[A new method of producing biologically active nanocomplexes by non-covalent conjugation of proteins with viral particles].

Currently, a range of biologically active molecules have been attached to plant and bacterial viras nanoscaffolds, yielding stable nanoparticles that display multiple copies of the desired molecule. In this paper we propose a new method of non-covalent attachment of peptides to the surface of virios. We have demonstrated that this method is efficient in a model system that includes tobacco mosaic virus particles, synthetic polycation (quaternized poly(4-vinylpyridine) carrying ethyl ethyl pendant radicals) and polypeptide of interest. This principle of step-by-step binding to the surface of virions was used for electrostatic association with hydrophilic fragment of influenza virus haemagglutinin.

[Development of immunochromatographic test systems for express detection of plant viruses].

Express immunochromatographic test-strip assays were developed for detection of five plant viruses varying in shape and size of virions: spherical carnation mottle virus, bean mild mosaic virus, rod-shaped tobacco mosaic virus, and filamentous potato viruses X and Y. Multimembrane composites (test strips) with immobilized polyclonal antibodies against viruses and colloidal gold-conjugated antibodies were used for the analysis. The immunochromatographic test strips were shown to enable the detection of viruses both in purified preparations and in leaf extracts of infected plants with a sensitivity from 0.08 to 0.5 microg/ml for 10 min. The test strips may be used for express diagnostics of plant virus diseases in field conditions.

[Translational regulation of potato virus X RNA-coat protein complexes: the key role of a coat protein N-terminal peptide].

The efficiency of in vitro translation of potato virus X (PVX) RNA within vRNP complexes assembled from genomic RNA and viral CP was examined. The vRNP particles contain the 5'-proximal RNA segments encapsidated by helically arranged CP head-like portions heterogeneous in length and the CP-free RNA tail. Translation of RNA is completely repressed upon incubation with PVX CP and is accompanied by vRNP particles production. By contrast, translation is activated in vRNPs in vitro assembled using two CP forms, differing in the principals of their N-terminal peptides modification. The N-terminal peptide of PVX CP represents the major phosphorylation site(s) for Thr/Ser-specific protein kinases. It was shown that: (i) CP phosphorylation results in a translational activation of vRNP; (ii) removal of N-terminal peptide from CP abolished activation and CP retains the translation repressing ability. It was suggested that substitution of Ser/Thr residues by non-phosphorylated Ala/Gly in N-terminal peptide of the mutant CP will led to a complete inhibition of vRNP translation. However, opposite results were obtained in our experiments: (i) RNA of such mutant virus (PVX-ST) was efficiently translated within the virions; (ii) RNA of a wild-type (wt) PVX also efficiently translated in mixedly assembled vRNP "wt PVX RNA + PVX-ST CP"; (iii) opposite result (repression of translation) was obtained with "mixed" vRNP (PVX-ST RNA + wtPVX CP). Therefore, the N-terminal peptide located at the surface of the particle and of the particles plays a key role in translation activation of the RNA encapsidated in vRNP and native virions.

[Synthesis of the tobacco mosaic virus in blocked spreading of infection]. / Sintez virusa tabachnoi mozaiki v usloviiakh bloka transporta infektsii.

The possibility of infection of tobacco upper leaves with tobacco mosaic virus (TMV) was examined in experiments where the inoculum was imbibed through the cut stem. The inoculum used were: a) a preparation of a virus-specific informosome-like ribonucleoproteins (vRNP) isolated from TMV-infected plants; b) a TMV preparation; or c) a mixture of TMV and vRNP. Multiplication of TMV in upper leaves was observed in neither of the variants; nevertheless in the vascular tissue and/or probably in adjoining parenchymal cells, two kinds of RNA were synthesized: of mol. w. (1.1--1.3) X 10(6) and (0.6--0.8) X 10(6). These RNA were not found in healthy plants in the presence of actinomycin D. The synthesis of genomic TMV RNA is suppressed under these conditions. Thus, some kind of abortive TMV infection takes place under the condition of experimental inoculation of plants through a cut stem. Molecular hybridization with the DNA of recombinant plasmid containing a nucleotide sequence complementary to the 3'-portion of genomic TMV RNA proves that short RNAs synthesized under the abortive infection conditions are TMV-specific. The experiments with differential temperature treatment of N-gene-containing plants under abortive infection conditions suggest that necrotization is not necessarily induced by genomic TMV RNA synthesis.

[Translation of the genomic and subgenomic RNAs of cucumber virus 3 in a cell free system from wheat embryos and in Xenopus laevis oocytes]. / Transliatsiia genomnoi i subgenomnoi RNK ogurechnogo virusa 3 v beskletochnoi sisteme iz zarodyshei pshenitsy i v ootsitakh Xenopus laevis.

The purified preparation of cucumber virus 3 (CV3) (one of the member of tobamovirus group) contains, besides full-length (2.0 X 10(6)) genomic RNA, a short (0.24 X 10(6)) subgenomic RNA coding for coat protein in Xenopus laevis oocytes as well as in the cell-free protein synthesizing system from wheat embryos. The similarity of the polypeptide coded by short CV3 RNA in vitro to natural CV3 coat protein is proved by the similarity of their molecular weights, antigenic specificity and by the similarity of the peptide maps. Individual CV3 genomic RNA coded in vitro for polypeptide 130 X 10(3), and was incapable of coding for the coat protein. The results of translation of an artificial mixture of short and full-length RNAs suggest that it is the short RNA that is preferentially translated.

[Virus-specific informosomes in tobacco cells infected with tobacco mosaic virus]. / Virusspetsificheskie informosomy v kletkakh tabaka, inifitsirovannykh virusom tabachnoi mozaiki.

We have previously detected in TMV-infected cells virus-specific informosome-like ribonucleoproteins (vRNP) that differed in the CsCl buoyant density from mature TMV particles. It is shown in the present work that [3H]uridine-labelled TMV-specific structures, when fractionated in Cs2SO4, produce three types of structures, i. e. with a buoyant density of 1.23 g/cm3 (the so-called 1.23 material), 1.29 g/gm3 (mature virus) and 1.34--1.49 g/cm3 (vRNP). The 1.23 material has been investigated. The incorporation of [3H]palmitic acid and the sensitivity of this material to 0.1% Na dodecyl sulphate was interpreted to mean the presence of membrane components. Treatment of the 1.23 material Na dodecyl sulphate induces the release of the mature virus, vRNP and free viral RNA. vRNP was shown to contain genome TMV RNA (mol. weight, 2.0 x 10(6)) and a considerable amount of subgenomic TMV RNA (mol. weight, 1.1--1.3 x 10(6) and 0.6--0.8 x 10(6)). It is demonstrated that RNA isolated from vRNP codes for TMV-specific proteins and is able to hybridize with recombinant plasmid containing DNA-copy of 3'-end RNA TMV fragment (about one half of genome).

[Analysis of polypeptides of virus-specific informosomes induced by tobacco mosaic virus and potato virus X]. / Analiz polipeptidov virusspetsificheskikh informosom, indutsiruemykh virusom tabachnoi mozaiki i X-virusom kartofelia.

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.

[Primary structure of RNA 3 of barley stripe mosaic virus and its variability]. / Pervichnaia struktura RNK 3 virusa shtrikhovatoi mozaiki iachemnia i ee variabel'nost'.

The complete nucleotide sequence was determined for three variants of the third genomic component of BSMV strain Argentina mild. The common variant, RNA 3 (2797 nucleotide), contains two open reading frames (ORFs) coding for two proteins with Mr of 74,229 (putative BSMV RNA polymerase) and Mr of 16,994. The second ORF is expressed from a subgenomic RNA. The extended variant RNA 3 differs from the common one only by the presence of a direct tandem repeat 351-363 nucleotides in length (with some variability) encompassing part of the leader sequence and the beginning of the first ORF. The resulting protein has a Mr of about 86,000. The defective variant, RNA 4, carries a deletion of 185 nucleotides in the 3'-end proximal part of the first ORF, which shortens the product to a Mr of 60,344.

[Primary structure of the potato virus X genome: the region preceding the capsid protein cistron]. / Izuchenie pervichnoi struktury genoma X-virusa kartofelia: oblast', predshestvuiushchaia tsistronu kapsidnogo belka.

DNA copies of the potato virus X (PVX) RNA corresponding to 2300 nucleotides at the 3'-end have been cloned. The cloned cDNA copies containing the nucleotides 445-1280 from the 3'-end have been sequenced. The 5'-terminal region of the PVX coat protein gene corresponds to residues 445-786 from the 3'-end. The amino acid sequences of two more open reading frames (ORF) have been deduced from the nucleotide sequence. The potential translation products of these ORF's would correspond to the nonstructural viral proteins. We have located the ORF1 within the region of residues 799-1009 preceding the coat protein cistron. The tentative protein is composed of 70 amino acids and has an aminoterminal segment which is markedly hydrophobic. ORF2 in the PVX sequence ends with UAG at nucleotides 942-944 and extends to the 5'-terminus for additional 340 nucleotides. The distant sequence homology exists between a carboxyterminal portion of PVX ORF2 and that of the nonstructural "30 K-proteins" of the plant tobamoviruses.

[Comparison of antigenic properties of the X-potato virus and its coat proteins. Effect of proteolytic cleavage on antigenic characteristics]. / Sravnenie antigennykh svoistv x-virusa kartofelia i ego belka obolochki. Vliianie proteoliticcheskogo rasshchepleniia na antigennye kharakteristiki.

Antigenic properties of intact potato virus X (PVX) particles and of PVX coat protein (CP) preparations were compared using different modifications of ELISA test. In the competitive ELISA test (reaction in solution) antibodies to intact virus react much stronger with PVX than with CP while antibodies to CP react much stronger with CP than with PVX. In the direct ELISA test (reaction on the solid support) the difference in reactions of antiCP antibodies with PVX and CP is eliminated while the one in reactions of antiPVX antibodies with these antigens remains. No difference was registered in reactivity of PVX absorbed directly on polystyrene or on immunoglobulin-coated wells (sandwich ELISA) to antiCP antibodies.

[The effect of proteolytic cleavage of potato virus X coat protein on its ability to self-assemble with RNA and viral infectivity]. / Vliianie proteoliticheskogo rasshchepleniia belka obolochki X-virusa kartofelia na ego sposobnost' k rekonstruktsii s RNK i na infektsionnost' virusa.

The process of proteolytic cleavage of potato virus X coat protein molecules inside the virions and in the dissociated state in the course of their purification and storage has been studied. In agreement with the previous reports, the intact form (Ps) of the coat protein in the viral particles was found to be gradually cleaved to three discrete lower molecular forms (Pi, Pf, Pu). During the storage of the dissociated coat protein preparations further cleavage was observed with formation of at least three additional lower molecular weight forms (Ppa, Ppb, Ppc). The location of proteolytic cleavage sites leading to formation of Ppa form was determined. The shortened forms Pi, Pf, Pu and Ppa (and possibly Ppc) were found to be incorporated into the viral particles in the course of reconstitution in vitro with the viral RNA. Infectivity of the virus containing only intact (Ps) form of the protein was found to be two to three folds higher than that of the virus containing only Pf form of the coat protein.

New viral vector for superproduction of epitopes of vaccine proteins in plants.

The novel viral vectors PVX-CP AltMV and PVXdt-CP AltMV are superexpressors of the capsid protein (CP). These viral vectors were constructed on the basis of the potato virus X (PVX) genome andAlternantheramosaic virus (AltMV) CP gene. The expression, based on the hybrid viral vectors, is genetically safe, since the systemic transport and formation of infective viral particles are blocked. CP AltMV can self-assemble into virus-like particles (VLPs) in the absence of genomic RNA. The vectors can be used for the presentation of foreign peptides (including epitopes of human pathogens) on the surface of the VLP. The N-terminal extracellular domain (M2e) of the influenza virus A M2 protein and its truncated variant (ΔM2e) were used as model heterologous peptides for the construction of the chimeric CP AltMV. Chimeric CP AltMV retains its ability to self-assemble into VLP. The epitopes of the M2 influenza virus protein were not eliminated during the process of accumulation, polymerization and purification of chimeric VLP AltMV, providing evidence of the stability of chimeric VLP with C-terminal heterologous epitopes. It appears that VLP produced by the vectors PVX-CP AltMV and PVXdt-CP AltMV can be used in the field of biotechnology for the presentation of the epitopes of vaccine proteins on their surfaces. The chimeric VLP AltMV with the presented foreign epitopes can be used as candidate vaccines.