Bean yellow mosaic virus subgroup; search for the group specific sequences in the 3' terminal region of the genome.

In order to examine relationships among viruses of the bean yellow mosaic subgroup of the Potyvirus genus, several isolates of bean yellow mosaic virus (BYMV) and clover yellow vein virus (C1YVV) were compared by amino acid sequence of the coat protein and nucleotide sequence of the 3' terminal non-coding region. The sequence comparisons showed that BYMV and C1YVV were distinct viruses but had close affinity to each other (85-95% homology among isolates of a virus but 70-77% homology between viruses), justifying establishment of the BYMV subgroup. There was an oligonucleotide consensus sequence present in the 3' terminal non-coding region of all potyviruses examined. This consensus sequence divided the potyviruses into three groups whose significance is not clear.

A simple, rapid method of nucleic acid extraction without tissue homogenization for detecting viroids by hybridization and RT-PCR.

A simple, rapid method of nucleic acid extraction on a microcentrifuge tube scale for detecting viroids is presented. Five distinct citrus viroids (CVds), chrysanthemum stunt viroid (CSVd), hop stunt viroid (HSVd), hop latent viroid (HLVd) and potato spindle tuber viroid (PSTVd) were detected in their natural host plants by hybridization using cRNA probes and reverse transcription-polymerase chain reaction (RT-PCR). Nucleic acids (NA) were liberated from tissues by incubation in a buffer containing potassium ethyl xanthogenate (PEX) without tissue homogenization, and then precipitated with ethanol (NA-PEX). All the viroids except CVd-IV could be detected clearly in NA-PEX by hybridization. HSVd, HLVd and PSTVd could also be detected in NA-PEX by RT-PCR. Although CVds and CSVd could not be detected in NA-PEX by RT-PCR, they were detected after further purification: differential precipitation with 2-butoxyethanol and HCl treatment followed by ethanol-precipitation. In addition, PCR in the presence of tetramethylammonium chloride specifically amplified the cDNA of all five distinct CVds under the same temperature and cycle conditions. Since all the viroids could be detected in NA liberated by PEX, the amount of NA extracted by the method described here is sufficient for detecting viroids, enabling the processing of a large number of samples.

Point mutations in helper component protease of clover yellow vein virus are associated with the attenuation of RNA-silencing suppression activity and symptom expression in broad bean.

Helper component protease (HC-Pro) is a potyvirus-encoded multifunctional protein and a major determinant of symptom expression in a susceptible plant. Here, we show the involvement of clover yellow vein virus (ClYVV) HC-Pro in necrotic symptom expression in broad bean (Vicia faba cv. Wase). In this host, lethal necrosis was induced by ClYVV no. 30, from which a spontaneous, mosaic-inducing mutant (MM) was obtained. Mapping with chimeric viruses between ClYVV no. 30 and MM attributed the symptom attenuation to two mutations at the HC-Pro positions 27 (threonine to isoleucine) and 193 (aspartic acid to tyrosine). Although neither mutant with the single amino acid substitution at position 27 or 193 (ClYVV/T27I or D193Y) induced the lethal necrosis, ClYVV/T27I still retained the ability to induce necrotic symptoms, but ClYVV/D193Y scarcely did so. The virus accumulation of ClYVV/D193Y was also lower than that of ClYVV no. 30. The mutations, T27I and D193Y, are located in a putative zinc finger domain and in one (N-terminal) of the two RNA binding domains, respectively, of HC-Pro. RNA-silencing suppression (RSS) activity of P1/HC-Pro in Nicotiana benthamiana was weakened by both mutations. Our results suggest a correlation between viral virulence and RSS function and the importance of the two domains in HC-Pro.

3C-like protease encoded by Rice tungro spherical virus is autocatalytically processed.

The 3C-protease of Rice tungro spherical virus (RTSV) was previously identified as a cis- and trans-acting protease. In vitro translation of the protease resulted in several protein products, demonstrating that the protease is cleaved by itself. The protease was then produced in Escherichia coli as a fusion protein with maltose-binding protein (MBP). Two forms of the protease were purified after MBP affinity chromatography in the column buffer. After analyses of the purified proteins, we speculated that a major internal cleavage site was in the C-terminal half. A point mutation was introduced at a potential major self-cleavage site (C(2763)). The mutation abolished the catalytic activity, suggesting that the mutation site is important for the recognition of the protease.

Restoration of the 3' end of potyvirus RNA derived from Poly(A)-deficient infectious cDNA clones.

Poly(A)-deficient full-length cDNA clones of clover yellow vein virus (ClYVV), a member of the genus Potyvirus, were found to be infectious when expressed from the CaMV 35S promoter. The poly(A) tail was replaced with different short sequences and the infectivities of the cDNA constructs were examined. Although the infectivity of the plasmid varied depending on the sequences introduced, all the constructs were infectious. In all cases, progeny viral RNAs from the cDNA clones had an authentic viral sequence at their 3' regions with poly(A) tails and the downstream nonviral sequences were completely lost. However, two minor mutations, a two-nucleotide deletion at the 3' end and a single-nucleotide addition at the second nucleotide position downstream of the poly(A) site, were also observed. The clones of the viral (-) strand RNAs had poly(U) tracts at their 5' ends, suggesting that their synthesis is primed by the poly(U) sequence. It furthermore suggests that the mutations were introduced during or after primary transcription from the cDNA and were maintained during authentic viral replication. Although the mechanism involved is not known, recovery of the poly(A) tail is an essential step for maintaining the infectivity of the viral cDNAs.

Hypothesis on particle structure and assembly of rice dwarf phytoreovirus: interactions among multiple structural proteins.

To study the morphogenesis and packaging of rice dwarf phytoreovirus (RDV), the interactions among multiple structural proteins were analysed using both the yeast two-hybrid system and far-Western blotting analysis. The following protein-protein interactions were observed. P3 (major core protein) bound to itself as well as to P7 (nucleic acid-binding protein) and P8 (major outer capsid protein). P7 bound to P1 (RNA-dependent RNA polymerase) and P8, in addition to P3. Based on these findings, we hypothesize that the core shell structure is based on P3-P3 interactions and that P7 has the ability to bind to multiple structural proteins as well as to genomic RNAs during viral particle assembly. Based on the observed protein-protein interactions and on computer-aided analysis of the numbers of structural proteins per particle, possible RDV assembly events are proposed.

The 3' terminal region is strictly required for clover yellow vein virus genome replication.

To identify the cis-element in the 3' terminal region of infectious cDNA required for replication of clover yellow vein virus (ClYVV), a series of mutants with duplications or deletions of the 3' terminal non-coding region (3'-NCR) of the genome that did not affect the ORFs in the genome was constructed. These were tested for infectivity, and the 3' terminal regions of their progeny RNAs were sequenced. Deletion mutants that lacked portions of the 3'-NCR were not infectious. Various mutants with duplicated 3' terminal sequences were infective only when the authentic 3' terminal sequence was restored, probably by recombination, and none of the constructs retained the original sequence in progeny viral RNA. When a coat protein gene sequence of bean yellow mosaic virus (BYMV) followed by a termination codon was introduced between the nuclear inclusion b and coat protein genes, infective progeny were generated. Sequence analyses of the progeny viruses showed that the coat protein gene was a chimera of the BYMV N-terminal and CIYVV C-terminal portions. These results suggest that the 3'-NCR of ClYVV contains cis-acting elements and is strictly required for genome replication.

Conserved terminal nucleotide sequences in the genome of rice black streaked dwarf virus.

The terminal regions of the dsRNA genome segments of rice black streaked dwarf virus (RBSDV) were sequenced. The individual dsRNAs, which were 32P-labelled at their 3' termini by incubation with [32P]pCp and T4RNA ligase, were separated by 5% PAGE, and the 10 dsRNA segments were sequenced by two-dimensional electrophoresis. The common 3'-terminal sequences ---GUC 3' and ---AAAAACUU 3' were found in the plus and minus strands, respectively. The strictly conserved terminal sequences (5' AAGUUUUU ... GUC 3') of the genome segments of RBSDV differ from those of the phytoreoviruses and rice ragged stunt virus.

Taxonomic characteristics of fijiviruses based on nucleotide sequences of the oat sterile dwarf virus genome.

Sequence determination of full-length cDNA clones of genomic segments 7-10 (S7-S10) of oat sterile dwarf fijivirus (OSDV) revealed that the 5' and 3' ends of the plus strands of these segments had the same conserved terminal sequences, 5' AACGAAAAA and UUUUUUUAGUC 3'. These sequences are similar, but not identical, to the conserved terminal nucleotide sequences of the genomic segments of rice black streaked dwarf fijivirus (RBSDV) and maize rough dwarf fijivirus (MRDV). The coding strands of S7 and S10 each contained two large nonoverlapping open reading frames (ORFs), as do RBSDV S7 and S9, MRDV S6 and S8 and Nilaparvata lugens reovirus (NLRV; a putative member of Fijivirus) S9. These results strongly suggest that the dicistronic nature of certain genomic segments is characteristic of fijiviruses. Computer analyses revealed sequence homology between RBSDV S7 ORF2, MRDV S6 ORF2 and OSDV S7 ORF2, suggesting that this protein is conserved among plant fijiviruses. No counterparts were found in the genome of NLRV, which is a nonphytopathogenic insect reovirus. Furthermore, phylogenetic trees derived from multiple sequence alignments of each of the homologous proteins from OSDV, RBSDV, MRDV and NLRV suggest that NLRV did not evolve from either Fijivirus group 2 (RBSDV and MRDV) or group 3 (OSDV).

Relatedness of the nucleotide sequence of the 3'-terminal region of clover yellow vein potyvirus RNA to bean yellow mosaic potyvirus RNA.

The sequence of the 3'-terminal 1,357 nucleotides of clover yellow vein potyvirus RNA was determined. A coat protein gene was identified and its predicted amino acid sequence deduced. It had 273 residues with a molecular weight of 31,019. The amino acid and nucleotide sequences of the virus were compared with those of five other potyviruses. The homology between these viruses indicated that clover yellow vein virus was a distinct virus, but had a closer affinity to bean yellow mosaic virus than to the four other potyviruses.

Viruses in the phytoreovirus genus of the Reoviridae family have the same conserved terminal sequences.

The 5'- and 3'-terminal nucleotide sequences of the dsRNA genome segments of rice dwarf virus (RDV) and rice gall dwarf virus (RGDV), members of the Phytoreovirus genus of the Reoviridae family, were determined and compared with those of wound tumour virus (WTV). The 3' tetranucleotides of the plus strand of all genome segments of RDV and RGDV were found to be the same (---UGAU 3'), except for segment 9 of RDV which had the 3'-terminal sequence---CGAU 3'. The conserved 3'-terminal sequence (---UGAU 3') was the same as that found in the genome segments of WTV, another member of the Phytoreovirus genus. On the other hand, the 5' termini of the plus strands of RDV and RGDV were found to have two or three types of common sequence. RDV had either 5' GGCAAA--- or 5' GGUAAA---, whereas RGDV had 5' GGCAUUUU---, 5' GGUAUUUU--- or 5' GGUAAUUU---. These conserved sequences were similar to the conserved 5'-terminal sequence of WTV (5' GGUAUU---). Although the three viruses differ in plant host range, tissue specificity, vector specificity and disease symptom expression, these results suggest that they have a common ancestral origin.

A cDNA clone to clover yellow vein potyvirus genome is highly infectious.

We obtained a highly infectious cDNA clone of clover yellow vein virus (CIYVV). The cDNA fragments, from which a full-length cDNA clone was constructed, were sequenced, and the complete nucleotide sequence of C1YVV RNA was determined. The viral genome is 9584 nucleotides (nt) in length excluding the poly(A) tail and contains one open reading frame (ORF) encoding a large polyprotein of 3072 amino acids. The non-coding region preceding the ORF is 190 nt long. The termination codon is followed by a 175-nt sequence. Seven potential protease NIa, one HC-pro and one P1 protease recognition sites were found in the C1YVV polyprotein by searching for cleavage consensus sequences among the potyvirus group. The cleavage dipeptides of C1YVV NIa protease are Q(E)/S(A,G). The F is conserved at the -2 position from the cleavage site except for at the P3/6K1 junction, and the V conserved at the -4 position among many potyviruses is not present at all. The genome organization of C1YVV was determined, and the amino acid sequence was compared with that of other potyviruses. The full-length cDNA clone of C1YVV was constructed by combining cDNA fragments and placed it under the control of the cauliflower mosaic virus 35S promoter. The full-length cDNA was constructed so that no extra nucleotide was present at the transcription initiation site and only 10 adenine residues were present at the 3' end of the C1YVV cDNA clone. Mechanical inoculation of a circular-formed plasmid DNA onto broad bean seedlings led to systemic infection, and the symptoms were similar to those caused by the wild-type virus but rather mild. Plasmid diluted as low as 500 pg/microl was able to induce symptoms, demonstrating that this full-length C1YVV cDNA is more infectious than any other infectious cDNAs so far reported. Filamentous particles reacting with the antiserum to C1YVV were observed in the crude sap of infected plants by immunoelectron microscopy, and genome replication was demonstrated by RT-PCR of 3' non-coding regions of C1YVV genome in total plant RNAs.

Inosine 5'-triphosphate can dramatically increase the yield of NASBA products targeting GC-rich and intramolecular base-paired viroid RNA.

Nucleic acid sequence-based amplification (NASBA) according to the standard protocol failed to amplify cRNA of viroids, probably because of their GC-rich and intramolecular base-paired structure. However, NASBA in the presence of inosine 5'-triphosphate successfully amplified the cRNAs to viroids in total nucleic acid extracts from citrus plants. As sequence specificity of the cRNA to viroids was confirmed by northern analysis, the amplification and fidelity of cRNAs are sufficient for the sensitive and specific detection of viroids.

The C-terminal region of the P3 structural protein of rice dwarf phytoreovirus is important for P3-P3 interaction.

Using a random peptide library designed for the yeast two-hybrid system, we identified a peptide that binds strongly to the P3 structural protein of rice dwarf phytoreovirus (RDV). The amino acid sequence of the peptide showed a high homology to the C-terminal region of P3. C-terminally truncated P3 lost its ability to interact with authentic P3. Our observations suggest that the C-terminal region of P3 is important for the P3-P3 interaction, which forms the core shell structure of RDV.

Characterization of RNA polymerase associated with rice ragged stunt virus.

The RNA polymerase associated with rice ragged stunt virus (RRSV) was characterized. Activity was optimum at 35-40 degrees in 0.1 MTris-HC1 (pH 8.5) and 6-8 mMMgCl2. S-Adenosyl-L-methionine stimulated the activity about 5- to 6-fold. It was also stimulated in the presence of chymotrypsin (200 micrograms/ml). The molecular weights of RNAs synthesized in vitro were calculated to be about half those of the respective genome segments. The synthesized RNAs hybridized to the genome RNAs, and the hybrids migrated identically to the genome RNAs in PAGE. These results indicate that RRSV particles transcribe full-length copies of the genome RNAs. The characteristics of the polymerase are discussed in relation to those of other members of the Reoviridae.

Detection and assignment of proteins encoded by rice black streaked dwarf fijivirus S7, S8, S9 and S10.

The proteins encoded by rice black streaked dwarf fijivirus (RBSDV) genomic segments 7-10 (S7-S10) were characterized. Open reading frames (ORFs) from these segments were expressed as fusion proteins in Escherichia coli. Antibodies raised against the expressed products were used as probes to determine whether the viral ORFs encode structural proteins. In Western blots, antibodies to the expressed S8 and S10 products reacted with a core capsid (65 kDa) and a major outer capsid (56 kDa) protein, respectively, while none of the antibodies to S7 and S9 products reacted with structural proteins. Antisera to RBSDV S7 ORF1 and S9 ORF1 each detected a single protein of the predicted size in total protein extracts from infected rice plants and viruliferous Laodelphax striatellus. Immunoelectron microscopy revealed that antibodies to RBSDV S7 ORF1 and RBSDV S9 ORF1 reacted with tubular structures and viroplasm, respectively, in sections of both infected maize plants and viruliferous L. striatellus. Antisera to ORF2 of S7 and S9 failed to detect any proteins in the infected tissue using either Western blotting or immuno-electron microscopic techniques.

Evolution of a quadripartite hybrid virus by interspecific exchange and recombination between replicase components of two related tripartite RNA viruses.

Cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) belong to the Cucumovirus genus. They have a tripartite genome consisting of single-stranded RNAs, designated 1, 2, and 3. Previous studies have shown that viable pseudorecombinants could be created in vitro by reciprocal exchanges between CMV and TAV RNA 3, but exchanges of RNAs 1 and 2 were replication deficient. When we coinoculated CMV RNAs 2 and 3 along with TAV RNAs 1 and 2 onto Nicotiana benthamiana, a hybrid quadripartite virus appeared that consisted of TAV RNA 1, CMV RNAs 2 and 3, and a distinctive chimeric RNA originating from a recombination between CMV RNA 2 and the 3'-terminal 320 nucleotides of TAV RNA 2. This hybrid arose by means of segment reassortment and RNA recombination to produce an interspecific hybrid with the TAV helicase subunit and the CMV polymerase subunit. To our knowledge, this is the first report demonstrating the evolution of a new plant or animal virus strain containing an interspecific hybrid replicase complex.