scholarly journals In virto and in vivo Phosphorylation of a Coat Protein of Potato Virus X

2021 ◽  
Vol 83 (5) ◽  
pp. 76-81
Author(s):  
L.O. Maksymenko ◽  
◽  
N.Y. Parkhomenko ◽  
Keyword(s):  
Coat Protein ◽  
Protein Phosphorylation ◽  
Potato Virus ◽  
Molecular Mechanisms ◽  
Potato Virus X ◽  
In Vitro Translation ◽  
Nitrocellulose Filter ◽  
Stage Of Development

At the present stage of development of plant virology the study of molecular mechanisms of regulation, translation and replication of viral RNA is of great interest. Potato virus X (PVX) RNA in viral particles is not available for in vitro translation, but acquires the ability to be translated as a result of shell protein phosphorylation. The aim of our study was to investigate the conditions of phosphorylation of the PVX coat protein in in vitro and in vivo systems, as well as the effect of EDTA and CaCl2 on the phosphorylation in vitro. Methods. The PVX coat protein was obtained by the guanidine chloride method. The kinase activity of PVX protein in vitro was determined in a standard reaction mixture containing Mn2+ ions, 0.8 mM EDTA, and 2 micro Ci 32P ATP (3000 Ci/mM). Phosphorylation of the protein in vivo was carried out by immersing Datura stramonium leaves with symptoms of PVX infection in water containing К3PO4 32P. After isolation of PVX from the leaves, the viral coat protein was fractionated by SDS-PAAG electrophoresis. Fractions of the protein were transferred from the gel by contact manner on a nitrocellulose filter. The PVX coat protein was detected by immunoblotting using immunoglobulins to PVX coat protein and rabbit antibodies labeled with peroxidase. The inclusion of labeled phosphorus in the PVX protein was detected by radioautography. Results. The PVX coat protein was phosphorylated in vitro in a standard incubation medium containing (gamma -32P) ATP. In contrast, the PVX coat protein cannot be phosphorylated in the same conditions in the presence of (alpha-32P) ATP. In vivo phosphorylated PVX coat protein was detected by exposing nitrocellulose filter with immunoblot on X-ray film. Additionally, it was found that the presence of 10 mm EDTA and 10 mm CaCl2 inhibited the process of the PVX coat protein phosphorylation in vitro. Conclusions. The coat protein of potato virus X is able to phosphorylate in vitro and in vivo systems. The terminal ATP phosphate plays a major role in the phosphorylation of the PVX coat protein. The presence of EDTA and Ca2+ influences on the process of protein phosphorylation in vitro. These agents are able to inhibit the process of phosphorylation of the PVX coat protein. Thus, the phenomenon of phosphorylation of the PVX coat protein apparently indicates about its participation in the regulation of the virus reproduction in the infected cell.

scholarly journals Potato virus X RNA-mediated assembly of single-tailed ternary ‘coat protein–RNA–movement protein’ complexes

2006 ◽  
Vol 87 (9) ◽  
pp. 2731-2740 ◽  
Author(s):  
O. V. Karpova ◽  
O. V. Zayakina ◽  
M. V. Arkhipenko ◽  
E. V. Sheval ◽  
O. I. Kiselyova ◽  
...  
Keyword(s):  
Coat Protein ◽  
Potato Virus ◽  
Rna Binding ◽  
Movement Protein ◽  
Infected Plant ◽  
Potato Virus X ◽  
Translation System ◽  
Transport Form

Different models have been proposed for the nature of the potexvirus transport form that moves from cell to cell over the infected plant: (i) genomic RNA moves as native virions; or (ii) in vitro-assembled non-virion ribonucleoprotein (RNP) complexes consisting of viral RNA, coat protein (CP) and movement protein (MP), termed TGBp1, serve as the transport form in vivo. As the structure of these RNPs has not been elucidated, the products assembled in vitro from potato virus X (PVX) RNA, CP and TGBp1 were characterized. The complexes appeared as single-tailed particles (STPs) with a helical, head-like structure composed of CP subunits located at the 5′-proximal region of PVX RNA; the TGBp1 was bound to the terminal CP molecules of the head. Remarkably, no particular non-virion RNP complexes were observed. These data suggest that the CP–RNA interactions resulting in head formation prevailed over TGBp1–RNA binding upon STP assembly from RNA, CP and TGBp1. STPs could be assembled from the 5′ end of PVX RNA and CP in the absence of TGBp1. The translational ability of STPs was characterized in a cell-free translation system. STPs lacking TGBp1 were entirely non-translatable; however, they were rendered translatable by binding of TGBp1 to the end of the head. It is suggested that the RNA-mediated assembly of STPs proceeds via two steps. Firstly, non-translatable CP–RNA STPs are produced, due to encapsidation of the 5′-terminal region. Secondly, the TGBp1 molecules bind to the end of a polar head, resulting in conversion of the STPs into a translatable form.

Characteristics of Artificial Virus-like Particles Assembled in vitro from Potato Virus X Coat Protein and Foreign Viral RNAs

Acta Naturae ◽  
2011 ◽  
Vol 3 (3) ◽  
pp. 40-46 ◽  
Author(s):  
M V Arkhipenko ◽  
E K Petrova ◽  
N A Nikitin ◽  
A D Protopopova ◽  
E V Dubrovin ◽  
...  
Keyword(s):  
Coat Protein ◽  
Potato Virus ◽  
Potato Virus X ◽  
Viral Rnas ◽  
Virus Like Particles ◽  
Artificial Virus

scholarly journals Translational Activation of Encapsidated Potato Virus X RNA by Coat Protein Phosphorylation

Virology ◽  
2001 ◽  
Vol 286 (2) ◽  
pp. 466-474 ◽  
Author(s):  
J.G. Atabekov ◽  
N.P. Rodionova ◽  
O.V. Karpova ◽  
S.V. Kozlovsky ◽  
V.K. Novikov ◽  
...  
Keyword(s):  
Coat Protein ◽  
Protein Phosphorylation ◽  
Potato Virus ◽  
Potato Virus X ◽  
Translational Activation

Antiviral Activity of Lactoferrin against Potato virus x In vitro and In vivo

2015 ◽  
Vol 10 (2) ◽  
pp. 86-94 ◽  
Author(s):  
Soad H. Taha ◽  
Samah A. Mokbel ◽  
Mahmoud Abdel- Hamid ◽  
Ali H. Hamed
Keyword(s):  
Antiviral Activity ◽  
Potato Virus ◽  
Potato Virus X

scholarly journals cis-Acting sequences required for coat protein binding and in vitro assembly of Potato virus X

Virology ◽  
2005 ◽  
Vol 334 (1) ◽  
pp. 83-97 ◽  
Author(s):  
Sun-Jung Kwon ◽  
Mi-Ri Park ◽  
Ki-Woo Kim ◽  
Carol A. Plante ◽  
Cynthia L. Hemenway ◽  
...  
Keyword(s):  
Coat Protein ◽  
Protein Binding ◽  
Potato Virus ◽  
Potato Virus X ◽  
Cis Acting ◽  
In Vitro Assembly

scholarly journals Plastocyanin Transit Peptide Interacts with Potato virus X Coat Protein, While Silencing of Plastocyanin Reduces Coat Protein Accumulation in Chloroplasts and Symptom Severity in Host Plants

2009 ◽  
Vol 22 (12) ◽  
pp. 1523-1534 ◽  
Author(s):  
Y. Qiao ◽  
H. F. Li ◽  
S. M. Wong ◽  
Z. F. Fan
Keyword(s):  
Coat Protein ◽  
Potato Virus ◽  
Symptom Severity ◽  
Host Plants ◽  
Transit Peptide ◽  
Potato Virus X ◽  
Precursor Protein ◽  
Bimolecular Fluorescence Complementation ◽  
Protein Accumulation

Potato virus X coat protein (PVXCP) is, through communication with host proteins, involved in processes such as virus movement and symptom development. Here, we report that PVXCP also interacts with the precursor of plastocyanin, a protein involved in photosynthesis, both in vitro and in vivo. Yeast two-hybrid analysis indicated that PVXCP interacted with only the plastocyanin transit peptide. In subsequent bimolecular fluorescence complementation assays, both proteins were collocated within chloroplasts. Western blot analyses of chloroplast fractions showed that PVXCP could be detected in the envelope, stroma, and lumen fractions. Transmission electron microscopy demonstrated that grana were dilated in PVX-infected Nicotiana benthamiana. Furthermore, virus-induced gene silencing of plastocyanin by prior infection of N. benthamiana using a Tobacco rattle virus vector reduced the severity of symptoms that developed following subsequent PVX infection as well as the accumulation of PVXCP in isolated chloroplasts. However, PVXCP could not be detected in pea chloroplasts in an in vitro re-uptake assay using the plastocyanin precursor protein. Taken together, these data suggest that PVXCP interacts with the plastocyanin precursor protein and that silencing the expression of this protein leads to reduced PVXCP accumulation in chloroplasts and ameliorates symptom severity in host plants.

scholarly journals Evidence that the Linker between the Methyltransferase and Helicase Domains of Potato Virus X Replicase Is Involved in Homologous RNA Recombination

2009 ◽  
Vol 83 (15) ◽  
pp. 7761-7769 ◽  
Author(s):  
Heidrun-Katharina Draghici ◽  
Mark Varrelmann
Keyword(s):  
Coat Protein ◽  
Potato Virus ◽  
Fluorescent Protein ◽  
Triple Gene Block ◽  
Leaf Tissue ◽  
Potato Virus X ◽  
Wild Type Virus ◽  
Rna Recombination ◽  
Gene Block

ABSTRACT Recombination in RNA viruses, one of the main factors contributing to their genetic variability and evolution, is a widespread phenomenon. In this study, an in vivo assay to characterize RNA recombination in potato virus X (PVX), under high selection pressure, was established. Agrobacterium tumefaciens was used to express in Nicotiana benthamiana leaf tissue both a PVX isolate labeled with green fluorescent protein (GFP) containing a coat protein deletion mutation (ΔCP) and a transcript encoding a functional coat protein +3′-ntr. Coexpression of the constructs led to virus movement and systemic infection; reconstituted recombinants were observed in 92% of inoculated plants. Similar results were obtained using particle bombardment, demonstrating that recombination mediated by A. tumefaciens was not responsible for the occurrence of PXC recombinants. The speed of recombination could be estimated by agroinfection of two PVX mutants lacking the 3′ and 5′ halves of the genome, respectively, with an overlap in the triple gene block 1 gene, allowing GFP expression only in the case of recombination. Ten different pentapeptide insertion scanning replicase mutants with replication abilities comparable to wild-type virus were applied in the different recombination assays. Two neighboring mutants affecting the linker between the methyltransferase and helicase domains were shown to be strongly debilitated in their ability to recombine. The possible functional separation of replication and recombination in the replicase molecule supports the model that RNA recombination represents a distinct function of this protein, although the underlying mechanism still needs to be investigated.

Alterations in the pattern of gene expression following heat shock in the nematode Caenorhabditis elegans

1983 ◽  
Vol 61 (6) ◽  
pp. 480-487 ◽  
Author(s):  
Terry P. Snutch ◽  
David L. Baillie
Keyword(s):  
Caenorhabditis Elegans ◽  
Heat Shock ◽  
Elevated Temperatures ◽  
In Vitro Study ◽  
In Vitro Translation ◽  
Nematode Caenorhabditis Elegans ◽  
Stage Of Development ◽  
Heat Shock Responses

Exposure of the nematode Caenorhabditis elegans to elevated temperatures induces the preferential synthesis of eight major polypeptides of approximate molecular weights 81 000, 70 000, 41 000, 38 000, 29 000, 19 000, 18 000, and 16 000. In pulse-labelled worms these peptides first appear at 29 °C and continue to be synthesized up to lethal temperatures. They are heat inducible at every stage of development. While temperature elevation induces the synthesis of the heat-shock polypeptides, the in vivo synthesis of most other proteins present before heat shock is suppressed. In contrast, in vitro translation of mRNA from heat-shocked worms shows no alteration from the pattern of normal 20 °C mRNAs except for the appearance of the heat-shock mRNAs. An in vitro study of RNA from control and heat-shocked dauer larvae shows that this developmental variant possesses little translatable mRNA but, upon heat shock, synthesizes a set of messages corresponding to the heat-shock polypeptides. The low background of this system will be especially useful in the analysis and purification of heat-shock mRNA for molecular cloning experiments. Extensive similarities between the Drosophila and C. elegans heat-shock responses are shown, including homology between the 70-kdalton heat-shock genes of the two organisms.

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