An RNA-dependent RNA polymerase is packaged within the virions of purified vesicular stomatitis virus, a nonsegmented negative-strand RNA virus, which carries out transcription of the genome RNA into mRNAs both in vitro and in vivo. The RNA polymerase is composed of two virally encoded polypeptides: a large protein L (240 kDa) and a phosphoprotein P (29 kDa). Recently, we obtained biologically active L protein from insect cells following infection by a recombinant baculovirus expressing L gene. During purification of the L protein from Sf21 cells, we obtained in addition to an active L fraction an inactive fraction that required uninfected insect cell extract to restore its activity. The cellular factors have now been purified, characterized, and shown to be ␤ and ␥ subunits of the protein synthesis elongation factor EF-1. We also demonstrate that the ␣ subunit of EF-1 remains tightly bound to the L protein in the inactive fraction and ␤␥ subunits associate with the L(␣) complex. Further purification of L(␣) from the inactive fraction revealed that the complex is partially active and is significantly stimulated by the addition of ␤␥ subunits purified from Sf21 cells. A putative inhibitor(s) appears to co-elute in the inactive fraction that blocked the L(␣) activity. The purified virions also package all three subunits of EF-1. These findings have a striking similarity with Q␤ RNA phage, which also associates with the bacterial homologue of EF-1 for its replicase function, implicating a possible evolutionary relationship between these host proteins and the RNA-dependent RNA polymerase of RNA viruses.Vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand RNA viruses, has long been a paradigm for studying gene expression of this class of RNA viruses that infect vertebrates, invertebrates, and plants (1). Some of the most common human pathogens that belong to this category are rabies, measles, mumps, and human parainfluenza. A hallmark of all negative strand RNA viruses is the obligate packaging of an RNA-dependent RNA polymerase within the mature virions (2) that transcribes the genome RNA into mRNAs both in vitro and in vivo (3). For VSV, the virion-associated RNA polymerase is generally thought to consist of two virally encoded protein subunits, L (240 kDa) and P (29 kDa), which remain tightly complexed within the virion (3). Studies on the structure and function of VSV RNA polymerase have been greatly aided by the ability to isolate the polymerase subunits from the virions in a relatively pure form (4, 5). Active reconstitution of transcription is achieved by mixing the genome RNA enwrapped with the nucleocapsid protein (N) (referred to as N-RNA template) and purified L and P proteins (4, 5). From a large body of evidence, it appears that the L protein possesses the catalytic activity for RNA synthesis and the P protein is a transcription factor essential for L function (3); no cellular protein(s) has so far been shown to be required for the RNA polymerase activity. Only recently, ...

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 ...

The phosphorylation of the P protein of vesicular stomatitis virus by cellular casein kinase II (CKII) is essential for its activity in viral transcription. Recent in vitro studies have demonstrated that CKII converts the inactive unphosphorylated form of P (P0) to an active phosphorylated form P1, after phosphorylation at two serine residues, Ser-59 and Ser-61. To gain insight into the role of CKII-mediated phosphorylation in the structure and function of the P protein, we have carried out circular dichroism (CD) and biochemical analyses of both P0 and P1. The results of CD analyses reveal that phosphorylation of P0 to P1 significantly increases the predicted ␣-helical structure of the P1 protein from 27 to 48%. The phosphorylation defective double serine mutant (P59/ 61), which is transcriptionally inactive, possesses a secondary structure similar to that of P0. P1, at a protein concentration of 50 g/ml, elutes from a gel filtration column apparently as a dimer, whereas both P0 and the double serine mutant elute as a monomer at the same concentration. Interestingly, unlike wild-type P1 protein, the P mutants in which either Ser-59 or Ser-61 is altered to alanine required a high concentration of CKII for optimal phosphorylation. We demonstrate here that phosphorylation of either Ser-59 or Ser-61 is necessary and sufficient to transactivate L polymerase although alteration of one serine residue significantly decreases its affinity for CKII. We have also shown that P1 binds to the N-RNA template more efficiently than P0 and the formation of P1 is a prerequisite for the subsequent phosphorylation by L protein-associated kinase. In addition, mutant P59/61 acts as a transdominant negative mutant when used in a transcription reconstitution assay in the presence of wild-type P protein.The RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV) 1 consists of two proteins: the large protein L (241 kDa) and the phosphoprotein P (29 kDa). Together, these proteins are needed to transcribe the linear, single-stranded viral RNA genome of negative polarity, which is tightly wrapped with the nucleocapsid N protein (N-RNA template) (1, 3). Genetic and biochemical studies have suggested that the L protein encodes all the basic transcription activities, whereas the P protein appears to be an RNA virus transcription factor (1, 2, 7) with properties similar to many well studied eucaryotic transcription factors/activators (31). The P protein contains ␣-helical coiled structure and is highly acidic, with Asp and Glu residues constituting one-third of the first 100 amino acid residues in the N-terminal half (domain I) of the polypeptide (17, 18). The acidic domain is also phosphorylated by cellular protein kinase (14, 15). The possible contribution of the Nterminal acidic domain I in the function of P protein seems to transactivate the L protein for transcription similar to those observed for eucaryotic acidic transactivators (30 -32). The Cterminal end, on the other hand, serves as the binding site for the L protein (domain ...