Banana bunchy top nanovirus DNA-1 encodes the 'master' replication initiation protein.
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Banana bunchy top nanovirus has a multicomponent, circular single-stranded DNA genome comprising at least six integral components, BBTV DNA-1 to -6, which have been consistently associated with bunchy top disease worldwide. At least three other components, BBTV S1, S2 and Y, which have been isolated from Taiwanese BBTV isolates, do not appear to be integral components. We show here that both BBTV DNA-1 and S1, which encode replication initiation (Rep) proteins, were capable of self-replication when bombarded into banana embryogenic cell suspensions. However, only BBTV DNA-1 was capable of directing the replication of two other BBTV genomic components, namely BBTV DNA-3 which encodes the coat protein, and DNA-5 which encodes a retinoblastoma binding-like protein. These results indicate that (i) BBTV DNA-1 is the minimal replicative unit of BBTV and encodes the 'master' viral Rep and (ii) BBTV S1 is possibly a satellite DNA which is unable to replicate integral BBTV components. (+info)
Tobravirus 2b protein acts in trans to facilitate transmission by nematodes.
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Analysis of RNA2 of TRV PaY4 showed it to be recombinant, carrying 3'-terminal sequences derived from RNA1. Virus produced using an infectious cDNA clone of PaY4 RNA2 was nematode transmissible, demonstrating that natural TRV recombinant isolates are not necessarily defective. Mutations introduced into PaY4 RNA2 showed that the 2b gene, but not the 2c gene, is required for transmission by both Paratrichodorus pachydermus and P. anemones nematodes. Experiments examined whether infection of plants with two different virus clones would impact upon nematode transmission of either virus. Simultaneous inoculation with TRV clones expressing green or red fluorescent proteins revealed that mixing of the two virus populations did not occur, although, in roots, adjacent cells were found containing green- or red-tagged viruses. Subsequently, in similar experiments it was found that a TRV PaY4 2b mutant was transmitted when combined with wild-type TRV PaY4. Also, transmission of a 2b mutant of an in vitro TRV/PEBV recombinant virus (TRV-C1) occurred after coinfection with wild-type virus. Thus, the tobravirus 2b transmission protein is trans-acting. Although TRV PaY4 and TRV PpK20 are both transmitted by P. pachydermus, a 2b mutant of TRV PaY4 was not transmitted when coinoculated to plants with TRV PpK20. (+info)
A novel strategy for the expression of foreign genes from plant virus vectors.
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Potato virus X (PVX)-based vector constructs were generated to investigate the use of an internal ribosome entry site (IRES) sequence to direct translation of a viral gene. The 148-nucleotide IREScp sequence from a crucifer-infecting strain of tobacco mosaic virus was used to direct expression of the PVX coat protein (CP). The IRES was inserted downstream of the gene encoding green fluorescent protein (GFP) and upstream of the PVX CP, in either sense or antisense orientation, such that CP expression depended on ribosome recruitment to the IRES. Stem-loop structures were inserted at either the 3'- or 5'-end of the IRES sequence to investigate its mode of action. In vitro RNA transcripts were inoculated to Nicotiana benthamiana plants and protoplasts: levels of GFP and CP expression were analysed by enzyme-linked immunosorbent assay and the rate of virus cell-to-cell movement was determined by confocal laser scanning microscope imaging of GFP expression. PVX CP was expressed, allowing cell-to-cell movement of virus, from constructs containing the IRES sequence in either orientation, and from the construct containing a stem-loop structure at the 5'-end of the IRES sequence. No CP was expressed from a construct containing a stem-loop at the 3'-end of the IRES sequence. Our results suggest that the IRES sequence is acting in vivo to direct expression of the 3'-proximal open reading frame in a bicistronic mRNA thereby demonstrating the potential of employing IRES sequences for the expression of foreign proteins from plant virus-based vectors. (+info)
Radioactive labelling of borme mosaic virus.
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Brome mosaic virus (BMV) can be radioactively labelled by allowing virus synthesis to take place in barley leaves whose cut ends are placed in solutions of radio active precursors of BMV. When barley plants are infected by inoculation of the first emerging leaf, the rate of incorporation of both [14C]-leucine and [32P]-ortho-phosphate into virus particles was found to be maximal in the second leaf, cut and labelled 4 to 5 days after inoculation. The rate of incorporation declined when the labelling or the cutting and labelling were delayed, even though assembly of particles still took place from pre-formed components. An exposure of 72 h to an appropriate amount of 32P yielded highly radioactive BMV RNA for possible sequencing studies. A specific radioactivity (32P) of the order of 1 mCi/mg RNA can be obtained readily. (+info)
HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal.
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Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation mechanism that is widespread in eukaryotic organisms. It is often associated with methylation of the transcribed region of the silenced gene and with accumulation of small RNAs (21 to 25 nucleotides) homologous to the silenced gene. In plants, PTGS can be triggered locally and then spread throughout the organism via a mobile signal that can cross a graft junction. Previously, we showed that the helper component-proteinase (HC-Pro) of plant potyviruses suppresses PTGS. Here, we report that plants in which PTGS has been suppressed by HC-Pro fail to accumulate the small RNAs associated with silencing. However, the transgene locus of these plants remains methylated. Grafting experiments indicate that HC-Pro prevents the plant from responding to the mobile silencing signal but does not eliminate its ability to produce or send the signal. These results demonstrate that HC-Pro functions downstream of transgene methylation and the mobile signal at a step preceding accumulation of the small RNAs. (+info)
Assembly of a rod-shaped virus. Disk aggregate of cucumber green mottle mosaic virus protein and its function.
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Although it is established that in the assembly of tobacco mosaic virus (TMV) in vitro, a 20 S disk aggregate of protein is essential for the initiation of the reaction, there is no agreement as to whether subsequent rod elongation proceeds by the addition of protein subunits or disks. Cucumber green mottle mosaic virus (CGMMV) is a rod-shaped virus closely related to TMV. It was observed directly by electron microscopy that CGMMV-protein also formed a single- or double-layer disk aggregate under conditions where reconstitution with the protein and TMV-RNA proceeds efficiently. Whichever forms, a single or double layer, a disk aggregate is required for the initiation of reconstitution, but cannot participate in rod elongation. These results obtained with CGMMV support our model proposed for the mechanism of assembly of TMV and rod-shaped viruses; i.e., the assembly occurs in two steps; disk aggregates of protein are essential only for initiation, and rod eleongation proceeds by subsequent addition of subunits. (+info)
Molecular characterization of the genome of Maize rayado fino virus, the type member of the genus Marafivirus.
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The complete nucleotide sequence of the single-stranded RNA genome of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus, is 6305 nucleotides (nts) in length and contains two putative open reading frames (ORFs). The largest ORF (nt 97-6180) encodes a polyprotein of 224 kDa with sequence similarities at its N-terminus to the replication-associated proteins of other viruses with positive-strand RNA genomes and to the papainlike protease domain found in tymoviruses. The C-terminus of the 224-kDa ORF also encodes the MRFV capsid protein. A smaller, overlapping ORF (nt 302-1561) encodes a putative protein of 43 kDa with unknown function but with limited sequence similarities to putative movement proteins of tymoviruses. The nucleotide sequence and proposed genome expression strategy of MRFV is most closely related to that of oat blue dwarf virus (OBDV). Unlike OBDV, MRFV RNA does not appear to contain a poly(A) tail, and it encodes a putative second overlapping open reading frame. (+info)
SDE3 encodes an RNA helicase required for post-transcriptional gene silencing in Arabidopsis.
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Post-transcriptional gene silencing (PTGS) provides protection in plants against virus infection and can suppress expression of transgenes. Arabidopsis plants carrying mutations at the SDE3 locus are defective in PTGS mediated by a green fluorescent protein transgene. However, PTGS mediated by tobacco rattle virus (TRV) was not affected by sde3. From these results we conclude that SDE3, like the previously described RNA polymerase encoded by SDE1, acts at a stage in the mechanism that is circumvented when PTGS is mediated by TRV. The product of SDE3 is similar to RNA helicase-like proteins including GB110 in mouse and other proteins in Drosophila and humans. These proteins are similar to, but clearly distinct from Upf1p and SMG-2, which are required for nonsense-mediated mRNA decay in yeast and Caenorhabditis elegans and, in the case of SMG-2, for PTGS. (+info)