Necrotic Yellow Vein Virus RNA Research Articles

Beet necrotic yellow vein virus (benyvirus)

<i>Beet necrotic yellow vein virus</i> (<i>benyvirus</i>)

Effects of structural modifications upon the accumulation in planta of replicons derived from beet necrotic yellow vein virus RNA 3.

Beet necrotic yellow vein virus (BNYVV) RNA 3 from which all but the 3' and 5' 'core' replication origins (promoters) have been deleted replicates when coinoculated to Chenopodium quinoa with viral RNAs 1 and 2. The resulting 'replicon' can be used to express inserted heterologous sequences in planta. The effects of alterations of replicon structure on its efficiency of accumulation in planta were examined. Inclusion of up to approximately 240 nucleotides of sequence from the region immediately upstream of the core 3'-promoter sequence increased replicon accumulation, suggesting that this region contains specific replication enhancer elements. Insertion of non-viral 'spacer' sequences between the core promoters also increased replicon accumulation, provided that no strong secondary structure was present. The highly homologous 3'-terminal core promoters of BNYVV RNAs 1, 2 and 4 could substitute for the RNA 3 core promoter but were generally somewhat less effective. Co-inoculation of full-length RNA 3 but not RNA 4 interfered with accumulation of the RNA 3-based replicons.

Detection and Characterization of a Distinct Type of Beet Necrotic Yellow Vein Virus RNA 5 in a Sugarbeet Growing Area in Europe

A fifth beet necrotic yellow vein virus (BNYVV) RNA species has been detected in Europe in sugarbeet infected with P-type BNYVV. Very little sequence variation was found between two European sources of this RNA 5*, but considerable differences were detected between these two European sources on the one hand and the four Japanese sources recently analysed by Kiguchi et al. on the other. The BNYVV RNA 5-encoded 26K proteins share a stretch of six amino acids (FRGPGN) with the BNYVV RNA 3-encoded 25K protein which may be of interest in view of the reported interactions between the two RNAs in pathogenicity.

Evidence for in vitro and in vivo autocatalytic processing of the primary translation product of beet necrotic yellow vein virus RNA 1 by a papain-like proteinase.

Beet necrotic yellow vein virus RNA 1 contains a single long ORF corresponding to the theoretical translation product of 237 kDa which contains the information necessary for replication of the viral genome. This ORF contains a putative papain-like proteinase domain which has been localized, on the basis of sequence alignments, between the helicase and polymerase domains. Here we show that the RNA 1 primary translation product can be cleaved autocatalytically in vitro into two species of 150 kDa and 66 kDa, the latter of which probably contains the entire polymerase domain. A 66 kDa protein was detected immunologically in infected C. quinoa protoplasts using an antiserum specific for the C-terminal region of the RNA 1 primary translation product, confirming that processing also occurs in vivo.

Complete nucleotide sequence of the Japanese isolate S of beet necrotic yellow vein virus RNA and comparison with European isolates.

The complete nucleotide sequences of beet necrotic yellow vein virus RNA-1 to RNA-4 of the Japanese isolate S (BNYVV-S) were determined and compared with those of French isolate (BNYVV-F2). The nucleotide sequences of the two isolates were very similar, differing by only 1.7% (RNA-1), 4.1% (RNA-2), 2.9% (RNA-3) and 3.6% (RNA-4), respectively. The differences of the amino acid sequences of the two isolates depended upon the open reading frames (ORF) as follows: P237, 1.4%; P22 (coat protein), 2.1%; 54k ORF, 3.4%; P42, 0.5%; P13, 1.7%; P15, 3.0%; P14, 7.0% P25, 6.4%; P31, 3.5%. Comparison of the coat protein and triple gene block (P42, P13 and P15) regions of RNA-2 with other isolates revealed that BNYVV-S was much more similar to the Yugoslavian isolate (BNYVV-Yu2) than to BNYVV-F2. The nucleotide differences between BNYVV-S and BNYVV-Yu2 were less than 1%. Based upon the grouping of BNYVV variants reported by Kruse et al. [10], BNYVV-S is thus considered to belong to the A type along with BNYVV-Yu2, whereas BNYVV-F2 is classified in the B type. Our data suggest that the Japanese isolate S may have been derived from European countries other than France or Germany.

Beet necrotic yellow vein virus 42 kDa triple gene block protein binds nucleic acid in vitro

The triple gene block (TGB) of beet necrotic yellow vein virus RNA 2 is required for cell-to-cell movement of the virus RNA. The protein P42 encoded by the 5'-proximal gene of the TGB has consensus sequence motifs characteristic of an ATP/GTP-dependent helicase. P42 was over-expressed in Escherichia coli and shown to bind both single- and double-stranded RNA and DNA by Northwestern blotting. Site-directed mutagenesis located the nucleic acid-binding domain to the N-terminal 24 amino acids of the protein and a point mutation or deletions in the region of P42 containing the helicase consensus sequences did not affect nucleic acid-binding activity of the immobilized protein. Electrophoretic mobility-shift assays revealed that P42 also binds nucleic acids in solution and that deletion of the N-terminal region inhibits this binding. Mutations in both the N-terminal nucleic acid-binding domain and the helicase domain blocked infection of leaves, indicating that both regions of P42 are important for its activity in vivo.

In situ localization of the non-structural protein P25 encoded by beet necrotic yellow vein virus RNA 3.

The in situ localization of the non-structural protein P25 encoded by beet necrotic yellow vein virus (BNYVV) RNA 3 and of the BNYVV coat protein (CP) was studied by immunoelectron microscopy in infected leaf and root cells of Chenopodium murale and C. quinoa. The CP was detected in the cytoplasm of all cell types except xylem, sieve elements, and companion cells. P25 was detected in the cytoplasm and nuclei of the same cell types. The intensity of CP labelling varied depending upon the stage of infection of the cell, whereas the P25 labelling intensity was similar in newly infected cells and in cels at later stages of infection. These results suggest that P25 may be synthesized at an earlier stage of infection than CP. Its presence in the nuclei of newly infected cells may be related to the reported effect of P25 on leaf symptom development.

The secondary structure of the 5'-noncoding region of beet necrotic yellow vein virus RNA 3: evidence for a role in viral RNA replication.

Secondary structure-sensitive chemical and enzymatic probes have been used to produce a model for the folding of the first 312 residues of the long 5'-noncoding region of beet necrotic yellow vein virus RNA 3. The structure consists of two major domains, one of which includes long distance base-pairing interactions between two short sequence elements (Box I and Box II) situated between positions 237 and 292 and complementary elements (Box I' and II') near the 5'-terminus. Previous studies have shown that base pairing between these sequence elements (in either the plus-strand or minus-strand RNA) is important for RNA 3 accumulation during infection. RNA 3 transcripts were produced containing mutations which preferentially disrupted Box II-II' base pairing in either the plus- or minus-strand. In infection experiments, transcripts with mutations which disrupted the Box II-II' interaction in the plus-strand structure replicated less efficiently than mutants in which the Box II-II' interaction was disrupted in the minus-strand. These findings indicate that the complex 5'-proximal plus-strand structure to which the Box II-II' interaction contributes comprises at least part of the promoter for plus-strand RNA synthesis.

Mapping the promoter for subgenomic RNA synthesis on beet necrotic yellow vein virus RNA 3

During infection of Tetragonia expansa leaves, RNA 3 of the quadripartite genome of beet necrotic yellow vein virus directs synthesis of a subgenomic RNA (RNA 3sub) which corresponds to the 3′-terminal 600 residues of the RNA 3 molecule. Biologically active run-off transcripts have been prepared from full-length cDNA of RNA 3 cloned behind a bacteriophage T7 RNA polymerase promoter. RNA 3 transcripts carrying deletions in the vicinity of the RNA 3sub initiation site were produced by site-directed mutagenesis at the cDNA level and then tested for their capacity to direct RNA 3sub synthesis in infected leaves. The cis-acting domain essential for normal levels of RNA 3sub production in planta (the ‘core’ promoter) did not extend in the 5′-direction beyond position −16 relative to the RNA 3sub transcription initiation site. The 3′-boundary of the core promoter domain was located somewhere between positions +100 and +208. Displacement of the promoter domain to an upstream site in RNA 3 produced a new subgenomic RNA starting at or near the predicted upstream site.

In vitro mutagenesis of biologically active transcripts of beet necrotic yellow vein virus RNA 2: evidence that a domain of the 75-kDa readthrough protein is important for efficient virus assembly.

RNA 2 of the multipartite genome of beet necrotic yellow vein virus carries the cistron for 21-kDa viral coat protein at its 5' extremity. The amber termination codon of the coat protein cistron undergoes suppression approximately 10% of the time so that translation continues into an adjacent 54-kDa open reading frame, yielding a 75-kDa readthrough protein. The roles of coat protein and the readthrough protein in infection were investigated with biologically active transcripts of RNA 2. Much of the coat protein cistron of the RNA 2 transcript could be deleted without interfering with viral replication and local lesion formation on leaves, although formation of the rod-shaped virions did not occur. Mutants in which the amber coat protein termination codon was replaced with an ochre codon or a tyrosine codon were also viable. The ochre codon was suppressed both in vitro and in planta. The mutant containing the tyrosine substitution produced only the 75-kDa read-through protein and was deficient in viral assembly. Deletions in the 54-kDa readthrough domain were also viable in planta but had different effects on virus assembly. A deletion in the C-terminal portion of the readthrough domain did not interfere with RNA packaging but, unexpectedly, deletions in the N-terminal portion were assembly deficient, although 21-kDa coat protein was produced in planta. Thus, the 75-kDa protein can apparently intervene in virion assembly even though it has not been detected in purified virions.

Two proteins encoded by beet necrotic yellow vein virus RNA 3 influence symptom phenotype on leaves.

RNA 3 of the beet necrotic yellow vein virus (BNYVV) quadripartite RNA genome is not essential for virus multiplication on leaves of Tetragonia expansa but has dramatic effects on symptom expression. Virus isolates containing RNA 3 produce bright yellow local lesions while isolates lacking RNA 3 produce much milder symptoms. Using directed mutagenesis of cDNA clones followed by in vitro synthesis of biologically active transcripts, a 25 kDa open reading frame (ORF) of RNA 3 was shown to be responsible for the yellow local lesion phenotype. In addition, two deletion mutants of RNA 3 were found to elicit the appearance of severe necrotic local lesions. Analysis of one of these mutants revealed that necrosis was due to the overexpression of a second short ORF, N, overlapping the 3'-terminal portion of the 25 kDa ORF. As shown by gene fusion studies, gene N is not detectably expressed from full-length RNA 3 but is translationally activated by deletion of upstream sequences. Introduction of gene N into the genome of the unrelated DNA virus, cauliflower mosaic virus, elicits a necrotic response instead of the typical mosaic symptoms, demonstrating that gene N can induce necrosis outside of the context of a BNYVV infection.

Multiplication of beet necrotic yellow vein virus RNA 3 lacking a 3′ poly(A) tail is accompanied by reappearance of the poly(A) tail and a novel short U-rich tract preceding it

Beet necrotic yellow vein virus RNAs 1 and 2 but not RNAs 3 and 4 are required for viral multiplication in Chenopodium quinoa leaves. Elimination of the 3′ poly(A) tail from RNA 3 transcripts markedly attenuated their ability to be amplified when co-inoculated with RNAs 1 and 2 to this host. Successful multiplication of the tailless RNA 3 was accompanied by the reappearance of new 3′ poly(A) tails on the progeny. The evidence suggests that the newly acquired poly(A) sequence results from the action of a poly(A) polymerase rather than recombination with the homologous 3′ terminal domains of RNAs 1 or 2. An unexpected feature of these progeny RNA 3 molecules was the presence of a novel short heterogenous U-rich tract separating the poly(A) tail from the 3′ end of the heteropolymeric RNA 3 sequence proper.

Mapping sequences required for productive replication of beet necrotic yellow vein virus RNA 3

Of the four genome components of beet necrotic yellow vein virus only RNAs 1 and 2 are essential for viral replication in leaves. We have mapped cis-regulatory elements on RNA 3 by introducing deletions into expressible cDNA clones and inoculating leaves with the altered transcripts along with RNAs 1 and 2. Transcripts carrying internal deletions extending to within 69 residues of the 3′ poly(A) tail or to within about 300 residues of the 5′ terminus were efficiently amplified and encapsidated in vivo. The 3′ terminal cis-essential domain can be folded into a secondary structure which is conserved among all four genomic RNAs and which probably contains the minus-strand promoter. RNA 3 transcripts with 75% of the central core of the sequence deleted or replaced by the β-glucuronidase (GUS) gene were also viable. GUS activity was detected in infected tissue in the latter case.

In vitro synthesis of biologically active beet necrotic yellow vein virus RNA

Beet necrotic yellow vein virus (BNYVV) has a quadripartite plus-strand RNA genome in which the two smallest genome components, RNA 3 and 4, are not necessary for virus multiplication in leaves. Infectious transcripts of BNYVV RNA 3 and 4 have already been described ( V. Ziegler-Graff, S. Bouzoubaa, I. Jupin, H. Guilley, G. Jonard, and K. Richards (1988) J. Gen. Virol. 69, 2347–2357 ). In this paper we describe synthesis of a full-length RNA-1 transcript by bacteriophage T7 RNA polymerase-directed run-off transcription of cloned viral cDNA. A recombinant plasmid containing a full-length cDNA insert of RNA 2 could not be maintained in Escherichia coli. Therefore full-length transcript of RNA 2 was produced by transcription of cDNA ligation products without amplification in bacteria. When inoculated together to leaves of Chenopodium quinoa or Tetragonia expansa the RNA 1 and 2 transcripts were infectious; they also supported multiplication of the BNYVV RNA 3 and 4 transcripts, providing a totally synthetic inoculum of the virus. In one recombinant clone of RNA 2 a point mutation causing an arginine to serine substitution at position 119 of the viral coat protein was discovered. The mutation was detected because the resulting coat protein had altered electrophoretic mobility. RNA 2 transcripts containing this mutation were infectious but viral RNA was not encapsidated. The mutation also interfered with long distance movement of the virus in spinach, presumably as a consequence of the packaging deficiency.

Effect of beet necrotic yellow vein virus RNA composition on transmission by Polymyxa betae

Beet necrotic yellow vein virus (BNYVV) is naturally transmitted by the soil-borne fungus Polymyxa betae and usually remains confined to the roots of infected sugarbeets. In naturally infected sugarbeets the virion RNA always consists of four components which are uniform in size in different isolates but when BNYVV is propagated by mechanical inoculation to leaves of Chenopodium quinoa the two smallest RNA components, RNA-3 and -4, may undergo deletion or disappear from the isolate, suggesting that they are only essential for the natural mode of infection. To test this hypothesis, several C. quinoa isolates of BNYVV with different RNA-3 and -4 contents have been retransmitted to sugarbeet root via P. betae. The results show that the two isolates containing no detectable full-length RNA-3 and -4 are poorly transmitted and that cases of successful infection are associated with the reappearance of full-length RNA-3 and -4.

In vitro Translation of Beet Necrotic Yellow Vein Virus RNA and Studies of Sequence Homology among the RNA Species Using Cloned cDNA Probes

SUMMARY The four RNA species of a French strain of beet necrotic yellow vein virus (BNYVV) have lengths of 7100, 4800, 1800 and 1500 nucleotides, as measured by agarose gel electrophoresis in denaturing conditions. Translation of purified RNA in a reticulocyte cell-free system revealed that RNA-2 encodes the 22000 mol. wt. coat protein. Cloned cDNA copies of each of the four viral RNA species have been used to show that BNYVV RNA-3 and RNA-4 share no sequence homology with RNA-1 and RNA-2. Thus, RNA-3 and RNA-4 cannot be subgenomic messengers derived from the larger RNA species.