Structure and assembly of turnip crinkle virus. VI. Identification of coat protein binding sites on the RNA.

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.

A homolog of an Escherichia coli phosphate-binding protein gene from Xanthomonas oryzae pv. oryzae.

A Xanthomonas oryzae pv. oryzae gene with sequence similarity to an Escherichia coli phosphate-binding protein gene (phoS) produces a periplasmic protein of apparent M(r) 35,000 when expressed in E. coli. Amino terminal sequencing revealed that a signal peptide is removed during transport to the periplasm in E. coli.

Several symptom-modulating mutations in the coat protein of turnip crinkle carmovirus result in particles with aberrant conformational properties.

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.

Asp-->Asn substitutions in the putative calcium-binding site of the turnip crinkle virus coat protein affect virus movement in plants.

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.

Mutational analyses of the putative calcium binding site and hinge of the turnip crinkle virus coat protein.

The turnip crinkle carmovirus (TCV) coat protein (CP) is folded into R (RNA-binding), S (shell), and P (protruding) domains. The S domain is an eight-stranded beta barrel common to the coat protein subunits of most RNA viruses. A five-amino-acid hinge connects the S and P domains. In assembled particles, each pair of CP subunits is thought to bind a single calcium ion through interactions with three residues of one subunit and two residues of a neighboring subunit. These five residues comprise the putative calcium-binding site (CBS). The putative CBS and hinge are adjacent to one another. Mutations were introduced into the putative CBS or hinge in an effort to further determine the biological functions of TCV CP. One putative CBS mutant, TCV-M32, exhibited wild-type cell-to-cell movement but failed to move systemically in Nicotiana benthamiana, and particles were not detected. Another putative CBS mutant, TCV-M23, exhibited deficient cell-to-cell movement but particles accumulated in isolated protoplasts. Two other putative CBS mutants, TCV-M22 and -M33, showed wild-type cell-to-cell and systemic movement but elicited mild systemic symptoms that were somewhat delayed. All of the hinge mutants exhibited wild-type movement but some elicited non-wild-type symptoms. Point mutations in the putative CBS or hinge appear to alter virus-ion interactions, secondary structure, or particle conformation, thereby affecting interactions between the CP and plant hosts.

Structure of the M2 protein gene of sonchus yellow net virus.

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.

Turnip crinkle virus infection from RNA synthesized in vitro.

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.

Structure of the nucleocapsid protein gene of sonchus yellow net virus.

The structure of the gene adjacent to the "leader RNA" gene of sonchus yellow net virus (SYNV), a plant rhabdovirus, was deduced by dideoxyribonucleotide sequence analysis of SYNV genomic (g) RNA and a series of plasmids constructed from SYNV gRNA or polyadenylated [poly(A)+] RNA from SYNV-infected plants. Evidence that this gene encodes the nucleocapsid (N) protein was obtained by reaction of SYNV N protein with polyclonal antibodies raised against recombinant proteins derived from the cloned gene. Experiments in which defined oligodeoxyribonucleotides were used to initiate reverse transcription of poly(A)+ RNA from SYNV-infected tobacco revealed that the N protein messenger (m) RNA gene begins at position 147 from the 3' end of the SYNV genome. Inspection of the sequence shows that this mRNA has a 56 nucleotide (NT) untranslated region followed by a 1425 NT open reading frame that is terminated by tandem UAA stop codons at positions 1628 to 1633 relative to the 3' end of SYNV gRNA. Little direct sequence homology is evident between the 475 amino acid polypeptide predicted from the SYNV sequence and the nucleocapsid (N) proteins deduced from nucleotide sequences of the Indiana and New Jersey serotypes of vesicular stomatitis virus (VSV) and rabies virus. However, a short region of possible importance contains a small group of chemically similar amino acids common to all four N proteins.

Physical map of the genome of sonchus yellow net virus, a plant rhabdovirus with six genes and conserved gene junction sequences.

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.

Size and complexity of polyadenylated RNAs induced in tobacco infected with sonchus yellow net virus.

Hybridization of 32P-labeled sonchus yellow net virus (SYNV) RNA to polyadenylated (poly A+) RNA from infected tobacco reveals the presence of four electrophoretically distinct components. These components probably represent five discrete RNA species complementary to SYNV RNA (scRNAs). The scRNAs are smaller than the 13,000 nucleotide (NT) SYNV genome and range in size from 1200 to 6600 NT. Individual recombinant DNA clones derived from SYNV RNA hybridize to at least three and probably four of the scRNAs. These results suggest that each of the scRNAs contains unique sequences with a combined size representing more than 90% of the viral genome. Therefore, the size range and sequence complexity of the scRNAs are as expected for messenger RNAs encoding the four major SYNV polypeptides and the minor "L-protein."

Characterization and detection of sc4: a sixth gene encoded by sonchus yellow net virus.

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.

Point mutations in the turnip crinkle virus capsid protein affect the symptoms expressed by Nicotiana benthamiana.

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 genome structure of turnip crinkle virus.

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.

Structure of the gene encoding the M1 protein of sonchus yellow net virus.

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.

Turnip crinkle virus defective interfering RNAs intensify viral symptoms and are generated de novo.

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.

Structure of the L (polymerase) protein gene of sonchus yellow net virus.

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.

Detection and sequence of plus-strand leader RNA of sonchus yellow net virus, a plant rhabdovirus.

Tobacco infected with the plant rhabdovirus sonchus yellow net virus (SYNV) contains short, 139- to 144-nucleotide (nt) transcripts complementary to the 3' terminus of the negative-strand genomic RNA. These transcripts are similar to the leader RNAs associated with several animal rhabdovirus infections in that they are encoded by the same region of the genome, but the SYNV transcripts are nearly 3 times longer than the animal rhabdovirus leader RNAs. The SYNV leader RNAs differ markedly in sequence from the leader RNAs associated with strains of vesicular stomatitis virus and rabies virus, although the first 30 nt of all three transcripts are rich in adenylate residues. The nucleotide sequence determined directly from SYNV RNA and from recombinant DNA clones derived from SYNV RNA reveals a possible initiation site for transcription of the N-protein mRNA that is located 147 nt from the 3' end of genomic RNA. The sequence (UUGU) at this site is complementary to the first 4 nt of the N-protein mRNAs of animal rhabdoviruses. In SYNV, the first AUG codon in the putative N-protein mRNA is located 57 nt downstream (at positions 203-205 in the viral genome) and is followed by an open reading frame for the remainder of the 1020 nt determined in these experiments.

Structure of the glycoprotein gene of sonchus yellow net virus, a plant rhabdovirus.

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.

Analysis of the genome of satellite panicum mosaic virus.

The relatedness of the genomes of satellite panicum mosaic virus (SPMV) and its helper virus, panicum mosaic virus (PMV), were investigated by nucleic acid hybridization. The results show that the satellite and helper virus RNAs have no appreciable homology or complementarity as assessed by hybridization with cDNA probes derived from the genomes of PMV and SPMV and with a probe complementary to the 3' terminus of SPMV RNA. The complete nucleotide sequence of SPMV RNA reveals that the genome is 826 nucleotides (nt) long. The ability to label SPMV RNA with polynucleotide kinase only after phosphatase treatment suggests that the 5' terminus is phosphorylated, but the extent of phosphorylation was not determined. The first open reading frame (ORF), encountered after an 88-nt 5'-untranslated region, encodes a 17,000 mol wt protein of a size and amino acid composition that are consistent with analysis of SPMV coat protein. An additional short ORF, located near the 3' end of the RNA, could encode a 6300 mol wt polypeptide. The minus strand also contains two ORFs that could potentially encode polypeptides of 7100 and 11,000 mol wt. No evidence is available to determine whether the second positive-strand ORF or the two minus-strand ORFs are expressed. The data presented here clearly show the SPMV RNA is distinct from the RNAs of other satellite viruses, in both size and nucleotide sequence. However, the 5'-untranslated portions of SPMV and satellite tobacco mosaic virus RNAs share some structural features that may be important in initiation of translation.