The phosphoprotein of rabies virus is phosphorylated by a unique cellular protein kinase and specific isomers of protein kinase C.
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 S(63) and S(64), whereas PKC isomers phosphorylated at S(162), S(210), and S(271). 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.
Gupta
A K
AK
Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Blondel
D
D
Choudhary
S
S
Banerjee
A K
AK
D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D018832:Molecular Chaperones; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015678:Viral Structural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D018832:Molecular Chaperones; C103961:P phosphoprotein, Rabies virus; D010750:Phosphoproteins; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D011493:Protein Kinase C