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PubMed1991

Influence of the poly(A) tail and putative polyadenylation signal on the infectivity of white clover mosaic potexvirus.

A series of mutations has been constructed in a cDNA clone of white clover mosaic virus (WCIMV) which decreases the poly(A) tail length of run-off transcripts from 74 to 27, 10 or zero 3'-terminal (A) residues. Although transcripts with short poly(A) tails were less infectious than wild-type RNA, complete removal of the (A) tail did not abolish infectivity. Addition of nonviral nucleotides to the 3' terminus of transcripts with no 3'-terminal (A) residues eliminated infectivity. Heterogenous-length poly(A) tails, indistinguishable from the wild type, were synthesized de novo in plants inoculated with transcripts with no 3'-terminal (A) residues, demonstrating the presence of a poly(A) polymerase activity in WCIMV-infected tissue. Mutation of a putative polyadenylation motif found in the 3' noncoding region of WCIMV decreased the efficiency of polyadenylation of the progeny of transcripts with 10 3'-terminal (A) residues. The same mutation in transcripts with no 3'-terminal (A) residues abolished infectivity.

Cloning, Molecular↗

PubMed1999

Detection and serological relationships of cymbidium mosaic potexvirus isolates.

Twenty-two isolates of Cymbidium mosaic virus (CyMV) were isolated from 35 orchid plants suspected of being infected with CyMV. Among the three methods used for detecting CyMV, immunoelectron microscopy (IEM-1) was shown to be the most sensitive method, being able to detect the virus in 71.43% of suspected CyMV-infected plants while the electron microscopic method and the indexing plant method could detect 51.43 and 42.86%, respectively. Out of 12 symptomless plants investigated, 25% were found by IEM-1 method to be infected with the virus. Purified CyMV were flexuous rods having lengths between 470-490 nm. A few end-to-end aggregates were also observed and the 280 260 absorbance ratios were from 0.884 to 0.929. The yield of CyMV was 31.07 to 44.09 mg per kg of Datura leaves. Antibodies against purified CyMV D2 were produced in rabbits and hens. The antibody titers in the yolk and sera of hens indicated that 0.5 mg of virus per immunization efficiently generated an abundant supply of IgY in the yolk, however 1 mg of virus per immunization gave a stronger immune response in both sera and yolk. The average yields of IgY were 6.5 +/- 0.6 and 9.4 +/- 0.9 mg/ml of yolk in the group that received 0.5 mg and the group that received 1.0 mg of the virus, respectively. Positive ELISA reactions were observed in 18 and 20 of 22 CyMV isolates when detected with rabbit IgG and IgY, respectively, demonstrating that those isolates were serologically related and the ELISA reactions were shown to be stronger with IgY than those with rabbit IgG in most isolates. The degree of reaction between the CyMV isolates, O(2) and O(4), and IgY was less than that of the other isolates. The two isolates, D(6) and Cat(6), gave negative reactions to rabbit IgG. The results of ELISA assays showed that the homologous serological reaction was not consistently stronger than the heterologous one. Twelve isolates out of twenty-two gave stronger reactions than the homologous antigen (CyMV D(2)) when IgY was used as the detecting antibody while nine isolates gave stronger reactions when using rabbit IgG. No reactions were observed with other plant viruses and plant proteins from healthy Datura.

Journal Article↗

PubMed1988

Single- and double-stranded viral RNAs in plants infected with the potexviruses papaya mosaic virus and foxtail mosaic virus.

Three classes of viral RNA were recovered from polyribosomes purified from papaya leaves infected with papaya mosaic virus (PapMV) and from barley leaves infected with foxtail mosaic virus (FoMV): full-length viral RNAs [6.8 and 6.2 kilobases (kb), respectively]; less abundant intermediate subgenomic RNAs (2.2 and 1.9 kb), and abundant, small subgenomic RNAs (1 and 0.9 kb). Small amounts of the PapMV-specified 1.0-kb subgenomic RNA were encapsidated, whereas no encapsidated subgenomic RNAs could be found in preparations of FoMV. Immunoprecipitation of the products of in vitro translation of the small subgenomic RNA of both viruses showed that it codes for the corresponding viral coat protein. FoMV genomic RNA isolated from polyribosomes also directed the efficient synthesis of a 37- to 38-kilodalton protein which was immunoprecipitated by an antiserum raised against the coat protein. We presume this product to be a readthrough protein initiated to the 5' side of and in the same reading frame as the coat protein-coding sequences in FoMV RNA. The predominant double-stranded viral-specified RNAs in tissues infected with PapMV, FoMV, and clover yellow mosaic virus were genome sized (6.8, 6.2, and 7.0 kb pairs, respectively). If double-stranded RNAs corresponding to coat protein subgenomic RNAs exist, they must be present in much lower relative abundances.

Genes, Viral↗

PubMed2005

The complete nucleotide sequence of hydrangea ringspot virus. Brief report.

The complete nucleotide sequence of the genome of the potexvirus, hydrangea ringspot virus, has been determined. The sequence is 6,185 nt in length, excluding the poly (A) tail, and contains six ORFs coding for proteins of 156, 26, 12, 8, 24, and 16 kDa, respectively. ORF 6 is contained within ORF 5 and in this respect the virus is similar to the potexviruses CsCMV, NMV, and SMYEV. Phylogenetic analysis of the putative products of the ORFs and signature motifs contained within these products shows the virus to be most closely related to CsCMV. A similar analysis of data for the coat proteins of potexviruses did not support the previously reported serological relationships between HdRSV and other potexviruses. This is the first complete sequence published for the genome of a potexvirus infecting a dicotyledonous, temperate, deciduous woody species.

Amino Acid Sequence↗

PubMed2003

Complete nucleotide sequence and genome organization of a Cactus virus X strain from Hylocereus undatus (Cactaceae).

The complete nucleotide sequence of a strain of Cactus virus X (CVX-Hu) isolated from Hylocereus undatus (Cactaceae) has been determined. Excluding the poly(A) tail, the sequence is 6614 nucleotides in length and contains seven open reading frames (ORFs). The genome organization of CVX is similar to that of other potexviruses. ORF1 encodes the putative viral replicase with conserved methyltransferase, helicase, and polymerase motifs. Within ORF1, two other ORFs were located separately in the +2 reading frame, we call these ORF6 and ORF7. ORF2, 3, and 4, which form the "triple gene block" characteristic of the potexviruses, encode proteins with molecular mass of 25, 12, and 7 KDa, respectively. ORF5 encodes the coat protein with an estimated molecular mass of 24 KDa. Sequence analysis indicated that proteins encoded by ORF1-5 display certain degree of homology to the corresponding proteins of other potexviruses. Putative product of ORF6, however, shows no significant similarity to those of other potexviruses. Phylogenetic analyses based on the replicase (the methyltransferase, helicase, and polymerase domains) and coat protein demonstrated a closer relationship of CVX with Bamboo mosaic virus, Cassava common mosaic virus, Foxtail mosaic virus, Papaya mosaic virus, and Plantago asiatica mosaic virus.

Cactaceae↗

PubMedDate not supplied

[Nucleotide sequence and structural organization of the 3'-terminal region of potato virus M].

The sequence of 2630 3'-terminal nucleotides has been determined for the genomic RNA of potato virus M (PVM), a type member of the carlavirus group. Analysis of this nucleotide sequence revealed five open reading frames coding for proteins of mol. wt. 25, 12, 7, 34 and 11 kDa (in 5'----3' direction). The PVM genome organization has been shown to be basically analogous to that of potexviruses, except that the latter at their 3' end lack the gene encoding 11K protein. Amino acid sequence comparison between the PVM 34K protein and the coat proteins of potexviruses has shown a high extent of homology in the C-terminal amino acids. Homology has also been found between the 25, 12 and 7K proteins encoded by PVM RNA and corresponding potexvirus proteins. All this gave grounds for suggestion that carlaviruses and potexviruses belong to the same subgroup of phytoviruses.

Amino Acid Sequence↗

PubMed2003

Molecular evidence supporting the classification of Hosta virus X as a distinct species of the genus.

Apotexvirus, Hosta virus X (HVX-Kr), causing mosaic and mottle symptoms was isolated from hosta plants ( Hosta spp.) in Korea. The 3'-terminal 2,711 nucleotides excluding the poly (A) tail were determined and shown to include the partial viral replicase, triple gene block (TGB) 1 (26 kDa), TGB2 (13 kDa), TGB3 (8 kDa), and 23 kDa coat protein (CP) and the 3'-nontranslated region (NTR), typical of potexviruses. The CP gene of the type isolate of HVX (HVX-U) was amplified by RT-PCR and its nucleotide sequence was determined. The CPs of HVX-Kr and HVX-U had 100% and 98.9% identical amino acids and nucleotides, respectively. Most of the regions of the genome HVX had over 50% nucleotide identical to other sequenced potexviruses. This is the first report of sequence information of HVX and molecular evidence supporting the virus as a distinct species of the genus Potexvirus.

3' Untranslated Regions↗

PubMed1996

Characterization of cassava common mosaic virus and a defective RNA species.

The genome of cassava common mosaic potexvirus (CsCMV) has been sequenced from cDNA clones and consists of 6376 nucleotides (nt). A 76 nt untranslated region (UTR) at the 5' terminus was followed by ORF1 which potentially encodes a protein of 1449 amino acids (aa). ORFs 2, 3, and 4 were predicted to encode proteins of 231, 112 and 97 aa, respectively. ORF5 potentially encodes a 229 aa protein of 25 kDa that is similar to the coat proteins of other potexviruses. The 3'-terminal UTR of 114 nt was followed by a poly(A) tail. The genomic organization of the CsCMV genome is similar to that of other potexviruses. A cDNA clone that was apparently obtained from a defective RNA species contained both the 5' and 3' UTRs and an ORF that potentially encodes the first 263 aa of ORF1 and the last 33 aa of the coat protein. Defective RNA species were found both in purified preparations of the virus and in total nucleic acid isolated from CsCMV-infected plants.

Amino Acid Sequence↗

PubMed1993

Signal for potyvirus-dependent aphid transmission of potato aucuba mosaic virus and the effect of its transfer to potato virus X.

A British isolate of potato aucuba mosaic potexvirus (PAMV) was transmitted by aphids (Myzus persicae) which had fed previously on a source of potato Y potyvirus (PVY). Nucleotide sequence analysis of the PAMV coat protein gene indicated that amino acid residues 14 to 16 from the N terminus of the coat protein have the sequence DAG, which is also found in the coat proteins of potyviruses and is required for their aphid transmissibility. A recombinant virus isolate (TXPA7) was produced in which a segment of the coat protein gene of PAMV encoding the 40 N-terminal amino acids was inserted in the genome of potato X potexvirus (PVX) in place of the segment encoding the 28 N-terminal amino acids of PVX coat protein. This isolate, and a second similar recombinant (TXPA5) in which the DAG motif was changed to YTS, were mechanically transmissible to intact plants, in which they caused slightly milder symptoms than PVX. Particles of TXPA7 reacted in immunosorbent electron microscopy with PVX- and PAMV-specific antibodies and so were antigenically distinguishable from PAMV and PVX particles, which reacted only with their homologous antibody, and from TXPA5 particles, which reacted only with the PVX antibody. Recombinant TXPA7 was transmitted by aphids that had already fed on a source of PVY whereas TXPA5 and PVX were not. TXPA7 was not transmitted by aphids that had not fed on a PVY source. It is concluded that (i) the potyvirus-dependent aphid transmissibility of PAMV results from possession of a domain which includes the DAG motif and is located near the N terminus of the virus coat protein, and (ii) potyvirus-dependent aphid transmissibility can be conferred on PVX, a non-aphid-borne potexvirus, by substituting this domain for the N-terminal part of its coat protein.

Amino Acid Sequence↗

PubMed1993

Novel rod-shaped viruses isolated from garlic, Allium sativum, possessing a unique genome organization.

Rod-shaped flexuous viruses were partially purified from garlic plants (Allium sativum) showing typical mosaic symptoms. The genome was shown to be composed of RNA with a poly(A) tail of an estimated size of 10 kb as shown by denaturing agarose gel electrophoresis. We constructed cDNA libraries and screened four independent clones, which were designated GV-A, GV-B, GV-C and GV-D, using Northern and Southern blot hybridization. Nucleotide sequence determination of the cDNAs, two of which correspond to nearly one-third of the virus genomic RNA, shows that all of these viruses possess an identical genomic structure and that also at least four proteins are encoded in the viral cDNA, their M(r)s being estimated to be 15K, 27K, 40K and 11K. The 15K open reading frame (ORF) encodes the core-like sequence of a zinc finger protein preceded by a cluster of basic amino acid residues. The 27K ORF probably encodes the viral coat protein (CP), based on both the existence of some conserved sequences observed in many other rod-shaped or flexuous virus CPs and an overall amino acid sequence similarity to potexvirus and carlavirus CPs. The 11K ORF shows significant amino acid sequence similarities to the corresponding 12K proteins of the potexviruses and carlaviruses. On the other hand, the 40K ORF product does not resemble any other plant virus gene products reported so far. The genomic organization in the 3' region of the garlic viruses resembles, but clearly differs from, that of carlaviruses. Phylogenetic analysis based upon the amino acid sequence of the viral capsid protein also indicates that the garlic viruses have a unique and distinct domain different from those of the potexvirus and carlavirus groups. The results suggest that the garlic viruses described here belong to an unclassified and new virus group closely related to the carlaviruses.

Amino Acid Sequence↗

PubMed1998

Molecular characterization of the garlic virus X genome.

The complete nucleotide sequence of the cDNA genome for garlic virus X (GVX), one of the major viruses infecting garlic plants, was determined. GVX is a single-stranded positive-sense RNA virus consisting of 8106 nucleotides excluding the 3'-end poly(A) tail and contains six open reading frames (ORFs) which encode putative proteins of 174 kDa (ORF1), 26 kDa (ORF2), 12 kDa (ORF3), 32 kDa (ORF4), 26 kDa (ORF5) and 15 kDa (ORF6). The putative viral proteins show similarity to those of carlaviruses and potexviruses but show the highest homology to shallot virus X (ShVX). Even though the GVX genome contains most of the structural elements common to carlaviruses and potexviruses, it is distinguished from them by the presence of an ORF4 which encodes an unusual protein. These results suggest that GVX may belong to an unassigned group of ShVX and GarV-type viruses rather than to the carlaviruses or potexviruses.

Amino Acid Sequence↗

PubMedDate not supplied

[The construction of a model of the envelope proteins of potex-group viruses].

Amino acid sequences of coat protein of potato X-virus, aucuba potato mosaic, papaya mosaic and white clover mosaic have been comparatively studied. The results of this comparative study have become the basis for prediction of the secondary structure of the viruses and for construction of a model of potexvirus coat protein. The potexvirus coat protein was based on the three-layered arrangement of five alpha spirals. It is supposed that RNA molecule is within the protein subunit. Availability of common amino acid sequences in alpha spirals of coat proteins of TMV and potexviruses is established and this permits supposing their common origin.

Amino Acid Sequence↗

PubMed2002

Complete nucleotide sequence of the genomic RNA of a French isolate of Pepino mosaic virus (PepMV). Brief report.

The complete nucleotide sequence of the genomic RNA of a French isolate of Pepino mosaic virus (PepMV) was determined. With a length of 6,425 nucleotides excluding the poly(A) tail at the 3' terminus, the PepMV genome contains five major open reading frames encoding a 164 kDa replicase, triple gene block proteins of 26 kDa, 14 kDa and 9 kDa, and a 25 kDa coat protein. This genome organization, as well as characteristic consensus motifs and high degrees of similarity of PepMV proteins with those of other potexviruses confirmed that PepMV belongs to the Potexvirus genus. Phylogenetic analyses carried out on replicase, triple gene block protein 1 and coat protein amino acid sequences revealed that PepMV is closely related to species Narcissus mosaic virus (NMV), Scallion virus X (SVX), Cymbidium mosaic virus (CymMV), and Potato aucuba mosaic virus (PAMV).

5' Untranslated Regions↗

PubMed2004

Strain diversity and conserved genome elements in Strawberry mild yellow edge virus.

The complete nucleotide sequence of Strawberry mild yellow edge virus isolate D74, and the sequences of a 878 nt region of the coat protein and flanking regions of twenty three isolates of SMYEV were obtained and analysed. The full sequence of the aphid transmissible strain D74 was deduced and found to have an 86% sequence identity to the non-transmissible Agrobacterium infectious MY18 strain. In contrast to isolate MY18 the 5' terminal nucleotides (GAAAAC) of D74 are typical of those from other potexviruses. However, both MY18 and D74 have a non-AUG initiation codon for ORF2 encoding the triple gene block protein 1 (TGB1), and an overlapping TGB3 and coat protein (CP), features unique in the Potexvirus genus. Other conserved features of the genome, including stem-loop structures in the untranslated regions, and motifs common to related viruses are described. The previously postulated ORF6 of MY18 is absent in twenty of the isolates sequenced, including D74. Phylogenetic analysis places all isolates in one of three distinct groups/strains named here I (type-D74), II (type-9Redland), and III (type-MY18), with the majority of isolates, including all European isolates tested, belonging to strain I (type-D74).

Amino Acid Motifs↗

PubMed2005

Two unique US isolates of Pepino mosaic virus from a limited source of pooled tomato tissue are distinct from a third (European-like) US isolate.

Three strains of Pepino mosaic virus (PepMV) found in the US have been cloned and sequenced by RT-PCR using total RNA from infected tissue as template, and degenerate potexvirus- and PepMV species- and isolate-specific primers. Despite limited source material, the complete nucleotide sequences (6413 and 6410 nts, respectively) of two isolates, PepMV-US1 and PepMV-US2, were obtained and analyzed using total RNA from less than 0.2 g of a pooled infected tomato leaf sample from Arizona. Sequence of the 3'-end of the third isolate from infected fresh tomato fruits from Maryland (PepMV-US3) was also determined. The genome organizations of PepMV-US1 and US2 were typical of the genus Potexvirus, with the following reading frame order: ORF 1, encoding a putative replicase; ORFs 2-4, triple gene block proteins (TGBp) 1-3; and ORF 5, coat protein (CP). Gene-for-gene comparison between PepMV-US1 and US2 revealed the following amino acid identities: 91% in replicase, 89% in TGBp1, 92% in TGBp2, 85% in TGBp3, and 93% in the CP; with an overall nucleotide identity of 86%. Nucleotide sequence comparisons between US1 and US2 and the European isolates showed only 79-82% identity, whereas the identity among the European isolates was over 99%. Sequence comparisons and phylogenetic analysis indicate that PepMV-US1 and US2 are distinctly different from the European isolates, while the CP of PepMV-US3 is nearly identical to the European isolates. The results presented also suggest that TGBp1 and TGBp3 are more suitable than either the replicase or coat protein gene products for discriminating PepMV isolates.

Amino Acid Sequence↗

PubMed1998

The GPRIME package: computer programs for identifying the best regions of aligned genes to target in nucleic acid hybridisation-based diagnostic tests, and their use with plant viruses.

The GPRIME (Group PRIMEr design) programs examine aligned sets of gene sequences to discover homologous regions to be targeted in diagnostic tests. The core program moves a 'window' over the aligned sequences and calculates, at each window position, a 'redundancy value', namely the number of sequences that would represent all permutations of the variable sequence positions within that window. Regions with minimal redundancy values may then be targeted in diagnostic tests based on oligonucleotide hybridisation. The likely specificity of tests targeting such regions can be assessed by searching the international databases with those regions using FASTA. The GPRIME programs, which include programs for designing primers to distinguish between two sub-sets of a group of aligned sequences, can be obtained from http://life.anu.edu.au/software.html. We have used GPRIME to design redundant primers for RT-PCR tests to detect all potexviruses and tobamoviruses, and then used these, together with a previously reported pair of primers for the Potyviridae, to screen some Australian orchid collections. Two orchid viruses previously reported from Australia were found; cymbidium mosaic potexvirus was common, but odontoglossum ringspot tobamovirus was not. In addition the recently described ceratobium mosaic potyvirus was found to be common, and three other novel potyviruses were also found.

DNA Primers↗

PubMed1998

Phylogenetic analysis of triple gene block viruses based on the TGB 1 homolog gene indicates a convergent evolution.

The complete nucleotide sequence of the triple gene block one (TGB 1) of cymbidium mosaic potexvirus (CymMV) was compared to those from other potex-, carla-, furo- and hordeiviruses. Seven conserved motifs in the TGB 1, including the ATP-GTP binding domain (P-Loop) consensus GXXGKTSTS, were found in all four virus genera. We propose that all TGBV can be classified into phylogenetic clusters based on their TGB 1 homolog genes. These clusters can be further delineated to form subgroups. The first cluster comprises the potexviruses which are further subdivided into three subgroups; BaMV, FMV, PlaMV and PapMV (subgroup Ia); CymMV, PAMV, NMV, SMYEaV and WC1MV (subgroup Ib) and PVX (subgroup Ic). The second cluster comprises carlaviruses with a dual subgrouping; CVB, LSV, PVM, PMV and ASPV (subgroup IIa) and LVX (subgroup IIb). The third cluster carries the most diverse of TGBV comprising furoviruses PCV, PMTV and BSBV (subgroup IIIa) and hordeiviruses PSLV, BSMV and LRSV (subgroup IIIb). The phylogenetic relationships of triple gene block viruses (TGBV) based on the TGB 1 homolog gene indicates a convergent evolution.

Amino Acid Sequence↗

PubMed1994

Disruption of virus movement confers broad-spectrum resistance against systemic infection by plant viruses with a triple gene block.

White clover mosaic virus strain O (WClMV-O), species of the Potexvirus genus, contains a set of three partially overlapping genes (the triple gene block) that encodes nonvirion proteins of 26 kDa, 13 kDa, and 7 kDa. These proteins are necessary for cell-to-cell movement in plants but not for replication. The WClMV-O 13-kDa gene was mutated (to 13*) in a region of the gene that is conserved in all viruses known to possess triple-gene-block proteins. All 10 13* transgenic lines of Nicotiana benthamiana designed to express the mutated movement protein were shown to be resistant to systemic infection by WClMV-O at 1 microgram of WClMV virions per ml, whereas all plants from susceptible control lines became systemically infected. Of the 13* transgenic lines, 3 selected for their abundant seed supply were shown to be resistant to systemic infection when challenged by inoculation with three different WClMV strains (O, M, and J) or with WClMV-O RNA at 10 micrograms/ml. Most plants were also resistant to systemic infection at inoculum concentrations up to 250 micrograms of WClMV virions per ml. In addition, the three 13* transgenic plant lines were found to be resistant to systemic infection with two other members of the Potexvirus group, potato virus X and narcissus mosaic virus, and the Carlavirus potato virus S but not to be resistant to tobacco mosaic virus of the Tobamovirus group. These results indicate that virus resistance can be engineered into transgenic plants by expression of dominant negative mutant forms of triple-gene-block movement proteins.

Base Sequence↗