Biomedical subjects

G Jonard

Publications and source records attributed to G Jonard.

At least 37 records · Page 2Linked to original sources

PubMed1995

Aphid transmission of beet western yellows luteovirus requires the minor capsid read-through protein P74.

Beet western yellows luteovirus is obligately transmitted by the aphid Myzus persicae in a circulative, non-propagative fashion. Virus movement across the epithelial cells of the digestive tube into the hemocoel and from the hemocoel into the accessory salivary glands is believed to occur by receptor-mediated endocytosis and exocytosis. Virions contain two types of protein; the major 22 kDa capsid protein and the minor read-through protein, P74, which is composed of the major capsid protein fused by translational read-through to a long C-terminal extension called the read-through domain. Beet western yellows virus carrying various mutations in the read-through domain was tested for its ability to be transmitted to test plants by aphids fed on agro-infected plants and semi-purified or purified virus preparations. The results establish that the read-through domain carries determinants that are essential for aphid transmission. The findings also reveal that the read-through domain is important for accumulation of the virus in agro-infected plants.

Amino Acid Sequence↗

PubMed1995

Nucleotide sequence of beet mild yellowing virus RNA.

The complete nucleotide sequence of the genomic RNA of beet mild yellowing virus, isolate 2ITB, is reported. The RNA consists of 5722 nucleotides and contains six long open reading frames which conform to the arrangement characteristic of Subgroup 2 luteoviruses. The three 3'-proximal open reading frames, which encode the viral coat protein, a putative movement protein and the Readthrough Domain, are highly homologous to the corresponding genes of beet western yellows luteovirus while the three 5'-proximal open reading frames are more closely related to the corresponding genes of cucurbit aphid borne yellows luteovirus. The sequence data thus indicate that beet mild yellowing virus should be considered a distinct virus rather than a strain of beet western yellows virus.

Amino Acid Sequence↗

PubMed1994

Nucleotide sequence of cucurbit aphid-borne yellows luteovirus.

The nucleotide sequence (5669 residues) of the genomic RNA of cucurbit aphid-borne yellows luteovirus (CABYV) is presented. Analysis of genome organization and sequence homologies indicate that CABYV is a member of luteovirus Subgroup 2 (other sequenced members: beet western yellows virus, potato leafroll virus, and barley yellow dwarf virus, RPV isolate) and appears to be most closely related to beet western yellows virus.

Animals↗

PubMed1994

Artificial defective interfering RNAs derived from RNA 2 of beet necrotic yellow vein virus.

Long internal deletions were introduced into cloned cDNA of beet necrotic yellow vein virus RNAs 1-4 and transcripts containing the deletions were tested for their ability to inhibit replication of viral RNA in Chenopodium quinoa protoplasts and plants. No inhibition was observed with the deletion mutants based on RNAs 1, 3 and 4 but the RNA 2 deletion mutants all provoked a dramatic inhibition of synthesis of viral RNAs 1 and 2.

Blotting, Northern↗

PubMed1994

Detection by immunogold labelling of P75 readthrough protein near an extremity of beet necrotic yellow vein virus particles.

RNA 2 of beet necrotic yellow vein virus carries the cistron for the 21 kd coat protein at its 5'-extremity. During translation, the coat protein cistron termination codon is suppressed about 10% of the time so that translation continues into the adjacent open reading frame to produce a 75 kd species, known as P75, which contains the coat protein sequence at its N-terminus. Immunoblotting experiments with a P75-specific antiserum showed that P75 is present in only trace amounts in purified virus preparations. Electron microscopic visualization of immunogold-labelled virions in crude tissue extracts has provided evidence for an association between P75 and at least a fraction of the BNYVV particles, with P75 being predominantly located near one end of the rod-shaped virions. This finding is discussed in the context of the current model for the role of P75 in virus assembly and vector transmission.

Capsid↗

PubMed1994

Complete nucleotide sequence of peanut clump virus RNA 1 and relationships with other fungus-transmitted rod-shaped viruses.

The complete nucleotide sequence of RNA 1 of the tentative furovirus peanut clump virus (PCV) has been determined by characterization of cloned cDNA and by direct RNA sequencing. The sequence is 5897 nucleotides in length and contains three long open reading frames (ORFs). The 5'-terminal proximal ORF has the potential to encode a polypeptide of M(r) 130942 (P131) containing methyltransferase and RNA helicase homologous domains and displaying homology with large nonstructural proteins of alpha-like viruses, which are known or thought to be involved in virus replication. The P131 ORF is followed in-frame by a second ORF which is probably expressed by partial readthrough of the UGA termination codon of the P131 ORF to produce a polypeptide of M(r) 191044 (P191). The readthrough region of P191 contains the characteristic 'core' RNA polymerase motif, indicating that the PCV replicase proteins are expressed as a pair of overlapping proteins as in the tobamoviruses, tobraviruses and the furovirus soil-borne wheat mosaic virus (SBWMV). Sequence comparisons indicate that P131 and P191 are most closely related to the replicase proteins of SBWMV and the hordeivirus barley stripe mosaic virus (BSMV) but are only distantly related to the replicase of the furovirus beet necrotic yellow vein virus (BNYVV). The 3'-terminal proximal ORF can encode a putative polypeptide of M(r) 14556 (P15) which displays homology to small cysteine-rich proteins of hordeiviruses and SBWMV. We have corrected four errors in the sequence of PCV RNA 2 published previously by Manohar et al. (Virology 195, 33-41, 1993). One of these changes causes two small ORFs near the 3' terminus of RNA 2 to be fused together to create an ORF for a putative polypeptide of M(r) 16833 (P17) which displays extensive homology with the third protein of the triple gene block of BSMV RNA beta.

Amino Acid Sequence↗

PubMed1993

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.

Base Sequence↗

PubMed1993

Nucleotide sequence and genetic organization of peanut clump virus RNA 2 and partial characterization of deleted forms.

The complete nucleotide sequence of peanut clump virus RNA 2 has been determined by characterization of cloned cDNA and by direct RNA sequencing. The sequence is 4503 residues in length and contains six long open reading frames (ORFs). The cistron for the coat protein (ORF 1) is located nearest the 5' end of the RNA and is separated from the 5' extremity by a noncoding region of 389 nucleotides. ORF 2 starts one nucleotide upstream of the first residue of the UGA termination codon of the coat protein cistron and encodes a polypeptide of 39300 M(r). Four additional ORFs with the potential to encode polypeptides with approximate M(r)'s of 51,000 (ORF 3), 13,800 (ORF 4), 9400 (ORF 5), and 6800 (ORF 6) are found on the 3' proximal half of the RNA. ORFs 3, 4, and 5 resemble the Triple Gene Block found in potexviruses, carlaviruses, a hordeivirus, and a furovirus. The 276 nucleotide 3' noncoding region of RNA 2 displays extensive sequence homology with the 3' noncoding region of RNA 1. Several peanut clump virus isolates propagated on Nicotiana benthamania contain RNA 2's significantly shorter than that of the type isolate. Partial characterization of two such isolates reveals that their RNA 2's have undergone deletions within ORF 2.

Amino Acid Sequence↗

PubMed1993

Identification of beet western yellows luteovirus genes implicated in viral replication and particle morphogenesis.

The roles in replication and viral assembly of different beet western yellows luteovirus gene products were investigated in Chenopodium quinoa protoplasts using mutated transcripts. Of the six long open reading frames (ORFs) present on the viral RNA, only ORFs 2 and 3, which encode proteins containing conserved putative replicase domains, were essential for replication. Various deletions in the 3' part of the genome within ORFs 4, 5, and 6 did not affect viral replication. Analysis of the progeny of those mutants capable of replication showed that virus particles were produced in protoplasts infected with transcripts modified in ORFs 1, 5, or 6 but not with transcripts unable to produce coat protein, encoded by ORF 4.

Base Sequence↗

PubMed1993

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.

Base Sequence↗

PubMed1992

Agroinfection as an alternative to insects for infecting plants with beet western yellows luteovirus.

Beet western yellows luteovirus, like other luteoviruses, cannot be transmitted to host plants by mechanical inoculation but requires an aphid vector, a feature that has heretofore presented a serious obstacle to the study of such viruses. In this paper we describe use of agroinfection to infect hosts with beet western yellows virus without recourse to aphids. Agroinfection is a procedure for introducing a plant virus into a host via Agrobacterium tumefaciens harboring a Ti plasmid, which can efficiently transfer a portion of the plasmid (T-DNA) to plant cells near a wound. The viral genome must be inserted into the T-DNA in such a way that it can escape and begin autonomous replication, a requirement that has, so far, limited agroinfection to pathogens with a circular genome. We have cloned cDNA corresponding to the complete beet western yellows virus RNA genome between the cauliflower mosaic virus 35S promoter and the nopaline synthase transcription termination signal. In one construct, a self-cleaving (ribozyme) sequence was included so as to produce a transcript in planta with a 3' extremity almost identical to natural viral RNA. When inoculated mechanically to host plants, the naked plasmid DNA was not infectious but, when introduced into T-DNA and agroinfected to plants, both the construct with and without the ribozyme produced an infection. This approach should be applicable to virtually any plant virus with a linear plus-strand RNA genome.

Animals↗

PubMed1992

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.

Base Sequence↗

PubMed1992

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.

Amino Acid Sequence↗

PubMed1992

Synthesis of full-length transcripts of beet western yellows virus RNA: messenger properties and biological activity in protoplasts.

Full-length cDNA of beet western yellows virus genomic RNA has been cloned behind the bacteriophage T7 RNA polymerase promoter of the transcription vector BS(-). The in vitro run-off transcription product obtained in the presence of T7 RNA polymerase and m7GpppG cap has the same messenger properties as natural viral RNA in in vitro translation systems. The full-length transcript was also able to infect Chenopodium quinoa protoplasts inoculated by electroporation. Infection could be followed by the appearance of viral coat protein in the inoculated protoplasts and the de novo synthesis of viral RNA. Site-directed mutagenesis experiments revealed that expression of beet western yellows virus open reading frame 1 and the C-terminal portion of open reading frame 6 were not required for infection of protoplasts. Additional experiments with these mutants and mutants in the other viral open reading frames should provide information concerning the requirements for beet western yellows virus replication and, ultimately, the role of virus genes in other important steps in the virus infection cycle, such as aphid transmission.

Base Sequence↗

PubMed1992

Efficient cell-to-cell movement of beet necrotic yellow vein virus requires 3' proximal genes located on RNA 2.

RNA 2 of beet necrotic yellow vein virus (BNYVV) carries six open reading frames. The four 3' proximal frames encode the proteins P42, P13, P15, and P14. The first three species present homologies to proteins encoded by three overlapping open reading frames (the triple gene block) in potexviruses, carlaviruses, and barley stripe mosaic virus. P14 does not display homology with other known plant viral proteins. The functions of P42, P13, P15, and P14 were investigated by site-directed mutagenesis. Full-length transcripts of wild-type BNYVV RNAs 1 and 2 were infectious when coinoculated to protoplasts or leaves of Chenopodium quinoa. RNA 2 transcripts in which P42, P13, and P15 were prematurely terminated by frameshift mutations replicated in protoplasts (when inoculated with wild-type RNA 1) but were not infectious to leaves, indicating that the triple gene block proteins of BNYVV are essential for viral cell-to-cell spread. Mutations in P14 were not lethal in leaf infections but smaller local lesions and lesser amounts of viral RNA were produced. RNA 2-related subgenomic RNA species of 2.6, 1.4, and 0.7 kb were detected; they presumably direct synthesis of P42, P13, and P14. No species of the length predicted for a P15-specific subgenomic RNA was detected.

Base Sequence↗

PubMed1992

cis-active sequences near the 5'-termini of beet necrotic yellow vein virus RNAs 3 and 4.

RNAs 3 and 4 of the multicomponent genome of beet necrotic yellow vein virus are dispensable for infection of Chenopodium quinoa leaves. We have used mutagenesis of biologically active RNA 3 transcripts to identify 5'-proximal sequences essential in cis for RNA 3 amplification. One such element, Box I, (nucleotides 283-292) was complementary to the first 10 residues (Box I') following the 5'-terminal cap. A second cis-active element (Box II) was identified between nucleotides 237-244 and was complementary to nucleotides 16-23 (Box II'). Other cis-active sequences exist between Box II' and II but have not been mapped to fine scale. Most sequence substitutions in Boxes I and II or in the 5'-proximal complementary sequences were lethal but compensatory mutations designed to restore Box I/I' or Box II/II' base pairing restored viability, suggesting that secondary structure involving these elements rather than their exact sequence is the critical feature. Transcripts bearing short deletions near residue 200 were replicated but did not assemble into virions, indicating that this region contains or contributes to a cis-active encapsidation signal. Similar experiments with RNA 4 transcript have shown that 5'-proximal cis-essential elements are limited to the first 400 residues of this RNA. Essential subdomains within this region have not been mapped but there are no structures obviously homologous to Boxes I/I' and II/II' of RNA 3.

Base Sequence↗

PubMed1991

Shortened forms of beet necrotic yellow vein virus RNA-3 and -4: internal deletions and a subgenomic RNA.

Beet necrotic yellow vein virus RNA-3 and RNA-4, produced as full-length biologically active transcripts in vitro, can undergo spontaneous internal deletions when inoculated onto Chenopodium quinoa leaves along with RNA-1 and -2. The deletion process is specific, giving rise to only a few major species, and can be rapid; deleted forms appear after only one or two passages in leaves. In one of the shortened forms of RNA-4, the deletion precisely eliminated one copy of a 15 nucleotide (nt) direct sequence repeat from the full-length prototype sequence, suggesting that 'copy-choice' switching of the replicase-template complex from one repeat to the other during RNA replication was responsible for the generation of this deletion. The deletion found in a major shortened form of RNA-3, on the other hand, did not occur near sequence repeats but began with GU and ended with AG like a nuclear intron sequence. Thus it is possible that the deleted sequence has been removed by splicing. However, two other deletions that were characterized were not associated with either of these types of sequence feature. An approximately 600 nt 5'-terminally truncated non-encapsidated form of RNA-3 was also detected in infected plant tissue. The evidence suggests that it is a subgenomic RNA derived from RNA-3.

Base Sequence↗

PubMed1990

Immunodetection in vivo of beet necrotic yellow vein virus-encoded proteins.

Open reading frames identified on the four genomic RNAs of beet necrotic yellow vein virus were cloned into bacterial expression vectors and resulting cl-fusion proteins expressed in Escherichia coli were used to raise polyclonal antibodies. This set of antisera was used to show the presence of 7 of 9 predicted viral proteins in mechanically inoculated Chenopodium quinoa leaves by the Western blot technique. Viral coat protein (p22) and its readthrough protein p85 encoded by RNA-2 could be detected in all subcellular fractions. Two other RNA-2-encoded proteins, p42 and p13, are predominantly associated with membranous structures. Another RNA-2-encoded protein, p14, as well as the two polypeptides p25 and p31, encoded by RNA-3 and -4, respectively, are soluble proteins. The viral proteins could first be detected about the time lesions became visible and increased thereafter except for p85, in which case the amount of the soluble form decreased with time. No protein could be detected corresponding to the RNA-1-encoded p237 protein or to the p15 species encoded by open reading frame V of RNA-2.

Antibodies, Viral↗