Search

Particles of several symptom-modulating TCV coat protein (CP) mutants were pretreated at pH 5.5, 7.5 or 8.5 and their conformations compared by agarose gel electrophoresis to those of wild-type particles. Particles of two mutants were swollen under conditions in which wild-type particles remained contracted; particles of one mutant were contracted under conditions in which wild-type particles were swollen; a portion of the particles of one mutant was contracted and another portion swollen under conditions in which wild-type particles remained contracted; and particles of one mutant, that elicited wild-type symptoms, comigrated with wild-type particles under all conditions tested. The results of in vitro translation experiments with mutant particles were essentially similar to those with wild-type particles, despite conformational differences at pH 5.5 and 8.5. These results suggest that more than the swollen conformation is required for in vitro translation, and that particle conformation may play a role in symptom elicitation.

The nucleotide sequence of a sixth gene (sc4) of the plant rhabdovirus sonchus yellow net virus (SYNV) was determined from viral genomic and poly(A)+ cDNA clones. The sc4 gene is 1196 nucleotides (nt) and has an open reading frame of 972 nt that is capable of encoding a protein of 324 amino acids. Primer extension analyses of poly(A)+ RNA isolated from infected plants indicate that the 5' end of the sc4 mRNA corresponds to nucleotide 2840, relative to the 3' end of the minus-sense genomic RNA and extends to nucleotide 4035. A 43-nt untranslated leader sequence precedes the predicted first AUG codon and a 181-nt untranslated sequence follows the translational stop codon. This gene is similar to the other SYNV genes in that it is flanked on each side by a conserved gene junction sequence. Polyclonal antibodies raised to an sc4 fusion protein react with a 37-kDa protein in virus-infected plants that is close to the predicted size of the sc4 protein. Western blot analyses of cellular fractionations from infected plants show that sc4 is membrane associated and sucrose density gradient analyses demonstrate that sc4 sediments in the same fractions as SYNV virions. Analysis of the sc4 open reading frame reveals that 16% of the amino acids are serine or threonine residues and that the protein has four potential consensus casein kinase II phosphorylation sites. The deduced amino acid sequence of sc4 also contains a motif related to alpha amylases and aspartic proteases. This completes the sequence determination of the 13,720-nt SYNV genome.

Many plant virus coat proteins have binding sites for divalent cations, particularly calcium. It has been speculated that the purpose of such sites is to ensure that plant viruses release their RNA only in the host cytoplasm which has a low calcium concentration. By comparison to tomato bushy stunt virus, two of the amino acids that probably interact directly with calcium ions in turnip crinkle virus (TCV) capsids, Asp155 and Asp157, were substituted with Asn residues by oligonucleotide-directed mutagenesis to create the mutant TCV-M18. TCV-M18 coat protein, and virions accumulated to wild-type levels in isolated protoplasts. Mutant virions were able to uncoat and to initiate de novo replication in protoplasts although no symptoms were observed in plants inoculated with TCV-M18. Mutant RNA accumulated to much lower levels than wild-type RNA in inoculated leaves and was not detected in upper, uninoculated leaves. The lower infectivity of TCV-M18 in plants may be due to a decreased capacity to move efficiently from cell to cell, and we suggest the possibility that TCV coat protein plays an active, pivotal role in cell-to-cell movement interactions.

The complete nucleotide sequence of the L protein gene of sonchus yellow net virus (SYNV), a plant rhabdovirus, was determined by dideoxynucleotide sequencing of cloned cDNAs derived from the negative-strand genomic RNA. The L protein gene is composed of 6401 nucleotides (nt) located between positions 7158 and 13558 relative to the 3' end of the genomic RNA. Sequence analysis suggests that the complementary mRNA contains a 44 nt untranslated 5' leader sequence preceding an open reading frame of 6348 nucleotides that is capable of encoding a polypeptide of 2116 amino acids with a deduced molecular weight of 241,569 Da. The L protein is positively charged, has a high proportion of the amino acids Leu and Ile, and contains putative polymerase and RNA binding domains. Extended alignment of the SYNV L protein amino acid sequence with those of other nonsegmented negative-strand RNA virus polymerases reveals conservation of sequences within 12 blocks that appear sequentially along the protein. A cluster dendrogram derived from the L protein alignments indicates that SYNV is more closely related to animal rhabdoviruses than to the paramyxoviruses and that the animal rhabdoviruses have diverged less from each other than from SYNV.

The nucleotide sequence of the glycoprotein (G) gene of sonchus yellow net virus (SYNV), a plant rhabdovirus, was determined from viral genomic and mRNA cDNA clones. The G cistron is 2045 nucleotides (nt) long and the G protein mRNA open reading frame (ORF), as determined from the cDNA sequence, contains 1896 nt and encodes a protein of 632 amino acids. Immunoblots with antibodies elecited against the purified glycoprotein from virus particles reacted with a fusion protein produced in Escherichia coli, indicating that the cloned ORF encodes the G protein. The 5' end of the G protein mRNA corresponds to nt 5111, relative to the 3' end of the viral (minus sense) genome, as determined by primer extension from mRNA isolated from infected plants, and extends to nt position 7155 on the genomic RNA. A 34-nt untranslated 5' leader sequence and a 115-nt untranslated 3' end flank the ORF on the mRNA. The gene junctions on either side of the G gene on the genomic RNA are identical to those previously described for other SYNV genes and are similar to sequences separating genes of animal rhabdoviruses. The predicted molecular weight of the G protein is 70,215 Da, a value less than the 77,000 Da estimated for the glycosylated G protein from virus particles. The deduced amino acid sequence of the SYNV G protein shares little direct relatedness with the G proteins of other rhabdoviruses, but appears to contain a similar signal sequence, a transmembrane anchor domain, and glycosylation signals. In addition, the SYNV G protein contains a putative nuclear targeting site near the carboxy terminus, which may be involved in transit to the nuclear membrane prior to morphogenesis.

In an effort to determine the biological function(s) of the capsid protein protruding domains unique to the plant carmo- and tombusviruses, we constructed turnip crinkle virus (TCV) mutants in which tandem, in-frame translation terminators replaced the first two codons of the five-amino acid hinge between the shell and the protruding domains of the TCV capsid protein. One of the mutants replicated in inoculated leaves and protoplasts without detectable accumulation of capsid protein. The mutant lacked the capacity to move systemically in Brassica campestris and Nicotiana benthamiana. After 8 weeks, revertant virions that had regained the capacity to move systemically were purified and found to have sense codons at the positions of the introduced translation terminators. One of the revertants, with amino acid substitutions in the hinge, elicited milder symptoms than those elicited by the wild-type virus, and another elicited more severe symptoms. Oligonucleotide-directed mutagenesis was used to show that the hinge mutations were sufficient to elicit the milder, but not the more severe, symptom syndrome. Single amino acid substitutions were also shown to be sufficient to elicit the milder, but not the more severe, symptoms.

The gene encoding the M1 protein of sonchus yellow net virus (SYNV), a plant rhabdovirus, has been sequenced and identified by Western blot analysis of SYNV proteins using antibodies directed against a fusion protein derived from a portion of the sequenced gene. The M1 gene is positioned between nucleotides 4039 and 5109 relative to the 3' end of the viral RNA and is the fourth gene from the 3' end of the genome. The 1071-nucleotide (nt) M1 gene lies between a putative nonstructural gene of unknown function and the gene encoding the glycoprotein and is bordered on either side by the same GG intergenic dinucleotide that separates other genes in the SYNV genome. The M1 mRNA (scRNA 6) consists of a 71-nt untranslated region at the 5' terminus followed by an 858-nt open reading frame (ORF) capable of encoding a protein with a calculated molecular weight of 31,779. The amino acid sequence deduced from this ORF is not highly homologous to those of other rhabdovirus matrix proteins, but has some localized regions of similarity. The UGA codon that terminates the M1 ORF is followed by a 3' untranslated region of 142 nt. The viral RNA (minus-sense) sequence corresponding to the extreme 3' end of the mRNA contains a 9-nt tract (3'-AUUGUUUUU-5') that is identical to the sequences at the termini of other SYNV genes.

Structural studies of turnip crinkle virus have been extended to include the identification of high-affinity coat protein binding sites on the RNA genome. Virus was dissociated at elevated pH and ionic strength, and a ribonucleoprotein complex (rp-complex) was isolated by chromatography on Sephacryl S-200. Genomic RNA fragments in the rp-complex, resistant to RNase A and RNase T1 digestion and associated with tightly bound coat protein subunits, were isolated using coat-protein-specific antibodies. The identity of the protected fragments was determined by direct RNA sequencing. These approaches allowed us to study the specific RNA-protein interactions in the rp-complex obtained from dissociated virus particles. The location of one protected fragment downstream from the amber terminator codon in the first and largest of the three viral open reading frames suggests that the coat protein may play a role in the regulation of the expression of the polymerase gene. We have also identified an additional cluster of T1-protected fragments in the region of the coat protein gene that may represent further high-affinity sites involved in assembly recognition.

Genome-length cDNA clones of turnip crinkle virus (TCV) were constructed with SmaI and XbaI restriction sites engineered at the 5' and 3' termini, respectively. The genome-length cDNAs were positioned downstream of modified lambda and T7 phage promoters such that in vitro transcription resulted in RNAs with 5 extra nucleotides at the 3' end, and 1, 2, or 14 extra nucleotides at the 5' end depending on the construction. Transcripts with 14 extraviral 5' nucleotides were not infectious, while transcripts with 1 or 2 additional 5' nucleotides, with or without 5'-cap analog included in transcription reactions, were biologically active. These were approximately an order of magnitude less infectious than RNA extracted from TCV virions. The additional 5' nucleotides were not maintained in progeny viral RNAs isolated from plants.

The nucleotide sequence of turnip crinkle virus (TCV) genomic RNA has been determined from cDNA clones representing most of the genome. Segments were confirmed using dideoxynucleotide sequencing directly from viral RNA, and the 3' terminal sequence was confirmed by chemical sequencing of end-labeled genomic RNA. Three open reading frames (ORFs) have been identified by examination of the deduced amino acid sequences and by comparison with the ORFs found in the genome of carnation mottle virus. ORF 1 initiates near the 5' terminus of the genome and is punctuated by an amber termination codon. Translation of ORF 1 would yield a 28-kDa protein and an 88-kDa read-through product. The read-through domain possesses amino acid sequence similarities with putative viral RNA polymerases. ORFs 2 and 3 encode products of 38 (coat protein) and 8 kDa, respectively, which are expressed from subgenomic mRNAs. The organization of the TCV genome suggests that TCV is closely related to carnation mottle virus and distinct from members classified in other small RNA virus groups, such as the tombus- and sobemoviruses.

We provide evidence that a plant rhabdovirus, sonchus yellow net virus (SYNV), is similar to most animal rhabdoviruses in the order of structural genes and in the nucleotide sequences at the gene junctions but that it differs in the presence and location of a putative nonstructural gene. From the patterns of hybridization of a library of recombinant DNA clones, we have shown that the SYNV genome is transcribed into a short 3'-terminal "leader RNA" and six mRNAs. The proteins encoded by the SYNV mRNAs, in order of the appearance of their genes in the SYNV genome, are designated 3'-N-M2-sc4-M1-G-L-5' (N, nucleoprotein; M, matrix protein; sc, protein encoded by SYNV complementary RNA; G, glycoprotein; L, large protein). The intergenic and flanking gene sequences are conserved and consist of a central core of 14 nucleotides (3'-UUCUUUUUGGUUGU/A-5') whose sequence is similar to the sequence at the gene junctions of vesicular stomatitis and rabies viruses. The SYNV core consists of an 8-nucleotide (3'-UUCUUUUU-5') transcription termination signal at the 5' terminus of each gene, a dinucleotide (GG) spacer whose complement does not appear in mRNA, and a tetranucleotide (3'-UUGU/A-5') that is complementary to the first four nucleotides at the 5' terminus of the SYNV mRNAs. These results, when compared with structural information available on animal rhabdoviruses, suggest that organization of structural genes and maintenance of signals thought to play important roles in regulation of transcription have been conserved during evolution in plant, insect, and vertebrate hosts. However, differences in number and location of putative nonstructural genes reveal some flexibility in genome organization that may be important in deducing taxonomic and evolutionary relationships among viruses causing diseases in phylogenetically diverse hosts.

The nucleotide sequence of the gene immediately following the nucleocapsid protein gene of sonchus yellow net virus (SYNV), a plant rhabdovirus, is presented. Serological reactions of SYNV proteins with antibodies elicited by a fusion protein constructed from the sequenced gene indicate that this gene encodes an SYNV structural protein designated M2. The 5' end of the M2 protein mRNA appears to correspond to position 1700 relative to the 3' end of SYNV RNA, because an extension product that maps to this position was synthesized by reverse transcription of polyadenylated [poly(A)+] RNA from infected tobacco that had been primed with an SYNV-specific oligodeoxyribonucleotide. The 3' end of the gene encoding the M2 protein is defined by a recombinant DNA plasmid derived from poly(A)+ RNA from SYNV-infected plants. This plasmid contains an insert with a 3'-terminal region corresponding to a uridylate-rich sequence present at positions 2832 to 2836 on SYNV genomic (g) RNA. These data thus suggest that the M2 protein mRNA is 1132 nucleotides (NT) long, excluding the poly(A) tail, and consists of a 50-NT untranslated 5' region, a 1035-NT open reading frame (ORF), and a 47-NT untranslated 3'region. The ORF is capable of encoding a 345-amino acid protein with a calculated molecular weight of 38,332. A small region of the M2 protein appears to have some similarity to the phosphoproteins of other rhabdoviruses. An identical 14-NT region occurs at the two sequenced gene junctions on SYNV gRNA and shares homology with regions separating the genes of some animal rhabdoviruses.

Defective interfering (DI) RNAs have been isolated from a broad spectrum of animal viruses and have recently been identified in plant virus infections. Because of their ubiquitous nature, DIs are thought to play an important role in virus replication and yields. DI RNAs have now been found in association with a natural isolate of turnip crinkle virus (TCV-B) and are generated de novo after inoculation of turnip with virus derived from cloned transcripts. DI RNA G, naturally found in the TCV-B isolate, is a mosaic molecule with 5' and 3' viral segments and a repeat of 36 nucleotides at the beginning of the 3' segment. The 5'-terminal 21 nucleotides of DI RNA G were not similar to genomic TCV sequences but did resemble sequences found at the 5' end of other small RNAs associated with TCV (satellite RNAs). DI RNA G interferes with the accumulation of TCV genomic RNA and, unlike other DI RNAs, intensifies the symptoms of its helper virus. Infection of turnip with virus derived from cloned transcripts of TCV-B resulted in de novo generation of a DI RNA, DI1 RNA. DI1 RNA differed from DI RNA G by containing exact 5' and 3' ends of TCV as well as an internal virus segment.