Analyses on mutation patterns, detection of population bottlenecks, and suggestion of deleterious-compensatory evolution among members of the genus Potyvirus.

Viruses of the family Potyviridae exhibited a robust single-nucleotide polymorphism profile at the between-species level, conforming to the neutral theory rule. However, the ratios of nonsynonymous to synonymous mutations (Ka/Ks) were relatively greater between-species than within-species in viral cistrons examined from members of the genus Potyvirus, indicating a relaxation on constraint. Judged by the McDonald and Kreitman's test, the fixation frequencies for nonsynonymous mutations across the genomes of closely related potyviruses were greater than expected, suggesting population bottlenecks at speciation. These mutation patterns are best explained by a deleterious-compensatory model.

The complete genome sequence, organization and affinities of carrot red leaf virus.

A sequence of 5723 nucleotides (GenBank accession number: AY695933) is reported for the RNA genome of an isolate of Carrot red leaf virus (CtRLV). The sequence is predicted to contain six large open reading frames and non coding sequences of 28 nucleotides at the 5' end, 110 nucleotides at the 3' end, and 215 nucleotides between the two main blocks of coding sequences. The 5' coding region encodes two polypeptides with calculated molecular masses (Mr) of 28.6 kDa (P0) and 68.2 kDa (P1) that overlap in different reading frames. Circumstantially, the third ORF in the 5' block is putatively translated by frameshift read-through to yield a polypeptide (P1 + P2) with a calculated Mr of 116.9 kDa. Frameshifting is predicted at a "shifty" sequence (GGGAAAC; nt 1523-1529) also found in most members of the genus Polerovirus. The C-terminal region of the 116.9 kDa polypeptide includes the consensus sequence for the viral RNA-directed RNA polymerase. The 3' block of coding sequence defines three putative polypeptides of: 23.0 kDa (P3), 21.3 kDa (P4, in a different reading frame) and 77.2 kDa (P3 + P5, by read-through of P3) respectively. From the genome structure of CtRLV, it is suggested that this virus belongs to the genus Polerovirus, rather than either the genus Luteovirus or the genus Enamovirus.

Construction and properties of a gene-silencing vector based on Poplar mosaic virus (genus Carlavirus).

A gene-silencing vector based on a full-length genomic clone of Poplar mosaic virus (PopMV) was constructed, with coat protein and movement protein genes removed, and containing instead, the coding sequence for green fluorescent protein (GFP). This paper demonstrates that the PopMV-derived gene-silencing vector was able to silence GFP expression in GFP transgenic Nicotiana benthamiana plants. The full-length genome of an Oxford isolate of PopMV (PV275) was cloned and sequenced. A full-length PopMV clone, under transcriptional control of the 35SCaMV promoter was then constructed, and the clone was able to replicate locally in Nicotiana species. Several autonomous plant RNA and DNA viruses have been converted into vectors and implemented for virus-induced gene-silencing (VIGS) of transgenes and endogenous genes [Burton, R., Gibeaut, D., Bacic, A., Findlay, K., Roberts, K., Hamilton, A., Baulcombe, D., Fincher, G., 2000. Virus-induced silencing of a plant cellulose synthase gene. Plant Cell 12, 691-706; Dalmay, T., Horsefield, R., Braunstein, T.H., Baulcombe, D.C., 2001. SDE3 encodes an RNA helicase required for post-transcriptional gene silencing in Arabidopsis. EMBO J. 20, 2069-2077; Gossele, V., Fache, I., Meulewaeter, F., Cornelissen, M., Metzlaff, M., 2002. SVISS--a novel transient gene silencing system for gene function discovery and validation in tobacco plants. Plant J. 32, 859-866; Holzberg, S., Brosio, P., Gross, C., Pogue, G.P., 2002. Barley stripe mosaic virus-induced gene silencing in a monocot plant. Plant J. 30, 315-327; Ratcliff, F., Martin-Hernandez, A., Baulcombe, D., 2000. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J. 25, 237-245; Ruiz, M., Vionnet, O., Baulcombe, D., 1998. Initiation and maintenance of virus-induced gene silencing. Plant Cell 10, 937-946]. The use of a virus that naturally infects trees as a gene-silencing vector has not been demonstrated before. The ability to systemically silence a plant transgene following the production of a gene-silencing signal from a locally replicating viral-construct derived from a carlavirus has not to our knowledge been shown before.

Complete sequences and phylogenetic analyses of a Singapore isolate of broad bean wilt fabavirus.

The complete nucleotide (nt) sequence of a Singapore isolate of broad bean wilt fabavirus from Megakepasma erythrochlamys L., designated BBWV-ME, was determined. Its bipartite genome consisted of two positive-sense single-stranded ribonucleic acids (RNA). RNA1 (5951 nt in length) encoded a putative protease cofactor, nucleotide triphosphate (NTP)-binding domain (helicase), viral genome-linked protein (VPg), protease and RNA-dependent RNA polymerase (RdRp). RNA2 (3607 nt in length) encoded a putative movement protein (MP) and coat proteins (CP). Genome organization of BBWV-ME was similar to other viruses in the Comoviridae family. Phylogenetic analyses showed that fabaviruses were more closely related to the comoviruses than the nepoviruses.

The prevalence and spatial distribution of viruses in natural populations of Brassica oleracea.

We report a survey of four viruses (beet western yellows luteovirus (BWYV), cauliflower mosaic caulimovirus (CaMV), turnip mosaic potyvirus (TuMV), turnip yellow mosaic tymovirus (TYMV)) in five natural populations of Brassica oleracea in Dorset (UK). All four viruses were common; 43% of plants were infected with BWYV, 60% with CaMV, 43% with TuMV and 18% with TYMV. For each virus there were significant differences in the proportion of infected plants among populations, which were not completely explained by differences in the age of plants. Multiple virus infections were prevalent, with 54% of plants having two or more virus types. There were statistically significant associations between pairs of viruses. The CaMV was positively associated with the other three viruses, and BWYV was also positively associated with TuMV. There was no detectable association between BWYV and TYMV, whereas TuMV and TYMV were negatively associated. We suggest these associations result from BWYV, CaMV and TuMV having aphid vectors in common, as aphids are attracted to plants that already have a virus infection. Infected plants were distributed randomly or were very weakly aggregated within populations. The implications of widespread multiple virus infections in natural plant populations are discussed with respect to the release of transgenic plants expressing virus-derived genes.

The genome organization and affinities of an Australian isolate of carrot mottle umbravirus.

The genomic sequence of an Australian isolate of carrot mottle umbravirus (CMoV-A) was determined from cDNA generated from dsRNA. This provides the first data on the genome organization and phylogeny of an umbravirus. The 4201-nucleotide genome contains four major open reading frames (ORFs). Analysis suggests that ORF2 encodes an RNA-dependent RNA polymerase, that ORF4 encodes a movement protein, and that the virus has no coat protein gene. The functions of ORFs 1 and 3 remain unknown. ORF2 is probably translated following ribosomal frameshifting. ORFs 3 and 4 are probably translated from a subgenomic mRNA. Sequence comparisons showed CMoV-A to be closely related to pea enation mosaic RNA2 (PEMV-RNA2), but also to have affinities with the Bromoviridae. These findings shed light on the relationships between the luteoviruses, PEMV, and the umbraviruses and on the relationships between the carmo-like viruses and the Bromoviridae.

A recombinational event in the history of luteoviruses probably induced by base-pairing between the genomes of two distinct viruses.

Alignments of luteovirus readthrough protein amino acid sequences show they consist of two distinct regions, here named the N domain and the C domain. N domain sequences were classified, and comparison of this gene phylogeny to phylogenies of other luteovirus genes revealed an anomaly in the relationships between beet western yellows luteovirus, cucurbit aphidborne yellows luteovirus (CABYV), and pea enation mosaic RNA1 (PEMV1). Together with alignments of virion protein and readthrough protein amino acid sequences, these gene phylogenies indicate the anomaly to be the result of two recombinational events, probably between ancestors of CABYV and PEMV1 and leading to the transfer of RNA coding for the N domain to an ancestor of CABYV. Two likely recombination sites were identified from the alignments, one at the 5' end of the readthrough protein gene and the other at the 5' end of the sequence coding for the C domain. Alignments of the nucleotide sequences encompassing the probable recombination sites suggest that base-pairing between the genomes of the two ancestral luteoviruses, resulting from local sequence similarity at the 5' end of the readthrough protein gene, probably induced one of the interspecies recombinational events.

Sequence analysis and location of capsid proteins within RNA 2 of strawberry latent ringspot virus.

The nucleotide sequence of the RNA 2 of a strawberry isolate (H) of strawberry latent ringspot virus (SLRSV) comprised 3824 nucleotides and contained one long open reading frame with a theoretical coding capacity of 890 amino acids equivalent to a protein of 98.8K. The N-terminal amino acid sequences of virion-derived proteins were determined by Edman degradation allowing the capsid coding regions to be located and serine/glycine cleavage sites to be identified within the polyprotein. The amino acid sequence in the capsid coding region of an isolate of SLRSV from flowering cherry in New Zealand was 97% identical to that of SLRSV-H. Except in the 3' and 5' terminal non-coding sequences, computer-based alignment and comparison algorithms did not reveal any substantial homologies between RNA 2 of SLRSV-H and the equivalent genomic segments in the nepoviruses arabis mosaic, cherry leaf roll, grapevine fanleaf, raspberry ringspot, grapevine hungarian chrome mosaic, tomato blackring, tomato ringspot, tobacco ringspot, or in the comoviruses cowpea mosaic and red clover mottle. Despite the similarities in overall genome organization, data from RNA 2 remain insufficient for unambiguous positioning of SLRSV in relation to species/genera in the Comoviridae.

The multiplication in plants of arabis mosaic virus satellite RNA requires the encoded protein.

Oligonucleotide-directed mutagenesis was used to create two mutations at each of three positions within the open reading frame (ORF) of a cDNA clone representing a satellite RNA from a lilac isolate of arabis mosaic nepovirus (ArMV). Three of the six mutants, in which stop codons were introduced at three different sites, did not direct synthesis of a translation product. The other three mutants, in which stop codons were not introduced, directed synthesis of a translation product (39K) although, in two of these, the mutation led to a single amino acid substitution. When Chenopodium quinoa plants were inoculated with in vitro transcripts from each of the six mutants together with the genomic RNA molecules (RNA-1 and RNA-2) of ArMV, progeny RNA was detected only with two of the three mutants in which the nucleotide changes did not introduce a stop codon to the coding region. To look for complementation, two deletion mutants were made. In these, 113 or 117 nucleotides were removed from two consecutive regions within the ORF. Two insertion mutants (in which the deleted sequences were replaced with a 130 nucleotide sequence from RNA-2 of cherry leaf roll nepovirus) were also made. Transcripts from none of these mutants retained messenger activity and none was detected either in C. quinoa plants or in virions, even in the presence of wild-type satellite RNA.

The nucleotide sequence of a satellite RNA associated with strawberry latent ringspot virus.

The nucleotide sequence of a satellite RNA associated with a strawberry isolate (H) of strawberry latent ringspot nepovirus (SLRSV) was determined from cDNA copies and the 5' end sequence was deduced from directly sequenced virion RNA. At the 3' end a poly(A) sequence was identified. A long open reading frame encoding a polypeptide of 331 amino acids (M(r) 36488) was determined. Sequence comparisons showed that SLRSV satellite RNA has no extensive homology with other sequences in the GenEmbl and Swiss-Prot databases.

The identification, cloning, and sequence analysis of the coat protein coding region of a birch isolate (I2) of cherry leaf roll nepovirus.

The coat protein gene of RNA-2 of cherry leaf roll nepovirus (CLRV) birch isolate I2 was cloned, identified, and sequenced. Transcripts derived from cDNA to the coat protein gene made a polypeptide of M(r) 51.5 k when translated in vitro. The predicted amino acid sequence of the coat protein showed little identity with nepoviruses having small RNA-2s. It did, however, have 27% sequence identity with the coat protein of tomato ringspot nepovirus which, like CLRV, has a relatively large RNA-2.

Partial nucleotide sequence of poplar mosaic virus RNA confirms its classification as a carlavirus.

The nucleotide sequence of the 3'-proximal 1328 nucleotides of poplar mosaic virus (PMV) was determined and shown to contain two large open reading frames (ORFs). The ORF nearer to the 3' terminus of the RNA is capable of encoding a polypeptide of 14K with a 'zinc-finger' motif, and is homologous to sequences in corresponding positions in five other carlaviruses. The other ORF encodes a protein of 36K which includes two sequences of amino acids identified in tryptic digests as virion capsid protein, and has amino acid sequences in common with both carlaviruses and potexviruses.

A 1.5 kb sequence homology in 3'-terminal regions of RNA-1 and RNA-2 of a birch isolate of cherry leaf roll nepovirus is also present, in part, in a rhubarb isolate.

A series of cDNA clones has been made from the birch isolate of cherry leaf roll nepovirus. Restriction enzyme analysis and sequencing showed that at the 3' end, RNA-1 and RNA-2 are identical for 1.5 kb. Also a 0.7 kb 3' end homology exists between the birch and rhubarb isolate. These sequences do not seem to code for any proteins; however, the sequence conservation points to a role in virus replication.

Biologically active transcripts of a large satellite RNA from arabis mosaic nepovirus and the importance of 5' end sequences for its replication.

Synthetic transcripts of a satellite RNA associated with a lilac isolate of arabis mosaic nepovirus (ArMV) were made from cDNA clones. Transcripts having either six (M1R) or 29 (M3R) extra nucleotides at their 5' ends replicated in the presence of ArMV genomic RNA in manually inoculated Chenopodium quinoa plants, even though M1R also differs from the native sequence at nucleotide position 2. Transcript 12R, which has 11 guanosyl residues and 27 other nucleotides not present in the natural satellite RNA at its 5' end, and also lacks the two 5'-terminal nucleotides (UA), replicated inefficiently, both in transformed tobacco plants and in plants that had been manually inoculated. Transcripts from another construct (M2R) lacking eight 5'-terminal bases of the native sequence did not multiply in plants. Each of these transcripts directed the in vitro synthesis of a protein (Mr 39K) encoded by satellite RNA, although 12R was the least efficient message. Analysis of the 5'-terminal sequences in progeny RNA from M1R showed that the non-native bases were removed and the second nucleotide corrected, suggesting that VPg plus a few initial 5'-terminal bases might serve as a primer for plus-strand synthesis of this satellite RNA. When M1R was inoculated with genomic RNAs from ArMV of ash or ivy, the transcripts replicated and were encapsidated. However, when the same amounts of M1R were inoculated with genomic RNAs of ArMV from hop or sugar-beet, progeny of the transcripts were not detected either in virions or in plants. Less surprisingly, this RNA transcript did not multiply in the presence of dogwood mosaic, strawberry latent ringspot, grapevine fanleaf or cherry leaf roll nepoviruses.

Transgenic plants and insect cells expressing the coat protein of arabis mosaic virus produce empty virus-like particles.

The 3' end of the RNA-2 of arabis mosaic virus (ArMV) was cloned and sequenced. The N-terminal amino acid sequence of the virion coat protein was determined by Edman degradation and the corresponding coding region identified. This gene was modified at the 5' and 3' ends by use of mismatched primers in the polymerase chain reaction (PCR), in order to facilitate the cloning of the gene, and to provide it with a methionine initiation codon. The modified cloned gene was expressed in transgenic plants, recombinant baculovirus-infected insect cells and bacteria. Both the insect cells and the plants expressing the modified coat protein gene contained empty virus-like particles (VLPs) similar to the empty virus shells found in plants infected with ArMV. These VLPs were not detected in the Escherichia coli expressing the coat protein. Analysis of the primary amino acid sequence in the ArMV coat protein revealed extensive regions of identity with that of grapevine fanleaf virus. Patterns in these identities may reflect a three-domain organization of the proteins.

Synthesis and proteolytic processing of arabis mosaic nepovirus, cherry leaf roll nepovirus, and strawberry latent ringspot nepovirus proteins in reticulocyte lysate.

The genomic RNA components of three nepoviruses, arabis mosaic (ArMV), cherry leaf roll (CLRV), and strawberry latent ringspot (SLRV), were translated in rabbit reticulocyte lysate. Each component (except the RNA-2 of CLRV) directed the synthesis of proteins that corresponded in size to their theoretical coding capacity. The RNA-1 components of all three viruses were translated to yield polyproteins of Mr 250k, which were autocatalytically processed to yield up to five cleavage products. The primary products of translation of the RNA-2 components of ArMV (Mr 115k and 105k), CLRV (Mr 165k) and SLRV (Mr 99k and 96k) were polyproteins that were stable on incubation, but which underwent proteolytic processing in the presence of the corresponding RNA-1 and its translation products. These polyproteins were immunoprecipitated using antisera to appropriate virions indicating that the RNA-2 sequences encode the coat protein cistrons.

The nucleotide sequence of a satellite RNA associated with arabis mosaic nepovirus.

The nucleotide sequence of a satellite RNA (satRNA) associated with a lilac isolate of arabis mosaic virus (ArMV) was determined from cDNA copies. The sequence was 1104 nucleotides in length excluding the poly(A) tail, contained a long open reading frame which encodes a polypeptide of 360 amino acids, with an Mr of 39K. Nucleotide sequence comparisons revealed that the ArMV-associated satRNA shared 83% nucleotide identity with a satRNA from grapevine fanleaf nepovirus, but no extensive sequence homology was observed with other nepoviral satRNAs.

Plant virus detection using a new form of indirect ELISA.

A novel form of indirect enzyme-linked immunosorbent assay (ELISA) has been devised for the detection of viruses in plants. The method uses protein A in two applications to sandwich antibody-antigen-antibody layers. The first applied layer of protein A prepares the plate for the coating antibody layer. The second layer of protein A is conjugated to the enzyme and detects the second antibody layer. The orientation of the IgG induced in the coating layer of antibody prevents later unwanted reaction with the conjugated protein A. Using seven antisera, protein A sandwich ELISA (PAS-ELISA) detected homologous virus isolates in standard dilutions of infected plant homogenates at A405 values which were at least one absorbance unit greater than those of healthy controls. The PAS-ELISA method was more sensitive than the direct double antibody sandwich form of ELISA (DAS-ELISA), e.g. not only were A405 values for homologous reactions greater in PAS-ELISA but also an antiserum to a birch isolate of cherry leaf roll virus detected four related isolates with the new method against only one with DAS-ELISA. However, dilution end points for the homologous virus were about the same in both methods. In a practical application, PAS-ELISA detected prune dwarf virus in 18-36% of tested Prunus avium seeds.