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Journal of Virology, January 2000, p. 91-98, Vol. 74, No. 1
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The Phosphoprotein of Rabies Virus Is Phosphorylated by a Unique Cellular Protein Kinase and Specific Isomers of Protein Kinase C

Ashim K. Gupta,1 Danielle Blondel,2 Suresh Choudhary,1 and Amiya K. Banerjee1,*

Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195,1 and Laboratoire de Genetique des Virus, CNRS, 91198 Gif sur Yvette cedex, France2

Received 10 June 1999/Accepted 28 September 1999

The phosphoprotein (P) gene of rabies virus (CVS strain) was cloned and expressed in bacteria. The purified protein was used as the substrate for phosphorylation by the protein kinase(s) present in cell extract prepared from rat brain. Two distinct types of protein kinases, staurosporin sensitive and heparin sensitive, were found to phosphorylate the P protein in vitro by the cell extract. Interestingly, the heparin-sensitive kinase was not the ubiquitous casein kinase II present in a variety of cell types. Further purification of the cell fractions revealed that the protein kinase C (PKC) isomers constitute the staurosporin-sensitive kinases alpha , beta , gamma , and zeta , with the PKCgamma isomer being the most effective in phosphorylating the P protein. A unique heparin-sensitive kinase was characterized as a 71-kDa protein with biochemical properties not demonstrated by any known protein kinases stored in the protein data bank. This protein kinase, designated RVPK (rabies virus protein kinase), phosphorylates P protein (36 kDa) and alters its mobility in gel to migrate at 40 kDa. In contrast, the PKC isoforms do not change the mobility of unphosphorylated P protein. RVPK appears to be packaged in the purified virions, to display biochemical characteristics similar to those of the cell-purified RVPK, and to similarly alter the mobility of endogenous P protein upon phosphorylation. By site-directed mutagenesis, the sites of phosphorylation of RVPK were mapped at S63 and S64, whereas PKC isomers phosphorylated at S162, S210, and S271. Involvement of a unique protein kinase in phosphorylating rabies virus P protein indicates its important role in the structure and function of the protein and consequently in the life cycle of the virus.

* Corresponding author. Mailing address: Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., NC20, Cleveland, OH 44195. Phone: (216) 444-0625. Fax: (216) 444-0512. E-mail: banerja{at}ccf.org.

Journal of Virology, January 2000, p. 91-98, Vol. 74, No. 1
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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