RESUMEN
A highly sensitive express immunochromatography method for molecular diagnosis of plant virus infections was elaborated on the example of a model object - tobacco mosaic virus (TMV). The analysis time does not exceed 5 min, and the lower limit of TMV detection in non-clarified leaf extract (2-4 ng/ml) is comparable with the sensitivity of the enzyme-linked immunosorbent assay of the virus. A single measurement requires 0.1-0.2 ml tested solution (extract from 10-20 mg of leaf material). The sensitivity of TMV determination in the leaf tissue extract was increased by more than one order of magnitude using signal enhancement by silver and is 0.1 ng/ml. In this case, analysis time did not exceed 25 min. The simplicity of this method makes it especially convenient in express diagnosis of numerous analyzed specimens. The prototype of a diagnostic kit for serial analyses of plant viral infections both in laboratory and field conditions was elaborated.
Asunto(s)
Cromatografía DEAE-Celulosa/métodos , Enfermedades de las Plantas/virología , Virus del Mosaico del Tabaco/aislamiento & purificación , Anticuerpos Antivirales/análisis , Ensayo de Inmunoadsorción Enzimática , Nanopartículas del Metal , Virus del Mosaico del Tabaco/inmunologíaRESUMEN
We propose that therapy of patients with anticancer drugs that poison DNA topoisomerases induces formation of covalent complexes of cellular RNAs and DNA topoisomerases. The appearance of these complexes can be detected with antibodies against a synthetic hapten mimicking the covalent linkage unit Tyr-pU(p) of picornavirus RNA and VPg. We synthesized hapten [N(Ac),CO(NH2)]Tyr-(5 P --> O)Up-O-(CH2)6NH2, conjugated it with BSA, and immunized rabbits with the antigen obtained. The raised polyclonal antibodies were purified by successive affinity chromatography on BSA-Sepharose and hapten-Sepharose columns. Target antibodies recognized hapten and encephalomyocarditis virus RNA-VPg complex specifically as found using the dot-immunogold method. We believe that these antibodies might be useful to study mechanism of picorna and similar virus RNA synthesis. The discovery and qualitative determination of the cellular RNA-DNA topoisomerases covalent complexes with these antibodies might be useful to monitor therapy efficacy by drugs "freezing" dead-end complexes of DNA topoisomerases and nucleic acids and to understand the mechanism of DNA topoisomerase poisoning in situ.
Asunto(s)
Anticuerpos/inmunología , ARN Viral/inmunología , Proteínas Virales/inmunología , Animales , Anticuerpos/metabolismo , Especificidad de Anticuerpos , Crotalus/inmunología , Crotalus/metabolismo , Virus de la Encefalomiocarditis/inmunología , Virus de la Encefalomiocarditis/metabolismo , Haptenos/inmunología , Haptenos/metabolismo , Humanos , Inmunohistoquímica , Picornaviridae/enzimología , Picornaviridae/inmunología , ARN Viral/metabolismo , Tirosina/inmunología , Tirosina/metabolismo , Proteínas Virales/metabolismoRESUMEN
VPg unlinkase is an unusual eukaryotic enzyme that catalyzes hydrolysis of the phosphodiester bond between residues of the unique tyrosine of VPg (viral protein genome-linked) and the 5;-terminal uridylic acid of picornavirus RNA. Cellular targets of the VPg unlinking enzyme are yet unknown. To determine an essential nucleic part of the covalent linkage unit that is necessary for the VPg unlinkase reaction, the following derivatives of the encephalomyocarditis virus (EMCV) VPg-RNA complex were used: [125I]Kp-pUpUpGp, [125I]Kp-pUp, and [125I]Kp-pU (Kp is residual peptides bound to RNA after proteinase K treatment of VPg-RNA). [125I]K-peptides were unlinked from [125I]Kp-pUpUpGp and [125I]Kp-RNA with similar velocity, but [125I]Kp-pUp was split much slower. Under the same conditions [125I]Kp-pU was not dissociated at all. Thus, pUp is a minimal part of picornavirus RNA that is necessary for VPg unlinkase. We speculate that cellular substrates of the enzyme are phosphodiesters of oligo(poly)ribonucleotides and tyrosine or tyrosine peptides. In no case [125I]VPg-pU, [125I]VPg-pUp, and [125I]VPg-pUpUpGp were hydrolyzed by VPg unlinkase, in contrast with [125I]VPg-RNA and [125I]VPg-pUpUpGpApApApGp. We conclude that the whole VPg, when bound to trinucleotide (but not to heptanucleotide), protects the inter-polymeric phosphodiester bond against hydrolysis of the covalent linkage unit. We speculate that VPg unlinkase might repair covalent complexes of RNA and topoisomerases and trigger degradation process of the picornavirus RNA.