S.V. Kozlovsky

AFM Study of Potato Virus X Disassembly Induced by Movement Protein

Recently we have reported that a selective binding of potato virus X (PVX)-coded movement protein (termed TGBp1 MP) to one end of a polar coat protein (CP) helix converted viral RNA into a translatable form and induced a linear destabilization of the whole helical particle. Here, the native PVX virions, RNase-treated (PVX(RNA-DEG)) helical particles lacking intact RNA and their complexes with TGBp1 (TGBp1-PVX and TGBp1-PVX(RNA-DEG)), were examined by atomic force microscopy (AFM). When complexes of the TGBp1 MP with PVX were examined by means of AFM in liquid, no structural reorganization of PVX particles was observed. By contrast, the products of TGBp1-dependent PVX degradation termed beads-on-string were formed under conditions of AFM in air. The AFM images of PVX(RNA-DEG) were indistinguishable from images of native PVX particles; however, the TGBp1-dependent disassembly of the CP-helix was triggered when the TGBp1-PVX(RNA-DEG) complexes were examined by AFM, regardless of the conditions used (in air or in liquid). Our data supported the idea that binding of TGBp1 to one end of the PVX CP-helix induced linear destabilization of the whole helical particle, which may lead to its disassembly under conditions of AFM.

Mutagenic analysis of Potato Virus X movement protein (TGBp1) and the coat protein (CP): in vitro TGBp1–CP binding and viral RNA translation activation

Previously, we have shown that encapsidated Potato virus X (PVX) RNA was non-translatable in vitro, but could be converted into a translatable form by binding of the PVX movement protein TGBp1 to one end of the virion or by coat protein (CP) phosphorylation. Here, a mutagenic analysis of PVX CP and TGBp1 was used to identify the regions involved in TGBp1-CP binding and translational activation of PVX RNA by TGBp1. It was found that the C-terminal (C-ter) 10/18 amino acids region was not essential for virus-like particle (VP) assembly from CP and RNA. However, the VPs assembled from the CP lacking C-ter 10/18 amino acids were incapable of TGBp1 binding and being translationally activated. It was suggested that the 10-amino-acid C-ter regions of protein subunits located at one end of a polar helical PVX particle contain a domain accessible to TGBp1 binding and PVX remodelling. The non-translatable particles assembled from the C-ter mutant CP could be converted into a translatable form by CP phosphorylation. The TGBp1-CP binding activity was preserved unless a conservative motif IV was removed from TGBp1. By contrast, TGBp1-dependent activation of PVX RNA translation was abolished by deletions of various NTPase/helicase conservative motifs and their combinations. The motif IV might be essential for TGBp1-CP binding, but insufficient for PVX RNA translation activation. The evidence to discriminate between these two events, i.e. TGBp1 binding to the CP-helix and TGBp1-dependent RNA translation activation, is discussed.

Regulation of RNA Translation in Potato Virus X RNA-Coat Protein Complexes: The Key Role of the N-Terminal Segment of the Protein

The efficiency of in vitro translation of the potato virus X (PVX) RNA was studied for viral ribonucleoprotein complexes (vRNP) assembled from the genomic RNA and the viral coat protein (CP). In vRNP particles the 5′-proximal RNA segments were encapsidated into the CP, which formed helical headlike structures differing in length. Translation of the PVX RNA was completely suppressed upon incubation with PVX CP and was activated within vRNPs assembled in vitro with two CP forms, differing in the modification of the N-terminal peptide containing the main phosphorylation site(s) for Thr/Ser protein kinases. It was shown that CP phosphorylation activates RNA translation within vRNPs and that the removal of the N-terminal peptide of CP suppresses activation, but CP still acts as a translational suppressor. This fact made it possible to suppose that the replacement of Ser/Thr by amino acid residues that are not subject to phosphorylation in the N-terminal peptide of CP of the mutant PVX (PVX-ST) completely inhibits RNA translation within vRNP. However, experiments disproved this assumption: PVX-ST RNA was efficiently translated within native virions, RNA of the wild-type (wt) PVX was efficiently translated in heterogeneous vRNP (wtRNA + PVX-ST CP), and the opposite result (repression of translation) was obtained for another heterogeneous vRNP (PVX-ST RNA + wtCP). Therefore, the N-terminal CP peptide located on the surface of the PVX virion or vRNP particles plays a key role in the activation of viral RNA translation.

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.