Biomedical subjects

S Bouzoubaa

Publications and source records attributed to S Bouzoubaa.

12 recordsLinked to original sources

PubMed2005

Subcellular localization of the Triple Gene Block movement proteins of Beet necrotic yellow vein virus by electron microscopy.

The Triple Gene Block proteins TGBp1, TGBp2, and TGBp3 of Beet necrotic yellow vein virus (BNYVV) are required for efficient cell-to-cell spread of the infection. The TGB proteins can drive cell-to-cell movement of BNYVV in trans when expressed from a co-inoculated BNYVV RNA 3-based 'replicon'. TGBp2 and TGBp3 expressed from the replicon were nonfunctional in this assay if they were fused to the green fluorescent protein (GFP), but addition of a hemagglutinin (HA) tag to their C-termini did not incapacitate movement. Immunogold labeling of ultrathin sections treated with HA-specific antibodies localized TGBp2-HA and TGBp3-HA to what are probably structurally modified plasmodesmata (Pd) in infected cells. A similar subcellular localization was observed for TGBp1. Large gold-decorated membrane-rich bodies containing what appear to be short fragments of endoplasmic reticulum were observed near the cell periphery. The modified gold-decorated Pd and the membrane-rich bodies were not observed when the TGB proteins were produced individually in infections using the Tobacco mosaic virus P30 protein to drive cell-to-cell movement, indicating that these modifications are specific for TGB-mediated movement.

Beta vulgaris↗

PubMed2001

Beet necrotic yellow vein virus particles localize to mitochondria during infection.

Fluorescent beet necrotic yellow vein virus (BNYVV) particles were produced by replacing part of the readthrough domain of the minor coat protein P75 with the green fluorescent protein (GFP). The recombinant virus was functional in plants and P75-GFP was incorporated at one end of the rod-shaped virions. Laser scanning confocal microscopy and transmission electron microscopy showed that virus-like particles, almost certainly authentic BNYVV virions, localized to the cytoplasmic surface of mitochondria at early times postinfection but relocated at later times to semiordered clusters in the cytoplasm. This is the first report of specific targeting of plant virus particles to the mitochondria in vivo.

Capsid↗

PubMed2000

P42 movement protein of Beet necrotic yellow vein virus is targeted by the movement proteins P13 and P15 to punctate bodies associated with plasmodesmata.

Cell-to-cell movement of Beet necrotic yellow vein virus (BNYVV) is driven by a set of three movement proteins--P42, P13, and P15--organized into a triple gene block (TGB) on viral RNA 2. The first TGB protein, P42, has been fused to the green fluorescent protein (GFP) and fusion proteins between P42 and GFP were expressed from a BNYVV RNA 3-based replicon during virus infection. GFP-P42, in which the GFP was fused to the P42 N terminus, could drive viral cell-to-cell movement when the copy of the P42 gene on RNA 2 was disabled but the C-terminal fusion P42-GFP could not. Confocal microscopy of epidermal cells of Chenopodium quinoa near the leading edge of the infection revealed that GFP-P42 localized to punctate bodies apposed to the cell wall whereas free GFP, expressed from the replicon, was distributed uniformly throughout the cytoplasm. The punctate bodies sometimes appeared to traverse the cell wall or to form pairs of disconnected bodies on each side. The punctate bodies co-localized with callose, indicating that they are associated with plasmodesmata-rich regions such as pit fields. Point mutations in P42 that inhibited its ability to drive cell-to-cell movement also inhibited GFP-P42 punctate body formation. GFP-P42 punctate body formation was dependent on expression of P13 and P15 during the infection, indicating that these proteins act together or sequentially to localize P42 to the plasmodesmata.

Amino Acid Sequence↗

PubMed1999

The first triple gene block protein of peanut clump virus localizes to the plasmodesmata during virus infection.

The subcellular localization of the first triple gene block protein (TGBp1) of peanut clump pecluvirus (PCV) was studied by subcellular fractionation and immunogold cytochemistry using TGBp1-specific antibodies raised against a fusion protein expressed in and purified from bacteria. In the inoculated and apical leaves of virus-infected Nicotiana benthamiana, TGBp1 localized to the cell wall and P30 fractions. Electron microscopy of immunogold-decorated ultrathin sections of the infected leaf tissue revealed TGBp1-specific labeling of the plasmodesmata joining mesophyll cells. In longitudinal sections of the plasmodesmata, the TGBp1-specific labeling was most commonly associated with the plasmodesmal collar region. In transgenic N. benthamiana, which constitutively expressed TGBp1, no TGBp1-specific immunogold labeling of plasmodesmata was observed, but plasmodesmata were gold decorated when the transgenic plants were infected with a TGBp1-defective PCV mutant, indicating that factors induced by the virus infection target and/or anchor the transgene TGBp1 to the plasmodesmata.

Arachis↗

PubMed1998

Identification of genes involved in replication and movement of peanut clump virus.

The genome of peanut clump pecluvirus (PCV) consists of two messenger RNA components which contain, respectively, three and five open reading frames (ORFs). Inoculation of transcripts from full-length cDNA clones derived from the PCV RNAs showed that RNA-1 is able to replicate in the absence of RNA-2 in protoplasts, but both RNAs are necessary for plant infection. To investigate the role of different gene products in viral RNA replication and movement, transcripts from mutant cDNA clones were inoculated to protoplasts and to Chenopodium quinoa or Nicotiana benthamiana plants, and progeny RNA was detected by Northern blot analysis. The protein P15, encoded by the third ORF of RNA-1, is essential for efficient replication of the viral genome. The three proteins, P51, P14, and P17, of the triple gene block contained in RNA-2 are involved in localized movement of the viral genome, whereas the coat protein (P23) is also required for vascular movement. Insertion of the beta-glucuronidase reporter gene (GUS) in place of the P23 or P39 genes (the first and the second genes of RNA-2) allows visualization of the virus infection in inoculated leaves. Although the presence of the GUS gene resulted in a lower accumulation of progeny RNA and, despite instability of the construct in planta, histochemical detection of PCV multiplication was more sensitive than Northern blot detection.

Arachis↗

PubMed1998

Cell-to-cell movement of beet necrotic yellow vein virus: I. Heterologous complementation experiments provide evidence for specific interactions among the triple gene block proteins.

Cell-to-cell movement of beet necrotic yellow vein virus (BNYVV) requires three proteins encoded by a triple gene block (TGB) on viral RNA 2. A BNYVV RNA 3-derived replicon was used to express movement proteins to functionally substitute for the BNYVV TGB proteins was tested by coinoculation of TGB-defective BNYVV with the various replicons to Chenopodium quinoa. Trans-heterocomplementation was successful with the movement protein (P30) of tobacco mosaic virus but not with the tubule-forming movement proteins of alfalfa mosaic virus and grapevine fanleaf virus. Trans-complementation of BNYVV movement was also observed when all three TGB proteins of the distantly related peanut clump virus were supplied together but not when they were substituted for their BNYVV counterparts one by one. When P30 was used to drive BNYVV movement in trans, accumulation of the first TGB protein of BNYVV was adversely affected by null mutations in the second and third TGB proteins. Taken together, these results suggest that highly specific interactions among cognate TGB proteins are important for their function and/or stability in planta.

Genes, Viral↗

PubMed1995

The small cysteine-rich protein P14 of beet necrotic yellow vein virus regulates accumulation of RNA 2 in cis and coat protein in trans.

The effect of null mutations of the small cysteine-rich protein P14 encoded by RNA 2 of beet necrotic yellow vein virus has been investigated using in vitro transcripts of viral RNA to infect Chenopodium quinoa protoplasts. The P14 mutations down-regulated RNA 2 accumulation by approximately 10- to 50-fold. Accumulation of minus-strand RNA 2 was also diminished but RNA 1 accumulation was much less affected. The inhibition of RNA 2 accumulation could not be complemented in trans by providing P14 from another source (either a second molecule of RNA 2 or an RNA 3-based replicon) containing and expressing the P14 gene. The P14 null mutations dramatically inhibited accumulation of viral coat protein, which is encoded by the 5'-proximal gene on RNA 2, but this effect could be complemented in trans, indicating that it occurs by a mechanism distinct from that affecting RNA 2 accumulation. Transient expression experiments were also carried out in which a plasmid expressing P14 and plasmids expressing a reporter gene placed downstream of potential translational control sequences (the 5'-noncoding sequences of RNAs 2, 3, or 4) were introduced into C. quinoa or Nicotiana tabacum leaves by microprojectile bombardment. Coexpression of P14 produced a 3- to 4-fold stimulation of reporter gene expression levels for all the constructs. The lack of sequence specificity suggests that this phenomenon is not directly related to the RNA 2-specific stimulation of coat protein accumulation observed in a viral infection.

Base Sequence↗

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↗

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↗

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

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.

Base Composition↗

PubMed1985

Chimeric vector construction for higher-plant transformation.

A chimeric vector pKR612B1 was developed containing the neomycin phosphotransferase (APH) gene from the Tn5 transposon under the control of the gene VI promoter of cauliflower mosaic virus (CaMV), and was used to transform higher plant protoplasts. Plasmid pDOB612, the parental vector of pKR612B1, has two unique restriction sites, SmaI and BamHI, positioned just downstream of the CaMV gene VI promoter sequence. These unique cloning sites can be used for any kind of gene insertion into this vector. Using the polyethylene glycol transformation procedure, a large number of turnip and tobacco protoplasts were transformed and proved to be resistant to kanamycin (Km). From tobacco protoplasts whole Km-resistant plants were regenerated and shown to contain the integrated foreign gene. APH activity was detected in both transformed calli and in regenerated plants. DNA from transformed clones was analysed by Southern blot hybridization, showing the presence of the Tn5-derived gene.

Chimera↗