Difference in the mechanism of self-assembly in vitro in tobacco mosaic virus and potato virus X

The effects of 254 nm UV-irradiation of tobacco mosaic virus (TMV) and potato virus X (PVX) RNA preparations on the RNA ability to self-assembly in vitro with the viral coat proteins were studied. It was found that while TMV RNA ability to assemble with the homologous protein is rapidly inactivated by the UV-irradiation, PVX RNA ability to be encapsidated by the PVX coat protein is quite resistant to the irradiation. More than that, the irradiation of TMV RNA with the dose strongly inhibiting its assembly with the homologous protein, did not result in any significant inhibition of this RNA ability to be coated with the PVX protein. The results testify to the profound differences in the mechanisms of RNA-protein interactions in the processes of self-assembly in vitro of tobamoviruses and potexviruses.     

Identification of a peptide of the membrane protein of tobacco mosaic virus, cross-linked with intraviral RNA under the effect of UV-radiation

As reported previously, UV-irradiation induces crosslinking between tobacco mosaic virus (TMV) coat protein molecules and intraviral RNA nucleotides. We have irradiated [3H]-uridine labeled TMV and isolated TMV coat protein subunits with the attached nucleotide label. These TMV protein subunits were hydrolyzed with trypsin. The tryptic peptides were separated by high-performance liquid chromatography and [3H]-labeled peptides were identified. The UV-irradiation of TMV was found to result in crosslinking to intraviral RNA of the T8 tryptic peptide (residues 93-112) of TMV coat protein.     

The coat protein of potato virus X is a strain-specific elicitor of Rx1-mediated virus resistance in potato

The Rx1 gene in potato confers extreme resistance to potato virus X (PVX). To investigate the mechanism and elicitation of Rx resistance, protoplasts of potato cv. Cara (Rx1 genotype) and Maris Bard (rx1 genotype) were inoculated with PVX and tobacco mosaic virus (TMV). At 24 h post-inoculation in Maris Bard protoplasts there was at least 100-fold more PVX RNA than in protoplasts of Cara. TMV RNA accumulated to the same level in both types of protoplast. However, when the TMV was inoculated together with PVX the accumulation of TMV RNA was suppressed in the Cara (Rx1 genotype) protoplasts to the same extent as PVX. The Rx1 resistance also suppressed accumulation of a recombinant TMV in which the coat protein gene was replaced with the coat protein gene of PVX. It is therefore concluded that Rx1-mediated resistance is elicited by the PVX coat protein, independently of any other proteins encoded by PVX. The domain of the coat protein with elicitor activity was localized by deletion and mutation analysis to the structural core of a non-virion form of the coat protein.     

Live-cell imaging of viral RNA genomes using a Pumilio-based reporter

We describe a method for localizing plant viral RNAs in vivo using Pumilio, an RNA-binding protein, coupled to bimolecular fluorescence complementation (BiFC). Two Pumilio homology domain (PUMHD) polypeptides, fused to either the N- or C-terminal halves of split mCitrine, were engineered to recognize two closely adjacent eight-nucleotide sequences in the genomic RNA of tobacco mosaic virus (TMV). Binding of the PUMHDs to their target sites brought the split mCitrine halves into close proximity, allowing BiFC to occur and revealing the localization of viral RNA within infected cells. The bulk of the RNA was sequestered in characteristic inclusion bodies known as viral replication complexes (VRCs), with a second population of RNA localized in discrete particles distributed throughout the peripheral cytoplasm. Transfer of the TMV Pumilio recognition sequences into the genome of potato virus X (PVX) allowed the PVX RNA to be localized. Unlike TMV, the PVX RNA was concentrated in distinctive 'whorls' within the VRC. Optical sectioning of the PVX VRCs revealed that one of the viral movement proteins was localized to the centres of the RNA whorls, demonstrating significant partitioning of viral RNA and proteins within the VRC. The utility of Pumilio as a fluorescence-based reporter for viral RNA is discussed.     

Use of a cell-free protein synthesizing system from cells of ascites carcinoma Krebs-2 for RNA translation of plant viruses

Cell-free translation in Krebs-2 extracts was optimized for RNAs of two plant viruses; potato virus X (PVX, potexvirus), and tobacco mosaic virus (TMV, tobamovirus). PVX and TMV RNAs programmed synthesis of similar sets of polypeptides in both the Krebs-2 extracts and the rabbit reticulocyte lysates, major virus-specific products being the same in molecular weight in both in vitro systems. PVX structural protein (p29) was absent among polypeptides synthesized in the Krebs-2 system but was readily identified by immuno-precipitation among the ones synthesized in the reticulocyte lysate system. The "cap" analog, m7Gpp, inhibited the synthesis of all the polypeptides programmed by PVX RNA in the Krebs-2 system. The synthesis of only a few of the most high molecular weight products in the reticulocyte lysate system was inhibited, the synthesis of a number of low molecular weight products (and among them p29) was even stimulated. Thus, the PVX capped messengers derived from PVX genomic RNA due to its fragmentation with endogenous nuclease activities. The use of the Krebs-2 system allows to avoid activation of internal PVX genes.     

RNA silencing-mediated resistance is related to biotic / abiotic stresses and cellular RdRp expression in transgenic tobacco plants

The discovery of RNA silencing inhibition by virus encoded suppressors or low temperature leads to concerns about the stability of transgenic resistance. RNA-dependent RNA polymerase (RdRp) has been previously characterized to be essential for transgene-mediated RNA silencing. Here we showed that low temperature led to the inhibition of RNA silencing, the loss of viral resistance and the reduced expression of host RdRp homolog (NtRdRP1) in transgenic T4 progeny with untranslatable potato virus Y coat protein (PVY-CP) gene. Moreover, RNA silencing and the associated resistance were differently inhibited by potato virus X (PVX) and tobacco mosaic virus (TMV) infections. The increased expression of NtRdRP1 in both PVX and TMV infected plants indicated its general role in response to viral pathogens. Collectively, we propose that biotic and abiotic stress factors affect RNA silencing-mediated resistance in transgenic tobacco plants and that their effects target different steps of RNA silencing.     

Localisation of a series of intra-RNA cross-links in 16S RNA, induced by ultraviolet irradiation of Escherichia coli 30S ribosomal subunits

Mild ultraviolet irradiation of E. coli ribosomal subunits leads to the formation of a number of intra-RNA cross-links, in addition to the RNA-protein cross-links already reported (see refs. 9, 10). After partial ribonuclease digestion of the RNA from irradiated subunits, complexes containing these intra-RNA cross-links can be isolated on a two-dimensional gel electrophoresis system, and subjected to sequence analysis. A series of these cross-linked complexes is described, and the cross-linked RNA regions are compared with the secondary structures derived for 16S RNA (see refs. 6, 7).     

马铃薯病毒一步法多重RT-PCR检测技术的构建

根据马铃薯病毒PVX、PVY、PVA、PLRV的CP基因序列设计4对特异性引物,通过对试剂浓度和反应条件进行优化,建立了能够同步检测PVX、PVY、PVA、PLRV的一步法多重RT-PCR检测方法。该方法对PVX、PVY、PVA、PLRV扩增出的靶带大小分别为732、422、132和336 bp,凝胶电泳易辨别区分。病毒RNA最低检测限度为7.8 pg/μL,对PVM、PVS、AMV、TMV及PSTVd的扩增为阴性。研究结果表明,该方法特异、灵敏,比两步法多重RT-PCR检测更加快速、简便,提高了检测效率,降低检测成本,为马铃薯病毒的高效检测提供了有效手段。     

RNA-protein cross-linking in Escherichia coli ribosomal subunits: localization of sites on 16S RNA which are cross-linked to proteins S17 and S21 by treatment with 2-iminothiolane.

Treatment of E. coli ribosomal subunits with 2-iminothiolane coupled with mild ultraviolet irradiation leads to the formation of a large number of RNA-protein cross-links. In the case of the 30S subunit, a number of sites on 16S RNA that are cross-linked to proteins S7 and S8 by this procedure have already been identified (see ref. 6). Here, by using new or modified techniques for the partial digestion of the RNA and the subsequent isolation of the cross-linked RNA-protein complexes, three new iminothiolane cross-links have been localized: Protein S17 is cross-linked to the 16S RNA within an oligonucleotide encompassing positions 629-633, and protein S21 is cross-linked to two sites within oligonucleotides encompassing positions 723-724 and positions 1531-1542 (the 3'-end of the 16S RNA).     

Precise localisation of three intra-RNA cross-links in 23S RNA and one in 5S RNA, induced by treatment of Escherichia coli 50S ribosomal subunits with bis-(2-chloroethyl)-methylamine.

Treatment of E. coli 50S ribosomal subunits with low doses of bis-(2-chloroethyl)-methylamine ("nitrogen mustard") leads to formation of a number of intra-RNA and RNA-protein cross-links. After partial digestion of the cross-linked subunits with cobra venom nuclease, followed by destruction of the protein moiety with proteinase K, complexes containing the intra-RNA cross-links were isolated by two-dimensional gel electrophoresis. The individual complexes were subjected to oligonucleotide analysis, either directly or after a second partial digestion procedure using ribonuclease T1, and the cross-link sites determined. In 23S RNA, the cross-links found were between bases 763 and 1567, 1210 and 1236, 1482 and 1501; in 5S RNA, base 69 was cross-linked to base 107. The significance of these cross-links in relation to the three-dimensional organization of the ribosomal RNA is discussed.     

Transgenic Plants Expressing Potato Virus X ORF2 Protein (p24) Are Resistant to Tobacco Mosaic Virus and Ob Tobamoviruses

The p24 protein, one of the three proteins implicated in local movement of potato virus X (PVX), was expressed in transgenic tobacco plants (Nicotiana tabacum Xanthi D8 NN). Plants with the highest level of p24 accumulation exhibited a stunted and slightly chlorotic phenotype. These transgenic plants facilitate the cell-to-cell movement of a mutant of PVX that contained a frameshift mutation in p24. Upon inoculation with tobacco mosaic virus (TMV), the size of necrotic local lesions was significantly smaller in p24+ plants than in nontransgenic, control plants. Systemic resistance to tobamoviruses was also evidenced after inoculation of p24+ plants with Ob, a virus that evades the hypersensitive response provided by the N gene. In the latter case, no systemic symptoms were observed, and virus accumulation remained low or undetectable by Western immunoblot analysis and back-inoculation assays. In contrast, no differences were observed in virus accumulation after inoculation with PVX, although more severe symptoms were evident on p24-expressing plants than on control plants. Similarly, infection assays conducted with potato virus Y showed no differences between control and transgenic plants. On the other hand, a considerable delay in virus accumulation and symptom development was observed when transgenic tobacco plants containing the movement protein (MP) of TMV were inoculated with PVX. Finally, a movement defective mutant of TMV was inoculated on p24+ plants or in mixed infections with PVX on nontransgenic plants. Both types of assays failed to produce TMV infections, implying that TMV MP is not interchangeable with the PVX MPs.     

Features of crossing-over in virus-infected tomato

The evidence of increased crossing over rate in tomato hybrids infected with TAV (Tomato aspermy virus), PVX (Potato virus X), TMV (Tobacco mosaic virus), TMV+PVX indicates the recombinogenic effect of viral infection. Cytological studies of the early diakinesis in healthy and virus-infected tomato revealed significant changes in chiasma number and position. The most significant changes were established for bivalents with two interstitial chiasmata and with one terminal and one interstitial. The data obtained indicate redistribution of the chiasmata position and induction of additional exchanges. The virus-induced recombination is segment-specific and depends on the host plant genotype, virus infection and the interaction between them.     

Analysis of polypeptides of virus-specific informosomes induced by tobacco mosaic virus and potato virus X

Informosome-like virus-specific ribonucleoprotein (vRNP) of tobacco mosaic virus (TMV) comprise a set of four major polypeptides having molecular weights of 17 500, 31 000, 37 000 and 39 000. Of the minor polypeptides, those of apparent molecular weights 25 000, 55 000, 68 000 and 70 000 had electrophoretic mobilities of polypeptides found in a ribonucleoprotein preparation from uninoculated plants. Polypeptide with mol.wt. 175 000 is TMV coat protein so far as: a) vRNP was precipitated with immunoglobulins against TMV and TMV coat protein; b) it had electrophoretic mobility similar to mobility of TMV coat protein; c) the peptide map of polypeptides with mol.wts 31 000, 37 000 and 39 000 are probably virus-specific-products. This is supposed because they are not present in cell informosomes protein, and they are not revealed in vRNP induced in cells after infection with potato virus X (PVX). Electrophoresis of vRNP-PVX protein reveals polypeptides of 23 000 (PVX coat protein), 55 000, 70 000, 78 000, 95 000, 120 000 and 145 000.     

The rye embryo system as an alternative to the wheat system for protein synthesis in vitro.

Isolated rye embryos are a readily available source for the preparation of very active, cell-free, protein-synthesizing systems. Incorporation levels up to 2000 pmol leucine per 50 mul assay are routinely obtained at saturating TMV (Tobacco mosaic virus) RNA concentrations; at limiting messenger RNA concentrations the incorporation exceeds 1000 leucine molecules per TMV RNA molecule. The characteristics of this cell-free system for the translation of TMV RNA are identical with those of a similarly prepared wheat germ system. The major advantage of the rye embryo system is its high reliability as compared to the unpredictable wheat germ system. Sucrose gradient analysis of the reaction mixture during the incubation shows an extensive polysome formation with TMV RNA and demonstrates efficient polypeptide chain release.     

四种广普性植物病毒高效mPCR检测方法的建立

本研究建立了能同时检测出烟草花叶病毒(TMV)、黄瓜花叶病毒(CMV)、马铃薯X病毒(PVX)和马铃薯Y病毒(PVY)的多重RT-PCR体系。TMV、CMV、PVX、PVY是四种广普性植物病毒,寄主范围广泛,并且常常发生复合侵染。本研究以上述四种病毒的CP基因部分序列设计引物,以反转录的cDNA为模板,建立多重RT-PCR反应体系,分别扩增出211～417bp的不同长度的基因片断,并通过序列测定来确认扩增序列的特异性。将反转录合成的cDNA进行浓度稀释,来对多重RT-PCR与单重RT-PCR的灵敏度进行比较,结果证明,多重RT-PCR体系能够同时快速检测这四种病毒,并且有很高的灵敏度。     

Observation of Potato Virus X in Tobacco Mosaic Virus (TMV)- Induced Local Lesions and Surrounding Zones in Leaves of Datura stramonium L. in Mixed Infection

In mixed infection by Tobacco Mosaic Virus (TMV) and Potato Virus X (PVX) of leaves of Datura stramonium L., PVX particles were observed in the developing local lesions in both the central part and on the periphery, in addition to TMV. PVX virions were found either separately or together with TMV. Sometimes in local lesions mainly in their periphery, PVX-specific laminar inclusion components were observed and, in certain cases, cylindrical bodies about 120—140 nm in diameter. In 2 mm surrounding zones from the edge of the lesions, TMV particles were not observed. However, in the majority of cells of these zones, PVX intensively accumulated, often forming large masses. In some cases, we observed parts of cells with relatively small amounts of dispersed PVX particles, associated with laminar inclusion bodies. In cell areas with large accumulations of PVX, laminar inclusions were not found.     

Formaldehyde-induced tight binding of protein to RNA in particles of rod-like virus

The formaldehyde-induced formation of tightly bound RNA-protein complexes of rod-like plant viruses was studied. The preparations of tobacco mosaic virus and closely related cucumber virus 4 were incubated with 1.5% formaldehyde for 20-50 hrs at 50 degrees C. Then the viral particles were disrupted, free protein was removed and viral RNA was centrifuged in the linear gradient of Cs2SO4. The RNAs from the formaldehyde-untreated viruses and RNA from the formaldehyde-treated tobacco masaic virus had the density of 1.65-1.66 g/cm3, while RNA from the formaldehyde-treated cucumber virus had the density of 1.57-1.42 g/cm3, depending on the incubation time. This is indicative of the protein binding to RNA. Treatment of the cucumber virus complex with pronase resulted in a liberation of free RNA with the density of 1.66 g/cm3; incubation for 2 min at 100 degrees C in a dissociating mixture (2% sodium dodecyl sulfate + 0.2% mercaptoethanol) did not cause the dissociation of the complex. Polyacrylamide gel electrophoresis showed that the most part of the protein molecules are bound within the complex not by covalent protein-protein cross-links.     

Single-Molecule Force Spectroscopy Study on the Mechanism of RNA Disassembly in Tobacco Mosaic Virus

To explore the disassembly mechanism of tobacco mosaic virus (TMV), a model system for virus study, during infection, we have used single-molecule force spectroscopy to mimic and follow the process of RNA disassembly from the protein coat of TMV by the replisome (molecular motor) in vivo, under different pH and Ca²⁺ concentrations. Dynamic force spectroscopy revealed the unbinding free-energy landscapes as that at pH 4.7 the disassembly process is dominated by one free-energy barrier, whereas at pH 7.0 the process is dominated by one barrier and that there exists a second barrier. The additional free-energy barrier at longer distance has been attributed to the hindrance of disordered loops within the inner channel of TMV, and the biological function of those protein loops was discussed. The combination of pH increase and Ca²⁺ concentration drop could weaken RNA-protein interactions so much that the molecular motor replisome would be able to pull and disassemble the rest of the genetic RNA from the protein coat in vivo. All these facts provide supporting evidence at the single-molecule level, to our knowledge for the first time, for the cotranslational disassembly mechanism during TMV infection under physiological conditions.