Mikrobiol. Z. 2021; 83(5):76-81.
In virto and in vivo Phosphorylation of a Coat Protein of Potato Virus X
L.O. Maksymenko, N.Y. Parkhomenko
Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Acad. Zabolotny Str., Kyiv, 03143, Ukraine
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.
Keywords: potato virus X, PVX protein, phosphorylation, protein kinase.
Full text (PDF, in English)
- Ivanov KI, Puustinen P, Merits A, Saarma M, Makinen K. Phosphorylation down-regulates the RNA binding function of the coat protein of potato virus A. J Biol Chem. 2001; 276(17):13530–40. https://doi.org/10.1074/jbc.M009551200
- Law LMJ, Everitt JC, Beatch MD, Holmes CF, Hobman TC. Phosphorylation of rubella virus capsid regulates its RNA binding activity and virus replication. J Virol. 2003; 77(3):1764–71. https://doi.org/10.1128/JVI.77.3.1764-1771.2003
- Makarov VV, Iconnicova AY, Kalinina NO, Guseunov MA, Vishnichenko VK. In vitro phosphorilation of the N-terminal half of hordeivirus movement protein. Biochemistry (Moscow). 2012; 77(9):1072–81. https://doi.org/10.1134/S0006297912090155
- Atabekov JG, Rodionova NP, Karpova OV, Kozlovsky SV, Novikov VK, Arkhipenko MV. Translation activation of encapsidated potato virus X RNA by coat protein phosphorylation. Virology. 2001; 286(2):466–74. https://doi.org/10.1006/viro.2001.1013
- Koenig H, Tremaine JH, Shepard JF. In situ degradation of the protein chain of potato virus X at the N- and C-termini. J Gen Virol. 1978; 38(2):329–337. https://doi.org/10.1099/0022-1317-38-2-329
- Wu G, Bruening G. Two proteins from cowpea mosaic virus. Virology.1971; 46(3):596–612. https://doi.org/10.1016/0042-6822(71)90063-8
- Ouchterlony O. Handbook of immunodiffusion and immunoelectrophoresis. Ann Arbor Science Publishers; 1968.
- Towbin H, Stalhelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and applications. Proc Nat Acad Sci USA. 1979; 76(9):4350–4. https://doi.org/10.1073/pnas.76.9.4350
- Menissier-de Murcia J, Geldreich A, Lebeurier J. Evidence for a protein kinase activity associated with purified particles of cauliflower mosaic virus. J Gen Virol. 1986; 67:1885–91. https://doi.org/10.1099/0022-1317-67-9-1885
- Laeimmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680–5. https://doi.org/10.1038/227680a0
- Oparka KJ, Roberts AG, Roberts IM, Prior DAM, Santa Crus S. Viral coat protein is targeted to, but does not gate, plasmodesmata during cell to cell movement of potato virus X. Plant J. 1996;10(5):805–13. https://doi.org/10.1046/j.1365-313X.1996.10050805.x
- Karpova OV, Rodionova NP, Ivanov KI, Kozlovsky SV, Dorokhov YuL, Atabekov IG. Phosphorylation of tobacco mosaic virus movement protein abolishes its translation repressing ability. Virology. 1999; 261(1):20–4. https://doi.org/10.1006/viro.1999.9842
- Didenko LF, Maksymenko LA, Parkhomenko NI. [In vitro phosphorylation of informosome proteins from leaves of Datura stramonium infected with potato X-virus]. Mikrobiol Z. 1993; 55(2):68–74. Russian.
- Fellers J, Wan J, Hong Y, Collins GB, Hunt AG. In vitro interactions between a potyvirus-encoded, genome linked protein and RNA-dependent RNA-polymerase. J Gen Virol. 1998; 79(8):2043–9. https://doi.org/10.1099/0022-1317-79-8-2043
- Watanabe Y, Ogawa T, Okada Y. In vivo phosphorylation of the 30 kDa protein of tobacco mosaic virus. FEBS Letters. 1992; 313(2):181–4. https://doi.org/10.1016/0014-5793(92)81440-W
- Cao L, Yu B, Kong D, Cong Q, Yu T, Chen Z. Functional expression and characterization of the envelope glycoprotein E1E2 heterodimer of hepatitis C virus. PLoS Pathog. 2019; 15(5):e1007759. https://doi.org/10.1371/journal.ppat.1007759
- Sokolova TM. [Hepatitis C virus (Flaviviridae: Hepacivirus:Hepacivirus C): regulation of signaling reactions of innate immunity]. Problems of Virology. 2020; 65(6):307–16. Russian. https://doi.org/10.36233/0507-4088-2020-65-6-1