The complete sequence of a UK strain of black raspberry necrosis virus.

The complete nucleotide sequence of a UK strain of the sadwavirus Black raspberry necrosis virus (BRNV) was obtained by amplification and sequencing of virus RNA from infected plants grown in a raspberry plantation in Aylth, Scotland. The RNA1 was 7,572 nucleotides (nt) in size and RNA2 was 6,350 nt in size, each excluding the 3' poly-A tail. The RNA1- and RNA2-encoded polyproteins are predicted to be processed into (RNA1) a protease cofactor, an RNA helicase, the VpG, a 3C-like protease, an RNA-dependent RNA polymerase and an AlkB protein, and (RNA2) a movement protein and two capsid proteins.

Rubus chlorotic mottle virus, a new sobemovirus infecting raspberry and bramble.

The complete nucleotide sequence of a new member of the unassigned genus Sobemovirus, isolated from raspberry and bramble plants in north east Scotland and given the name Rubus chlorotic mottle virus (RuCMV), was obtained. The virus has a single, positive-strand RNA genome of 3,983 nucleotides and, in common with other sobemoviruses, contains four open reading frames (ORFs) encoding, from 5' to 3', the P1 protein that is likely to be a suppressor of RNA silencing, ORF2a that has homology to serine-proteases, ORF2b that is the probable RNA dependent RNA polymerase, and ORF3 that is the coat protein. ORF2b protein is potentially expressed as a fusion with ORF2a protein by a -1 frameshift at the heptanucleotide sequence UUUAAAC. Phylogenetic analyses showed that RuCMV is a distinct virus not closely related to any of the other sequenced sobemoviruses. Based on the obtained sequence a full-length cDNA copy of RuCMV was cloned and in vitro transcripts derived from this clone were shown to be fully infectious.

Yeast two-hybrid study of tobacco rattle virus coat protein and 2b protein interactions.

The specificity of the interaction between the coat protein (CP) and 2b nematode-transmission helper protein of two isolates, PpK20 and PaY4, of Tobacco rattle virus (TRV) that differ in their transmission characteristics was investigated. Yeast two-hybrid experiments identified a central domain of the CP that promoted CP:CP interaction but inhibited CP:2b interaction. Deletion of a conserved coiled-coil domain from the 2b protein prevented its interaction with CP, whereas deletion of N- and C-terminal domains of the 2b protein greatly enhanced its interaction with CP. A C-terminal flexible domain of the CP was also shown to be important for interaction with the 2b protein. However, this domain was not sufficient to direct isolate-specific interaction of these proteins either in yeast or via a chimeric TRV in planta. Although these two TRV isolates are both transmitted by a shared vector trichodorid nematode, Paratrichodorus pachydermus, the CP from isolate PpK20 did not interact with the 2b protein from isolate PaY4, and vice versa, suggesting that in the field mixed infections of TRV are unlikely to cause promiscuous transmission by alternative trichodorid nematode species.

'Worms' that transmit viruses.

Plant-parasitic nematodes transmit viruses that hit hard on global agriculture. Both the nematodes and the viruses they carry persist in the soil for a long time with the only remedy being highly toxic chemicals. Fundamental new research may offer the hope of developing safer control strategies.

Tobravirus 2b protein acts in trans to facilitate transmission by nematodes.

Analysis of RNA2 of TRV PaY4 showed it to be recombinant, carrying 3'-terminal sequences derived from RNA1. Virus produced using an infectious cDNA clone of PaY4 RNA2 was nematode transmissible, demonstrating that natural TRV recombinant isolates are not necessarily defective. Mutations introduced into PaY4 RNA2 showed that the 2b gene, but not the 2c gene, is required for transmission by both Paratrichodorus pachydermus and P. anemones nematodes. Experiments examined whether infection of plants with two different virus clones would impact upon nematode transmission of either virus. Simultaneous inoculation with TRV clones expressing green or red fluorescent proteins revealed that mixing of the two virus populations did not occur, although, in roots, adjacent cells were found containing green- or red-tagged viruses. Subsequently, in similar experiments it was found that a TRV PaY4 2b mutant was transmitted when combined with wild-type TRV PaY4. Also, transmission of a 2b mutant of an in vitro TRV/PEBV recombinant virus (TRV-C1) occurred after coinfection with wild-type virus. Thus, the tobravirus 2b transmission protein is trans-acting. Although TRV PaY4 and TRV PpK20 are both transmitted by P. pachydermus, a 2b mutant of TRV PaY4 was not transmitted when coinoculated to plants with TRV PpK20.

Efficient expression of foreign proteins in roots from tobravirus vectors.

Viral vectors were constructed from infectious cDNA clones of each of the three tobraviruses, tobacco rattle virus (TRV), pea early-browning virus (PEBV), and pepper ringspot virus (PepRSV). RNA2 of each of the three viruses was modified to carry an additional coat protein subgenomic promoter and was used to express green fluorescent protein (GFP) when inoculated to plants. The tobravirus expression vectors have a wide host range and were able to express GFP in, for example, Nicotiana species, tomato, pea, arabidopsis, and sugar beet. The TRV vector was able to invade and express GFP very efficiently in roots, whereas the widely used PVX vector was not.

Immunogold Localization of Tobacco Rattle Virus Particles within Paratrichodorus anemones.

Unequivocal evidence of the viral nature of virus-like particles observed at the specific site of retention of tobacco rattle virus (TRV) in Paratrichodorus and Trichodorus nematodes has not previously been available. A new staining technique using safranin-O, which does not affect viral antigenicity, was used with an antiserum raised against the coat protein of TRV and prepared for use with immunogold labelling. Application of this method enabled the occurrence and localization of particles of TRV to be confirmed in the pharynx of the natural vector of the virus, Paratrichodorus anemones, and provided unequivocal evidence that the particles observed were TRV particles. The TRV particles were observed attached only to the cuticle lining the posterior tract of the pharyngeal lumen of the vector. Therefore, the specific site of retention of TRV particles in P. anemones is apparently more localized than reported to occur in other vector trichodorid species.

The E116 isolate of Dutch pea early-browning virus is a recombinant virus.

The complete nucleotide sequence of RNA2 of the E116 isolate of Dutch pea early-browning virus (PEBV-D) was obtained from overlapping cDNA clones. The RNA was found to encode three open reading frames corresponding to, in 5' to 3' order, the coat protein, the 2b nematode transmission protein and the C-terminal part of the cysteine-rich 1b protein derived from RNA1. The 3' non-coding region of PEBV-D RNA2 was also shown to be derived from RNA1. This is the first demonstration that recombination of PEBV occurs in nature. Comparison of the amino acid sequences of the PEBV-D RNA2 proteins with those of British PEBV and several isolates of tobacco rattle virus reveals complex patterns of mixing of the genomes of these two viruses.

Similarities in the genome organization of tobacco rattle virus and pea early-browning virus isolates that are transmitted by the same vector nematode.

Although sequence data have been obtained for several tobravirus isolates, only two of these isolates are nematode-transmissible. Tobacco rattle virus (TRV) PpK20 is transmitted by Paratrichodorus pachydermus, whereas pea early-browning virus (PEBV) TpA56 is transmitted by Trichodorus primitivus. To clarify whether differences in the genome structure of these isolates are relevant to the specificity of interactions with particular vector nematodes, or merely reflect a taxonomic difference between TRV and PEBV, we have sequenced RNA2 of a new isolate of TRV (TpO1) that is transmitted by the same vector nematode as PEBV TpA56 but is not transmitted by the nematode vector of TRV PpK20. TRV TpO1 RNA2 encodes, in 5' to 3' order, a coat protein (CP), a 9K protein, a 2b (29K) protein and a 2c (18K) protein. Amino acid sequence comparison shows that both the CP and 2b proteins of TRV TpO1 resemble more closely the analogous proteins from PEBV TpA56 than those from TRV PpK20. Also, the TRV TpO1 9K protein has similarities with the PEBV 9K protein whereas this protein is lacking in TRV PpK20.

Replication of in vitro tobravirus recombinants shows that the specificity of template recognition is determined by 5' non-coding but not 3' non-coding sequences.

Natural recombinant tobacco rattle tobravirus (TRV) isolates contain sequences from a different tobravirus, pea early browning virus (PEBV). To characterize the sequence requirements for viable recombinant formation hybrid cDNA clones of RNA2 of PEBV and TRV were assembled. Inclusion of 320 nt from the 5' terminus of PEBV or 335 nt from the 5' terminus of TRV in the hybrid RNAs was sufficient to permit their replication by, respectively, PEBV RNA1 or TRV RNA1 regardless of the origin of the 3' terminal region. However, PEBV RNA1 but not TRV RNA1 was sometimes able to support low level replication of RNA2 containing the heterologous 5' terminal region. In vitro translation of PEBV transcripts containing 5' noncoding region deletions supported the hypothesis that in vivo the PEBV coat protein (CP) is expressed from a subgenomic RNA and that, therefore, in the recombinants the CP subgenomic promoter probably is recognized by the replicase of the heterologous virus.

Viral determinants of pea early browning virus seed transmission in pea.

Pea early browning virus (PEBV) is a member of the genus, Tobravirus. It is transmitted by soil-inhabiting trichodorid nematodes and through seeds from diseased plants. By introducing mutations into the PEBV genome, we have studied the viral determinants of seed transmission in pea. Neither deleting a portion of the genome containing the three nonstructural genes in RNA2 nor the interuption of any of the three genes individually prevented PEBV seed transmission. However, a comparison of two PEBV isolates indicated a minor role for RNA2 or its products. In contrast, the removal of the coding sequence of the 12K gene in RNA1 almost completely abolished viral seed transmission. The virus lacking the 12K gene caused more severe symptoms on leaves and pods, and accumulated to a higher level than the wild-type virus in both types of tissues. However, the 12K deletion mutant accumulated poorly in anthers and carpels, and could not be detected in pollen grains and ovules. These results suggest that the 12K gene is involved in the infection of the gametic cells and hence the seed transmission of PEBV in pea.

Monoclonal antibodies detect a single amino Acid difference between the coat proteins of soilborne wheat mosaic virus isolates: implications for virus structure.

ABSTRACT Four monoclonal antibodies (MAbs) were prepared against an isolate of soilborne wheat mosaic furovirus from Oklahoma (SBWMV Okl-7). Three MAbs had different reactivities in tests on SBWMV isolates from Nebraska (Lab1), France, and Japan. One MAb (SCR 133) also reacted with oat golden stripe furovirus. None of the MAbs cross-reacted with other rod-shaped viruses including beet necrotic yellow vein furovirus, potato mop-top furovirus, and tobacco rattle tobravirus. Sequence analysis of nucleotides between 334 and 1,000 of RNA 2, the region that encodes the coat protein (CP) and the first 44 amino acids of a readthrough protein, of the four SBWMV isolates revealed up to 27 base changes from the published sequence of a Nebraska field isolate of SBWMV. Most changes were translationally silent, but some caused differences of one to three amino acids in residues located near either the N- or C-terminus of the CPs of the different isolates. Two further single amino acid changes were found at the beginning of the readthrough domain of the CP-readthrough protein. Some of these amino acid changes could be discriminated by MAbs SCR 132, SCR 133, and SCR 134. Peptide scanning (Pepscan) analysis indicated that the epitope recognized by SCR 134 is located near the N-terminus of the CP. SCR 132 was deduced to react with a discontinuous CP epitope near the C-terminus, and SCR 133 reacted with a surface-located continuous epitope also near the C-terminus. Predictions of CP structure from computer-assisted three-dimensional model building, by comparison with the X-ray fiber diffraction structure of tobacco mosaic virus, suggested that the three CP amino acids found to differ between isolates of SBWMV were located near the viral surface and were in regions predicted to be antigenic.

Single-strand conformation polymorphism analysis of RT-PCR products of UK isolates of barley yellow mosaic virus.

Single-strand conformation polymorphism (SSCP) analysis of the bipartite genomes of several UK isolates of barley yellow mosaic virus (Ba YMV) was done using fragments of cDNA amplified by RT-PCR. Isolates differed in their SSCP patterns in several regions, but in no case was the pattern able to distinguish between common and resistance-breaking strains. In regions where the nucleotide sequences of UK isolates had been determined, there was no simple relationship between numbers of nucleotide differences and SSCP patterns: differences of only 2 or 3 nucleotides (nt) gave different SSCP patterns, whereas differences of as many as 29 nt did not. Although SSCP analysis has some potential as a rapid and sensitive tool for distinguishing virus isolates, differences detected do not necessarily relate to biological properties and the results are highly dependent on gel conditions.

Multiple virus genes involved in the nematode transmission of pea early browning virus.

Mutations were introduced into four genes encoded by RNA2 of pea early browning virus (PEBV) to determine their possible involvement in the transmission of this virus by nematodes. Deletion of 28 amino acids from the C-terminus of the coat protein abolished the formation of virus particles. Deletion of 15 amino acids at the C-terminus did not affect particle formation but did abolish nematode transmission. In contrast, deletion of 13 amino acids immediately preceding the 16 C-terminal residues did not affect particle formation and decreased rather than abolished nematode transmission. A deletion in the gene encoding a 29-kDa protein and a frameshift mutation in the gene encoding a 23-kDa protein both abolished transmission without affecting virus particle formation. Mutations in an ORF encoding a 9-kDa protein, which is located on the genome between the coat protein gene and 29K gene, gave conflicting results. Removal of the AUG initiation codon from the 9K ORF had no effect on transmission, whereas the introduction of a frameshift mutation, which would prematurely terminate expression of the putative 9-kDa protein, decreased the frequency of transmission. The results show that the coat protein and probably all three of the other RNA2-encoded proteins play a role in the transmission of PEBV by vector nematodes.

Sequence analysis of wheat and oat furovirus capsid protein genes suggests that oat golden stripe virus is a strain of soil-borne wheat mosaic virus.

In northern blots, cDNA probes prepared to soil-borne wheat mosaic virus (SBWMV) RNA-1 and RNA-2 hybridized to RNA-1 and RNA-2, respectively, from a UK isolate of oat golden stripe virus (OGSV), as well as to their homologous RNAs. RT-PCR was used to amplify, clone and sequence a region of about 750 nucleotides spanning the capsid protein gene and part of the readthrough protein on RNA-2 from OGSV, a French isolate of SBWMV and two stable deletion mutants (Lab1 and Okl-7) of SBWMV isolates from Nebraska and Oklahoma respectively. There was very high (96.7-99.1%) nucleotide homology between all these sequences and the wild-type SBWMV sequences from Nebraska and Oklahoma. OGSV was more similar to SBWMV from France and Nebraska than were any of the isolates to SBWMV from Oklahoma. Of the few differences in the deduced amino acid sequences of the capsid proteins from the different isolates, OGSV differed from all SBWMV isolates only in one amino acid (isoleucine for valine at position 88). The high degree of similarity suggests that OGSV may best be classified as an oat strain of SBWMV.

Rapid cloning of uncharacterised tobacco rattle virus isolates using long template (LT) PCR.

A rapid method was developed using long template (LT)-PCR to amplify the complete RNA2 of isolates of TRV for which no sequence data are available. The amplification makes use of a 5' terminal oligonucleotide which contains degeneracies corresponding to the sequences of several different TRV isolates, and a 3' oligonucleotide which is complementary to a sequence present in all known isolates. This method was used to show the high degree of sequence homology existing in the terminal regions of two uncharacterised TRV isolates (TPO3 and PAY4), and revealed the deletion of an 80-nucleotide sequence in the 5' terminal region of TPO3 RNA2.

Molecular characterisation of UK isolates of barley yellow mosaic bymovirus.

Several isolates of barley yellow mosaic virus (BaYMV) from different sites in the UK, including some that were virulent on European resistant winter barley cultivars (resistance-breaking strain: BaYMV-2) and some that were not, were examined by RT-PCR, restriction mapping and sequencing of selected parts of the virus genome. Nucleotide and predicted amino acid sequences were determined for the 5'-terminal region, part of the NIa coding region and the coat protein coding region on RNA 1 and an area at the N-terminus of the 70-kDa protein coding region on RNA 2. The sequences differed from those previously reported for a BaYMV isolate from Japan and for two German isolates, one of which was of the BaYMV-2 strain. There were no strain-specific amino acid differences and the few, non-consecutive, nucleotide differences detected were probably not significant and were insufficient to develop a rapid diagnostic test to distinguish BaYMV-2 from other isolates. Restriction mapping of RNA 2 cDNA again showed no consistent strain-related differences. The differences previously reported between the two German isolates are probably not strain-related.

Sequence comparison of RNA2 of nematode-transmissible and nematode-non-transmissible isolates of pea early-browning virus suggests that the gene encoding the 29 kDa protein may be involved in nematode transmission.

A cDNA clone containing the complete coding region of RNA2 of the newly described TPA56 isolate of pea early-browning virus (English serotype, PEBV-E) has been obtained. A plasmid (pFLA56) containing this clone together with the 5' and 3' non-coding regions of PEBV isolate SP5 (the type isolate of PEBV-E) and flanked by the CaMV 35S promoter and NOS terminator is infectious when co-inoculated onto plants with pCaN1, an infectious clone of PEBV SP5 RNA1. Virus containing RNA2 derived from the cDNA clone of the TPA56 isolate is transmitted by Trichodorus primitivus nematodes, whereas virus containing RNA2 from a clone of the SP5 isolate is not transmitted. Sequencing revealed 11 differences out of 3374 nucleotides between the transmissible TPA56 and non-transmissible SP5 clones. However, only three of the base changes affected the amino acid sequences of virus gene products. A single, conservative amino acid change was present in the virus coat protein. Two non-conservative changes occurred in the protein of molecular mass 29.6 kDa expressed from an open reading frame located down-stream of the coat protein gene, suggesting that it has a function in nematode transmission and that changes in this protein prevent vector transmission of the SP5 isolate of PEBV.

An analysis of spontaneous deletion sites in soil-borne wheat mosaic virus RNA2.

Plants mechanically inoculated with soil-borne wheat mosaic virus (SBWMV, Oklahoma isolate) generated several deleted forms of RNA2, the smaller genomic RNA (3593 nt). Four naturally deleted forms were cloned by RT-PCR methods and the sequences around each deletion site were compared. SBWMV RNA2 molecules were deleted for 519, 759, 964, or 1030 nt, respectively, each within the coat protein-readthrough domain. No common sequences were found flanking the 5'- and 3'-sites of deletion; however, the 5'-site of each deletion lay in the region between genome coordinates 1417-1465. The delta 759nt clone isolated from virus-infected plants after a single mechanical passage was identical to a clone described previously (J. Chen, S. A. MacFarlane, and T. M. A. Wilson, 1994, Virology 202, 921-929) which became the dominant, stable form of SBWMV RNA2 after five or more serial mechanical transfers. In this study, a total of 33 independent, RT-PCR clones with an apparent deletion of 759 nt, isolated after one to seven mechanical passages, were also sequenced and their precise deletion sites were compared. Twenty-six clones contained the original "stable," 759-nt deletion, whereas 7 clones, which were found only during the first four passages, had deletions at one of four alternative sites. After the fifth mechanical passage the stable 759-nt deletion dominated. The results also show that, in general, the smaller deletions are not intermediates in the larger deletion process. The complete nucleotide sequence of SBWMV RNA2 (Oklahoma) is also reported.

Detection and sequence analysis of a spontaneous deletion mutant of soil-borne wheat mosaic virus RNA2 associated with increased symptom severity.

Repeated passage of soil-borne wheat mosaic virus (SBWMV; wild-type Oklahoma field isolate; designated Okl-0) by manual inoculation resulted in deletion of part of SBWMV RNA2. Deletion was apparent in the population of RNA2 molecules after only 11 weeks in primary inoculated wheat plants (called Okl-1) and after 5 passages (Okl-5; 20 weeks) no full-length RNA2 remained. The extent of deletion in the Oklahoma isolate was compared with that in the previously studied Lab 1 (Nebraska) isolate. RNA2 from Okl-1, Okl-7, and Lab 1 were analyzed by RT-PCR amplification using sets of primer pairs which spanned all portions of the intact molecule. Lab 1 and the new stable isolate, Okl-7, were found to be deleted for 1058 and 759 nt, respectively, within the region encoding the coat protein-readthrough domain. However, the Okl-7 deletion was in a different position from the Lab 1 deletion. This suggests that deletions of SBWMV RNA2 which occur during serial manual inoculation are not directed toward production of a conserved, truncated form of the 84-kDa extended coat protein, but might reflect an RNA sequence-dependent event.