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

Abstract

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 ␣, ␤, ␥, and , with the PKC␥ 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.Like viruses belonging to the rhabdovirus and paramyxovirus family, Rabies virus (RV), a member of Lyssavirus genus, contains a linear nonsegmented RNA genome of negative polarity. The ribonucleoprotein (RNP) complex contains the genome RNA enwrapped by the nucleocapsid protein (N) and the RNA polymerase, which contains a large protein (L) and the phosphoprotein (P) (1). Both L and P proteins of RV, like the corresponding proteins in rhabdoviruses and paramyxoviruses, are involved in the transcription and replication of the genome RNA (1, 46). Although the L proteins of this class of viruses show significant similarity in amino acid sequence, the P proteins appear to be highly divergent and nonhomologous. However, all P proteins are structurally similar in being highly acidic and phosphorylated and in playing a common vital role as transcription factors for the function of the corresponding L proteins. The P proteins, in addition to providing the transcription function of the L protein, appear to play an important role in the replicative process as well (19,22,28,34,36). In this role, the P protein forms a complex intracellularly with the N protein to impart an undefined replication-competent form to the latter which enables it to encapsidate nascent RNA chains during the replicative reaction ...

Genetic Analysis of Phosphoprotein and Matrix Protein of Rabies Viruses Isolated in Brazil

“…In addition, Ser 145 was found to be conserved predominantly carnivorarelated RV isolates while Ala 145 was predominant in chiropteran-related RV isolates. Phosphoacceptors associated with protein kinase C (PKC) or RV protein kinase (RVPK) were identified as Ser 63 , Ser 64 , Ser 162 , Ser 210 and Ser 271 in the P protein of the CVS strain [13]. The phosphoacceptors associated with PKC, Ser 162 , Ser 210 and Ser 271 , were retained in all Brazilian RV isolates, while those of RVPK, Ser 63 and Ser 64 , were not.…”

“…In five serine residues which constitute the phosphoacceptors in the P protein of the CVS strain [13], Ser 210 and Ser 271 within the PKC phosphoacceptor target were notably conserved in all lyssavirus genotypes (GT 1 to 7) [32], including the Brazilian RV variants examined in this study. Studies on VSV and paramyxovirus have shown that P gene-encoded multiple proteins are important in the viral replication cycle and pathogenicity of the virus [12,24,37].…”

Rabies virus P and small P products interact directly with PML and reorganize PML nuclear bodies

“…The major L-binding site resides within the first 19 residues of P (Chenik et al, 1998;Jacob et al, 2001). The rabies virus P protein is phosphorylated by two kinases: the unique cellular protein kinase, RVPK (rabies virus protein kinase), and protein kinase C (Gupta et al, 2000). Both kinases phosphorylate specific sites on the P protein, resulting in the formation of different phosphorylated forms of the P protein with different motilities in SDS -PAGE (Gupta et al, 2000).…”

“…The rabies virus P protein is phosphorylated by two kinases: the unique cellular protein kinase, RVPK (rabies virus protein kinase), and protein kinase C (Gupta et al, 2000). Both kinases phosphorylate specific sites on the P protein, resulting in the formation of different phosphorylated forms of the P protein with different motilities in SDS -PAGE (Gupta et al, 2000). In addition, four other aminoterminally truncated products (P2, P3, P4 and P5) translated from P mRNA have been found in the purified virus, in infected cells and in cells transfected with a plasmid encoding the complete P protein.…”

Studies on the Rabies Virus RNA Polymerase: 3. Two‐Dimensional Electrophoretic Analysis of the Multiplicity of Non‐Catalytic Subunit (P Protein)

“…In vitro phosphorylation assays strongly suggested that p37 serves as a precursor for p40 production, which was shown to occur even in the absence of the other viral gene products and was suggested to be catalyzed by cellular heparin-sensitive enzyme(s) (31). More recently, Gupta et al (14) suggested that a cellular heparin-sensitive 71-kDa kinase (termed the rabies virus protein kinase; RVPK) is involved in p40 production, while a staurosporine-sensitive kinase (PKC gamma isomer) is involved in the phosphorylation of p37 without affecting the electrophoretic mobility.…”