners with the hexanucleotide element in vivo. These cis-acting elements and interactions correlate in several ways to those previously observed for plus-strand RNA accumulation in vivo, suggesting that dynamic interactions between elements at both termini and the same subset of internal octanucleotide elements are required for both minus- and plus-strand RNA synthesis and potentially other aspects of PVX replication.

Base Sequence↗

PubMed1997

Coat protein gene and 3'-noncoding region of a new Korean isolate of cymbidium mosaic virus.

A new Korean isolate of cymbidium mosaic virus (denoted as CymMV-K2), a member of potexviruses, was identified and isolated from a Korean cultivar of Cymbidium species When the nucleotide sequence of the 3'-terminal region of the viral RNA was compared with those of the corresponding regions of two Singaporean isolates (denoted as CymMV-S1 and CymMV-S2) and a Korean isolate (denoted as CymMV-K1), the nucleotide sequence of the coat protein gene of CymMV-K2 was highly homologous to other cymbidium mozaic viruses (92.9%-96.7% homology). The coat protein of CymMV-K2 and CymMV-S2 consists of 223 amino acids, while the coat protein of CymMV-K1 and CymMV-S1 consists of 220 amino acids. This difference was caused by deletions of 5 nucleotides in the coat protein open reading frame (ORF) of CymMV-S1 and CymMV-K1, when compared with CymMV-K2 and CymMV-S2. These deletions result in changes of the deduced amino acid sequence and the length of the coat protein. The 3'-noncoding region of the CymMV-K2, which contains sequences involved in the replication and polyadenylation of viral RNA, was compared with those of other cymbidium mosaic viruses. No canonical polyadenylation signal was found in the 3'-noncoding region of CymMV-K2, whereas in other CymMVs AAUAAA boxes, are present at the end of RNA with their AAA portions as the first A residue of the poly(A) tail.

Amino Acid Sequence↗

PubMed1998

Genome characterization of a Korean isolate of cymbidium mosaic virus.

The complete nucleotide sequence of the genomic RNA of a Korean isolate of cymbidium mosaic virus (CymMV-K2) was determined. The genomic RNA is 6227 nucleotides in length, excluding the poly(A) tail. It contains a 5'-noncoding region (NCR) of 73 nucleotides, five open reading frames (ORFs 1 to 5) which encode proteins with M(r) 160 kDa RNA-dependent RNA polymerase (ORF1), 26 kDa movement protein 1 (ORF2), 13 kDa movement protein 2 (ORF3), 10 kDa movement protein 3 (ORF4), 24 kDa coat protein (OFR5), and a 3' NCR of 76 nucleotides. The 5'-end of the CymMV-K2 genome initiates with GGAAAA which contrasts to GAAAA at the 5'-ends of other potexviruses, including a Singapore isolate of CymMV (CymMV-S2). When compared with CymMV-S2, 171 base substitutions were observed in the CymMV-K2 genome. Substitutions in the overlapping ORFs (ORFs 2 to 4) occurred more frequently than those in 5' NCR, ORF1, and 3' NCR. In addition to substitutions, two single-base deletions, one in the intercistronic region between ORF1 and ORF2 and the other in the ORF2, were found on the CymMV-K2 genome. The deletion in the ORF2 induced a frameshift which altered the C-terminal domain of movement protein 1. ORF3 and ORF4 of the CymMV-K2 genome are partially different from those of another Singapore CymMV genome (CymMV-S1) which has four frameshifts due to nucleotide deletions within these ORFs. Interestingly, the frameshifts resulted in no change in the conserved sequences of the movement proteins but reconstructed their transmembrane domains.

Amino Acid Sequence↗

PubMed1993

Nucleotide sequence and genetic organization of peanut clump virus RNA 2 and partial characterization of deleted forms.

The complete nucleotide sequence of peanut clump virus RNA 2 has been determined by characterization of cloned cDNA and by direct RNA sequencing. The sequence is 4503 residues in length and contains six long open reading frames (ORFs). The cistron for the coat protein (ORF 1) is located nearest the 5' end of the RNA and is separated from the 5' extremity by a noncoding region of 389 nucleotides. ORF 2 starts one nucleotide upstream of the first residue of the UGA termination codon of the coat protein cistron and encodes a polypeptide of 39300 M(r). Four additional ORFs with the potential to encode polypeptides with approximate M(r)'s of 51,000 (ORF 3), 13,800 (ORF 4), 9400 (ORF 5), and 6800 (ORF 6) are found on the 3' proximal half of the RNA. ORFs 3, 4, and 5 resemble the Triple Gene Block found in potexviruses, carlaviruses, a hordeivirus, and a furovirus. The 276 nucleotide 3' noncoding region of RNA 2 displays extensive sequence homology with the 3' noncoding region of RNA 1. Several peanut clump virus isolates propagated on Nicotiana benthamania contain RNA 2's significantly shorter than that of the type isolate. Partial characterization of two such isolates reveals that their RNA 2's have undergone deletions within ORF 2.

Amino Acid Sequence↗

PubMed1996

Beet soil-borne virus RNA 3--a further example of the heterogeneity of the gene content of furovirus genomes and of triple gene block-carrying RNAs.

The complete nucleotide sequence of RNA 3 of the Ahlum isolate of beet soil-borne virus (BSBV) was determined from cDNAs obtained with immunocaptured virus particles and denatured preparations of dsRNA. BSBV RNA 3 is unique among the plant virus RNAs studied so far in containing apparently only the coding sequences of a triple gene block (TGB). The derived amino acid sequences of the three putative TGB-encoded proteins showed the highest level of sequence similarities with those of the corresponding proteins of potato mop top furovirus (PMTV) followed by those of peanut clump furovirus and barley stripe mosaic hordeivirus. Progressively fewer similarities were found with the TGB-encoded proteins of beet necrotic yellow vein virus (uncertain classification), potato X potexvirus, and potato M carlavirus. The 3'-terminal 78 nucleotides of BSBV RNA 3 can be folded into a tRNA-like structure and a high degree of sequence similarity exists between the 122 3'-terminal nucleotides of BSBV RNA 3 and PMTV RNA 2. In other regions, however, no pronounced sequence similarities were found between the two RNAs, and PMTV RNA 2 contains an additional putative gene for a cysteine-rich protein downstream of the TGB. The two viruses are unrelated serologically. BSBV RNA 3 adds a further variant to the heterogeneity of the gene content of furovirus genomes and of triple gene block-carrying RNAs.

Amino Acid Sequence↗

PubMed1996

Comparisons of the genomic cis-elements and coding regions in RNA beta components of the hordeiviruses barley stripe mosaic virus, lychnis ringspot virus, and poa semilatent virus.

Nucleotide sequences of the genomic RNA beta components of hordeiviruses poa semilatent virus (PSLV) and lychnis ringspot virus (LRSV) were determined. PSLV and LRSV closely resemble barley stripe mosaic virus (BSMV), type hordeivirus, in the gene arrangement of their RNAs beta, comprising 5'-proximal beta a (coat protein) gene and downstream triple gene block (TGB) coding for the beta b, beta c, and beta d putative transport proteins. The beta a, beta b, beta c, and beta d proteins of the three hordeiviruses showed significant sequence similarity, with the respective proteins of PSLV and BSMV being closer to each other than to their counterparts of LSRV. Comparisons of the TGB-encoded proteins of hordeiviruses, potexviruses, carlaviruses, and furoviruses indicate that the first and second TGB genes belong to the monophyletic groups, whereas the third gene may have multiple ancestry. LRSV, PSLV, and BSMV showed remarkable variation in the 3'-untranslated regions of their genomic RNAs. Among the three hordeiviruses, LRSV has the shortest 3'-noncoding region that lacks tentative pseudoknot-forming elements conserved upstream of the 3'-tRNA-like structure in the BSMV and PSLV genomes. On the other hand, LRSV RNA beta, like that of BSMV, contained the internal poly(A) sequence that is absent from PSLV RNA.

Adaptation, Physiological↗

PubMed2000

Pea embryonic tissues show common responses to the replication of a wide range of viruses.

The response of pea embryonic tissues to the replication of a range of different viruses was investigated using in situ hybridization to analyze changes in the expression of two host genes, heat shock protein 70 (hsp70) and lipoxygenase (lox1). Excised pea embryos were infected using microprojectile bombardment with a nonseed transmissible strain of Pea seed-borne mosaic potyvirus, or with Pea early browning tobravirus (PEBV), White Clover mosaic potexvirus, or Beet curly top geminivirus. Collectively, these examples represent families of viruses with differing genomic features, differing numbers of genomic components and differing replication strategies. In all cases, there was an induction of hsp70 associated with virus replication and, in most cases, a downregulation of lox1. Hence, either each virus has a direct inducer of these common responses or the induction is indirectly the result of a generic feature of virus infection. By exploiting the bipartite nature of the PEBV genome, the coat protein gene and genes involved in vector transmission were excluded as potential inducers.

Cotyledon↗

PubMed1989

Possible members of the potyvirus group transmitted by mites or whiteflies share epitopes with aphid-transmitted definitive members of the group.

There are at least ten viruses identified in the literature that resemble definitive potyviruses in having flexuous filamentous particles and inducing the formation of "pinwheel" cytoplasmic inclusions in infected cells but that are transmitted by eriophyid mites, whiteflies or soil fungi and not by aphids, the vectors of the definitive potyviruses. The taxonomic status of these viruses is uncertain at present. Using a broadly cross-reactive antiserum raised against the dissociated coat protein core (residues 68-285) of a definitive potyvirus (Johnsongrass mosaic virus), we have shown that wheat streak mosaic virus which is transmitted by mite and sweet potato mild mottle virus which is transmitted by whitefly have coat proteins that share epitopes with definitive potyviruses. This finding further supports their classification as definitive members of the potyvirus group. The cross-reactive antiserum used here had been shown previously to react with coat proteins of fifteen different definitive potyviruses. The antiserum did not react with coat proteins of potexviruses and tobamoviruses.

Animals↗

PubMed1991

Nucleotide sequence of the 3' terminal region of lettuce mosaic potyvirus RNA shows a Gln/Val dipeptide at the cleavage site between the polymerase and the coat protein.

DNA complementary to the 3' terminal 1651 nucleotides of the genome of the common strain of lettuce mosaic virus (LMV-O) has been cloned and sequenced. Microsequencing of the N-terminus enabled localization of the coat protein gene in this sequence. It showed also that the LMV coat protein coding region is at the 3' end of the genome, and that the coat protein is processed from a larger protein by cleavage at an unusual Q/V dipeptide between the polymerase and the coat protein. This is the first report of such a site for cleavage of a potyvirus polyprotein, where only Q/A, Q/S, and Q/G cleavage sites have been reported. The LMV coat protein gene encodes a 278 amino acid polypeptide with a calculated Mr of 31,171 and is flanked by a region which has a high degree of homology with the putative polymerase and a 3' untranslated region of 211 nucleotides in length. Percentage of homology with the coat protein of other potyviruses confirms that LMV is a distinct member of this group. Moreover, amino acid homologies noticed with the coat protein of potexvirus, bymovirus, and carlavirus elongated plant viruses suggest a functional significance for the conserved domains.

Amino Acid Sequence↗

PubMed1993

Nucleotide sequence and in vitro translation of the coat protein gene of cymbidium mosaic virus.

The nucleotide sequence of the coat protein gene of cymbidium mosaic virus (CyMV) has been determined from a cDNA clone that encompassed the 3'-region of the virus genome. The 0.75 kb coat protein open reading frame (ORF) codes for a 24kD polypeptide. The coat protein coding region was separated from the 3'-poly(A) terminal of the viral genome by an 80 base noncoding region. The derived amino acid sequence of this ORF showed considerable homology with other potexvirus coat protein sequences, notably with narcissus mosaic virus (NMV), potato virus X (PVX), and white clover mosaic virus (WClMV). In vitro expression of the CyMV coat protein gene was carried out by T7 RNA polymerase on the linearized cDNA clone template, followed by in vitro translation of the purified transcript in wheat germ extract. One of the translated products was found to comigrate with the coat protein of the virus at about 24 kD, as determined by NaDodSO4-PAGE. This translated product was also found to crossreact with antibodies raised against CyMV.

Amino Acid Sequence↗

PubMed2003

Nucleotide sequences of the coat protein gene of potyviruses infecting Ornithogalum thyrsoides.

Species of three viral genera infecting Ornithogalum thyrsoides plants showing mosaic symptoms were identified using RT-PCR and degenerate universal primers for each viral genus. The DNA fragments obtained encoded the coat protein (CP) gene and were sequenced. The plants were found to be infected with one or other of three potyvirus species, one of them was Ornithogalum mosaic virus (OrMV). The other two viruses were previously unrecorded and were named Ornithogalum virus 2 (OV-2) and 3 (OV-3). Direct comparison and phylogenetic analysis with published OrMV isolates revealed that the CP of the three OrMV-like clones were more similar to Pterostylis virus Y (PtVY) than to OrMV. No carlavirus or potexvirus was isolated.

Base Sequence↗

PubMed2003

Detection and classification of allexiviruses from garlic in China.

Degenerate primers for RT-PCR were designed and used to amplify genome fragments ( c. 750 nt in the coat protein-ORF6 region) of allexiviruses from a total of 28 garlic samples from 24 provinces in China. Many samples contained more than one distinct sequence. A total of 60 different sequences were obtained. Phylogenetic analysis and two-way comparisons were used to assess the status of the sequences and to re-examine the criteria for distinguishing species within the genus. Most of the sequences could be allocated to either Garlic virus D or Garlic virus X on the basis of sequence similarity but some appeared to be intermediate between existing species. There were no sequences of Garlic virus C or Shallot virus X. A comparison with the related genera Carlavirus, Foveavirus and Potexvirus suggests that the published allexivirus species demarcation criteria may have been drawn too tightly and should be re-examined.

China↗