Atabekov Ig

Reciprocal dependence between pectinmethylesterase gene expression and tobamovirus reproduction effectiveness in Nicotiana benthamiana.

The transport protein (TP) of tobacco mosaic virus (TMV) ensures intercellular transport of viral RNA through plasmodesms, apparently by interacting with the cell proteins of endoplasmic reticulum, cytoskeleton, and cell wall [1]. During the past years, several cell proteins that specifically interact with VTM TP have been identified. These are cytoskeletal tubulin, myosin, and actin [2, 3]; protein kinanses [4‐6]; transcriptional coactivator KELP [7]; and cytoskeletal protein MPB2C [8]. The functional role of these proteins in the transport of viral infection remains unknown.

Comparative analysis of protein kinases that phosphorylate tobacco mosaic virus movement protein in vitro

Phosphorylation is a widespread form of posttranslational modification of eukaryotic proteins. Phosphorylation affects the protein tertiary structure and functional activity. It is catalyzed by protein kinases (phosphotransferases) that use the γ -phosphate group of ATP (or GTP) and alcohol groups of serine (threonine) or the phenol group of tyrosine in the proteins to form phosphoresters. Phytoviral genomes do not contain the genes encoding protein kinases (PKs). For this reason, viral proteins are phosphorylated by cell enzymes. Thus, cell protein kinases may be regarded as host-cell proteins interacting with viral proteins (in particular, with the movement protein of tobacco mosaic virus (TMV), which is required for the virus transport in the infected plant [3–8]). It was shown that TMV movement protein is phosphorylated in vitro and in vivo at the C-terminal serine and threonine residues by PKs associated with the cell walls of the host plant [2, 3]. However, the phosphorylation of TMV movement protein in infected tobacco protoplasts, in which cell walls are absent, was also reported [4, 5]. The questions concerning determination of the phosphorylation sites of the TMV movement protein, PK types involved in phosphorylation, and its functional role remain open [4–9]. With the use of different PK inhibitors, it was shown that the PK that phosphorylates the TMV movement protein in vitro in the cytoplasmic fraction of tobacco leaves and suspension culture of the protoplasts is related to casein kinase II (CKII) [7]. We used this approach to characterize the PK associated with the cell walls isolated from different tobacco species ( Nicotiana glutinosa, N. benthamiana , and N. tabacum ). The recombinant movement protein was used as a substrate, and the fraction isolated from the cell walls served as a protein-kinase source. Cell-wall preparations from N. glutinosa, N. benthamiana , and N. tabacum were isolated as described earlier [8]. Phosphorylation of TMV movement protein with cell wallassociated PKs was performed in the standard reaction mixture containing 20 mM HEPES–KOH (pH 7.4), 5 mM MgCl 2 , 50 mM KCl, 0.5 μ l of [ γ 32 P ]ATP (5000 Ci/mol, 400 MBq/ml), 30 μ l of cell-wall extract, and 2 μ g of recombinant TMV movement protein. The reaction was performed for 15 min at room temperature. We used the following inhibitors of Ser/Thr PKs: suramine and quercetin (both were used at CKII-inhibiting concentrations [7]); genistein, an inhibitor of tyrosine PKs; staurosporine, an inhibitor of a broad spectrum of Ser/Thr PKs (including protein kinase C); and H-89, an inhibitor of cAMP-dependent PKs (protein kinase A). As shown in Figs. 1a–1e, two of the five inhibitors used—suramine and quercetin—markedly affected the efficiency of phosphorylation of TMV movement protein. However, in both cases, the PK activity was not suppressed completely. Thus, PKs from the cell wall fractions of N. glutinosa, N. benthamiana , and N. tabacum, which are able to phosphorylate TMV movement protein in vitro, may be classified with the CKII-type PKs.

Effect of the N-terminal domain of the coat protein of potato virus X on the structure of viral particles.

The cell-to-cell movement of the potexvirus genomic RNA through plasmodesmata is mediated by virions [1] or ribonucleoproteins containing coat protein (CP) and the virus-encoded movement protein (termed TGBp1) [2]. It was shown in our earlier works [3, 4] that, in contrast to the RNA of tobacco mosaic virus (TMV), the encapsidated genomic RNA of potato virus X (PVX) was inaccessible for translation in vitro. However, PVX RNA is converted into a translated form either after CP phosphorylation by serine/threoninespecific protein kinases or as a result of the formation of a PVX complex with TP1 [4, 5]. A hypothesis was put forward according to which there were two different mechanisms of the translational activation of PVX at different stages of infection development: during phosphorylation of parental virions by cell protein kinases in primarily infected cell or as a result of virion interaction with the movement protein TP1 at further stages of infection development [4, 5]. It is well known that PVX CP is a glycoprotein [6]; its molecular weight was calculated from the results of sequencing (25 kDs). Polyacrylamide gel electrophoresis (PAGE) of CP in the presence of sodium dodecyl sulfate (SDS) revealed abnormal electrophoretic mobility, which in different strains of PVX corresponded to molecular weights of 27–29 kDa [6, 7]. The N-terminal domain of PVX CP is enriched with serine and threonine residues and exposed on the surface of viral particles [8]. This domain can be removed from the surface of the viral particle as a result of treatment with plant tissue proteases or trypsin [7]. Comparative study of phosphorylation, translation activity, and infectivity of native PVX preparations and trypsinized PVX viral particles devoid of the N-terminal domain demonstrated that (1) treatment of PVX with trypsin removed the most part of the 32 P-radioactivity from the PVX CP phosphorylated in situ ; (2) a trypsin-treated virus with a removed N-terminal domain (PVXtr) lost the phosphorylation-induced ability to activate the translation of viral RNA; and (3) the specific infectivity of PVXtr was reduced [5]. However, PVX RNA remained intact after trypsinization, and its translation might be activated as a result of PVXtr incubation with TP1. Thus, the N-terminal domain of PVX CP is the main acceptor of phosphate during the phosphorylation of CP as a component of the PVX virion in vitro [5]. This domain may also contain hypothetical sites of PVX CP glycosylation, which can explain, at least partly, the abnormal mobility of PVX CP in SDS PAGE [6]. To test the hypothesis on the role of phosphorylation of PVX CP as an activator of translation in vivo , we constructed a PVX mutant in which the potentially phosphorylated amino acid residues serine and threonine in the N-terminal domain of CP were substituted by the neutral nonphosphorylated amino acid residues alanine and glycine.

Role of C- and N-terminal mutations of the movement protein of tobacco mosaic virus in activation of complexes between the transport protein and viral RNA that are not translated in vitro.

Most plant viruses encode one or more proteins required for intercellular and systemic transport of virus. The movement protein (MP) 30K of tobacco mosaic virus (TMV) ensures translocation of viral genome from primary infected cells to neighboring healthy cells through plasmodesms. In infected cells, as well as in transgenic plants, TMV MP is accumulated in plasmodesms, increasing their carrying capacity. The movement protein of TMV nonspecifically binds to single-stranded RNA in vitro in a cooperative manner, forming linear ribonucleoproteins (RNPs) [1, 2]. These observations have given rise to the idea that viral infection spreads from one cell to another in the form of RNP containing viral RNA in the complex with MP (viral RNA temporarily may be not involved in translation and replication). Earlier, we showed that TMV TP is a specific repressor of RNA translation in vitro [3]. It is known that protein kinases associated with tobacco cell walls can phosphorylate the C-terminal region (Ser258, Thr261, and Ser265) of TMV TP [4]. The phosphorylation of TMV TP by protein kinases of cell walls or protein kinases of animal origin converts the MP–RNA complexes of TMV into in vitro translated complexes, which are infective for isolated protoplasts [5, 6]. To determine the functional domains of TMV TP, which are responsible for its activity as a translational repressor, in this work we obtained (1) C-terminal TMV TP mutants CT11, CT33, and CT84 (containing deletions of amino acids 258–268, 236–268, and 185– 268, respectively); (2) N-terminal deletion mutants NT30, NT96, and NT133 (lacking 30, 96, and 133 Nterminal amino acids, respectively); (3) C-terminal MP mutants, in which alanine substituted for the following amino acid residues: Ser267 (mutant Sb1A); Ser258, Thr261, and Ser265 (mutant Sb3A); and Ser258, Thr261, Ser265, and Ser267 (mutant Sb4A).