The first 19 or 52 amino acids of P were fused to the GFP protein as described in Materials and Methods (Fig. to L. These results indicate that this major L binding site resides within the 19 first residues of the P protein. We also mapped the region of L involved in the conversation with P. Mutant L proteins consisting of the carboxy-terminal 1,656, 956, 690, and 566 amino acids all bound to the P protein, whereas deletion of 789 residues within the terminal region eliminated binding to P protein. This result demonstrates that this carboxy-terminal domain name of L is required for the conversation with P. Rhabdoviruses contain a single-stranded negative-sense RNA genome (11 to 15 kb) which is usually tightly encapsidated with the viral nucleoprotein (N) to form an RNP (nucleocapsid) template for transcription and Rabbit polyclonal to ATP5B replication. During transcription, a 47-nucleotide-long leader RNA and five capped and polyadenylated mRNAs are synthesized (32). The replication process yields nucleocapsids made up of full-length antigenome-sense RNA which in turn serve as templates for the synthesis of genome-sense RNA. The active virus-encoded RNA polymerase complex is composed of the large protein (L) and its cofactor, the phosphoprotein (P) (14). The L protein is usually a multifunctional enzyme and is the RNA-dependent RNA polymerase. This protein may carry out all enzymatic actions of transcription, including initiation and elongation of transcripts as well as cotranscriptional modifications of RNAs such as capping, methylation, and polyadenylation (1). The sequences of rhabdovirus L-protein amino acids have been compared with those of other negative-strand RNA viruses (9, 27, 33). Four motifs (A to D) constitute JNJ0966 the so-called polymerase module and are conserved in all viral RNA-dependent DNA and RNA polymerases (24). Part of the highly conserved motif C which is located within the amino-terminal half of the rabies virus L protein has recently been shown to be involved in the formation of the catalytic center of the protein (26). Functions of the P protein are not well defined. Studies with vesicular stomatitis virus (VSV), JNJ0966 the best-characterized rhabdovirus, have shown that this P protein is usually a noncatalytic cofactor and a regulatory protein: it associates with the JNJ0966 L protein in the polymerase complex and interacts with both soluble and genome-associated N protein (13, 21, 30). The P protein has different phosphorylation says and is believed to bind with different affinities to the RNP template and to have different transcription activities (2, 3, 17). Furthermore, the VSV P protein has been shown to form multimers, and multimerization seems to be necessary for binding both to the L protein and to the template (12, 17). Rabies virus and VSV are structurally comparable. Thus, by analogy, their RNA polymerase complexes may have comparable properties. In vitro and in vivo studies have shown that rabies virus P protein forms specific complexes with N proteins (8, 15). We have previously exhibited the presence of two N-protein binding sites around the P protein; one is located between amino acids 69 and 177, and another requires the carboxy-terminal region comprising the amino acids 268 to 297 (8). The rabies virus P protein has at least two differently phosphorylated forms (34). Four additional proteins (P2, P3, P4, and P5) translated from the P mRNA have been found in purified virus, in infected cells, and in cells transfected JNJ0966 with a plasmid encoding the complete P protein. Translation of these proteins is initiated from internal in-frame AUG initiation codons by a leaky scanning mechanism (7). To characterize functional domains of the rabies virus RNA polymerase, we have expressed P and L proteins from plasmids in cultured cells, and we show that they form a complex that can be immunoprecipitated. Analyses of P-protein deletion mutants reveal that this amino-terminal residues of the P protein are involved in the interaction with the L protein and that they are sufficient to mediate binding to L of a green fluorescent protein (GFP) fusion protein. Analysis of the P-L complex formation with truncated L proteins shows that binding to the P protein is usually mediated by the carboxy-terminal part of the L protein. MATERIALS AND METHODS Cells and virus. BSR cells, cloned from BHK-21 (baby hamster kidney) cells, were produced in Eagles minimal essential medium supplemented with 10% calf serum. The CVS strain of rabies virus was cultivated and purified as previously described (18). Recombinant vaccinia virus vTF7-3, made up of the T7 RNA polymerase gene, was kindly provided by B. Moss, National Institutes.