Enhanced competitiveness of tomato bushy stunt virus defective interfering RNAs by segment duplication or nucleotide insertion.

We have analyzed atypical tomato bushy stunt virus defective interfering (DI) RNA species which accumulated during a passage series in protoplasts. We present a rationale for the order of appearance of these molecules and show, using competition assays, that either segment duplication or single nucleotide insertion can enhance DI RNA competitiveness. Possible mechanisms for the introduction of the modifications observed in these DI RNAs are discussed.     

An internally located RNA hairpin enhances replication of Tomato bushy stunt virus RNAs

Defective interfering (DI) RNAs of Tomato bushy stunt virus (TBSV), a plus-sense RNA virus, comprise four conserved noncontiguous regions (I through IV) derived from the viral genome. Region III, a 70-nucleotide-long sequence corresponding to a genomic segment located 378 nucleotides upstream of the 3' terminus of the genome, has been found to enhance DI RNA accumulation by approximately 10-fold in an orientation-independent manner (D. Ray and K. A. White, Virology 256:162-171, 1999). In this study, a more detailed structure-function analysis of region III was conducted. RNA secondary-structure analyses indicated that region III contains stem-loop structures in both plus and minus strands. Through deletion analyses of a DI RNA, a primary determinant of region III activity was mapped to the 5'-proximal 35-nucleotide segment. Compensatory-type mutational analyses showed that a stem-loop structure in the minus strand of this subregion was required for enhanced DI RNA replication. The same stem-loop structure was also found to function in a position-independent manner in a DI RNA (albeit at reduced levels) and to be important for efficient accumulation within the context of the TBSV genome. Taken together, these observations suggest that the 5'-proximal segment of region III is a modular RNA replication element that functions primarily through the formation of an RNA hairpin structure in the minus strand.     

Integrity of nonviral fragments in recombinant Tomato bushy stunt virus and defective interfering RNA is influenced by silencing and the type of inserts

Recombinant plant viruses have the propensity to remove foreign inserts during replication. This process is virus-specific and occurs in a host-dependent manner. In the present study, we investigated the integrity of foreign inserts in recombinant plant viruses using a model system consisting of Tomato bushy stunt virus (TBSV) and its defective interfering RNA (DI). These were tested in Nicotiana benthamiana plants that were either wild type or transgenic for the green fluorescent protein (GFP) gene. GFP-derived inserts were retained in the recombinant TBSV and DI population that were inoculated onto GFP-transgenic N. benthamiana plants in which silencing of the GFP transgene was initiated, but they were removed from the virus and DIs that were maintained on wild-type plants. A foreign insert derived from an endogenous N. benthamiana gene encoding the H subunit of the magnesium chelatase (NbChlH) was deleted, whereas the fragment of an RNA-dependent RNA polymerase gene (NbRdRP1m) was retained in the recombinant TBSV population. These results demonstrate that the recombination of TBSV to remove nonviral fragments is influenced by silencing and the type of inserts.     

番茄丛矮病毒的分子生物学研究进展

近年来,多种植物RNA病毒载体被广泛地应用于外源基因的表达、植物病毒学和植物病理学基础理论的研究中.番茄丛矮病毒(Tomato bushy stunt virus,TBSV)是番茄丛矮病毒科(Tombusviridae)番茄丛矮病毒属(Tombusvirus)的典型成员.TBSV病毒基因组复制、转录和翻译等分子机制的研究取得了巨大的进展,使得利用TBSV构建稳定、高效的表达载体成为可能.     

Tomato bushy stunt virus (TBSV) infecting Lycopersicon esculentum

Tomato bushy stunt virus (TBSV) was detected in tomato crop (Lycopersicon esculentum) in Egypt with characteristic mosaic leaf deformation, stunting, and bushy growth symptoms. TBSV infection was confirmed serologically by ELISA and calculated incidence was 25.5%. Basic physicochemical properties of a purified TBSV Egh isolate were identical to known properties of tombusviruses of isometric 30-nm diameter particles, 41-kDa coat protein and the genome of approximately 4800 nt. This is the first TBSV isolate reported in Egypt. Cloning and partial sequencing of the isolate showed that it is more closely related to TBSV-P and TBSV-Ch than TBSV-Nf and TBSV-S strains of the virus. However, it is distinct from the above strains and could be a new strain of the virus which further confirms the genetic diversity of tombusviruses.     

A plant derived multifunctional tool for nanobiotechnology based on Tomato bushy stunt virus

Structure, size, physicochemical properties and production strategies make many plant viruses ideal protein based nanoscaffolds, nanocontainers and nano-building blocks expected to deliver a multitude of applications in different fields such as biomedicine, pharmaceutical chemistry, separation science, catalytic chemistry, crop pest control and biomaterials science. Functionalization of viral nanoparticles through modification by design of their external and internal surfaces is essential to fully exploit the potentiality of these objects. In the present paper we describe the development of a plant derived multifunctional tool for nanobiotechnology based on Tomato bushy stunt virus. We demonstrate the ability of this system to remarkably sustain genetic modifications and in vitro chemical derivatizations of its outer surface, which resulted in the successful display of large chimeric peptides fusions and small chemical molecules, respectively. Moreover, we have defined physicochemical conditions for viral swelling and reversible viral pore gating that we have successfully employed for foreign molecules loading and retention in the inner cavity of this plant virus nanoparticles system. Finally, a production and purification strategy from Nicotiana benthamiana plants has been addressed and optimized.     

Authentic replication and recombination of Tomato bushy stunt virus RNA in a cell-free extract from yeast

To study the replication of Tomato bushy stunt virus (TBSV), a small tombusvirus of plants, we have developed a cell-free system based on a Saccharomyces cerevisiae extract. The cell-free system was capable of performing a complete replication cycle on added plus-stranded TBSV replicon RNA (repRNA) that led to the production of approximately 30-fold-more plus-stranded progeny RNAs than the minus-stranded replication intermediate. The cell-free system also replicated the full-length TBSV genomic RNA, which resulted in production of subgenomic RNAs as well. The cell-free system showed high template specificity, since a mutated repRNA, minus-stranded repRNA, or a heterologous viral RNA could not be used as templates by the tombusvirus replicase. Similar to the in vivo situation, replication of the TBSV replicon RNA took place in a membraneous fraction, in which the viral replicase-RNA complex was RNase and protease resistant but sensitive to detergents. In addition to faithfully replicating the TBSV replicon RNA, the cell-free system was also capable of generating TBSV RNA recombinants with high efficiency. Altogether, tombusvirus replicase in the cell-free system showed features remarkably similar to those of the in vivo replicase, including carrying out a complete cycle of replication, high template specificity, and the ability to recombine efficiently.     

Structure of the intracellular defective viral RNAs of defective interfering particles of mouse hepatitis virus.

The intracellular defective RNAs generated during high-multiplicity serial passages of mouse hepatitis virus JHM strain on DBT cells were examined. Seven novel species of single-stranded polyadenylic acid-containing defective RNAs were identified from passages 3 through 22. The largest of these RNAs, DIssA (molecular weight [mw], 5.2 X 10(6)), is identical to the genomic RNA packaged in the defective interfering particles produced from these cells. Other RNA species, DIssB1 (mw, 1.9 X 10(6) to 1.6 X 10(6)), DIssB2 (mw, 1.6 X 10(6)), DIssC (mw, 2.8 X 10(6)) DIssD (mw, 0.82 X 10(6)), DIssE (mw, 0.78 X 10(6)), and DIssF (mw, 1.3 X 10(6)) were detected at different passage levels. RNase T1-resistant oligonucleotide fingerprinting demonstrated that all these RNAs were related and had multiple deletions of the genomic sequences. They contained different subsets of the genomic sequences from those of the standard intracellular mRNAs of nondefective mouse hepatitis virus JHM strain. Thus these novel intracellular viral RNAs were identified as defective interfering RNAs of mouse hepatitis virus JHM strain. The synthesis of six of the seven normal mRNA species specific to mouse hepatitis virus JHM strain was completely inhibited when cells were infected with viruses of late-passage levels. However, the synthesis of RNA7 and its product, viral nucleoprotein, was not significantly altered in late passages. The possible mechanism for the generation of defective interfering RNAs was discussed.     

On the rigidity of RNA in tomato bushy stunt virus

The motional state of RNA in tomato bushy stunt virus, both in the crystalline state and in solution, has been investigated using ³¹P nuclear magnetic resonance methods. It has been found that the RNA is highly immobile in the native virus and it is suggested that the lack of a high-resolution X-ray diffraction pattern for either the RNA or the N-terminal regions of the protein coat molecules (Harrison et al., 1978) is due to static disorder in the crystals. Dynamic disorder has been detected in the virus after treatment with EDTA, which causes a structural change and an increase in particle size.     

Natural infection of sugar beet with tomato bushy stunt virus

A virus transmissible toChenopodium quinoa was isolated from leaves of sugar beet showing large chlorotic ring spots and line pattern. The virus was serologically unrelated to tobacco necrosis virus and tomato black ring virus or to its beet ringspot strain either. A positive result was obtained with antiserum against tomato bushy stunt virus. Reactions of herbaceous indicators and properties of the virus in crude sap were in accordance with the serological diagnosis. A survey of natural hosts of tomato bushy stunt virus demonstrated recently by the authors is given.     

Chloroplast alterations induced by tomato bushy stunt virus inDatura leaves

Summary Peculiar chloroplast alterations were found in mesophyll cells ofDatura stramonium systemically infected with tomato bushy stunt virus. These alterations lead to complete rearrangement of the thylakoids.     

Tomato bushy stunt virus co-opts the RNA-binding function of a host metabolic enzyme for viral genomic RNA synthesis

Tomato bushy stunt virus (TBSV), a plus-stranded [(+)] RNA plant virus, incorporates the host metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) into the viral replicase complex. Here, we show that, during TBSV replication in yeast, the yeast GAPDH Tdh2p moves from the cytosol to the peroxisomal membrane surface, the site of viral RNA synthesis. In yeast cells lacking Tdh2p, decreasing the levels of its functionally redundant homolog Tdh3p inhibited TBSV replication and resulted in equivalent levels of (+) and minus-stranded [(-)] viral RNA, in contrast to the hallmark excess of (+)RNA. Tdh2p specifically bound an AU pentamer sequence in the (-)RNA, suggesting that GAPDH promotes asymmetric RNA synthesis by selectively retaining the (-)RNA template in the replicase complex. Downregulation of GAPDH in a natural plant host decreased TBSV genomic RNA accumulation. Thus, TBSV co-opts the RNA-binding function of a metabolic protein, helping convert the host cell into a viral factory.     

Conserved regions in defective interfering viral double-stranded RNAs from a yeast virus.

We have completely sequenced a defective interfering viral double-stranded RNA (dsRNA) from the Saccharomyces cerevisiae virus. This RNA (S14) is a simple internal deletion of its parental dsRNA, M1, of 1.9 kilobases. The 5' 964 bases of the M1 plus strand encode the type 1 killer toxin of the yeast. S14 is 793 base pairs (bp) long, with 253 bp from the 5' region of its parental plus strand and 540 bp from the 3' region. All three defective interfering RNAs derived from M1 that have been characterized so far preserve a large 3' region, which includes five repeats of a rotationally symmetrical 11-bp consensus sequence. This 11-bp sequence is not present in the 5' 1 kilobase of the parental RNA or in any of the sequenced regions of unrelated yeast viral dsRNAs, but it is present in the 3' region of the plus strand of another yeast viral dsRNA, M2, that encodes the type 2 killer toxin. The 3' region of 550 bases of the M1 plus strand, previously only partially sequenced, reveals no large open reading frames. Hence only about half of M1 appears to have a coding function.