JOURNAL OF VIROLOGY,0022-538X/98/$04.000Copyright © 1998, American Society for MicrobiologyMar. 1998, p. 1925–1930Vol. 72, No. 3Mapping the Interacting Domains between the Rabies VirusPolymerase and PhosphoproteinM. CHENIK,1M. SCHNELL,2K. K. CONZELMANN,2AND D. BLONDEL1*Laboratoire de Ge ´ne ´tique des Virus, CNRS, 91198 Gif sur Yvette Cedex, France,1and Federal Research Centre forVirus Diseases of Animals, D-7400 Tu ¨bingen, Federal Republic of Germany2Received 22 August 1997/Accepted 8 December 1997The RNA polymerase of rabies virus consists of two subunits, the large (L) protein and the phosphoprotein(P), with 2,127 and 297 amino acids, respectively. When these proteins were coexpressed via the vacciniavirus-T7 RNA polymerase recombinant in mammalian cells, they formed a complex as detected by coimmu-noprecipitation. Analysis of P and L deletion mutants was performed to identify the regions of both proteinsinvolved in complex formation. The interaction of P with L was not disrupted by large deletions removing thecarboxy-terminal halfofthe P protein. On the contrary, P proteins containing a deletion in the amino terminuswere defective in complex formation with L. Moreover, fusion proteins containing the 19 or the 52 first residuesof P in frame with green fluorescent protein (GFP) still bound to L. These results indicate that the major Lbinding site resides within the 19 first residues of the P protein. We also mapped the region of L involved inthe interaction with P. Mutant L proteins consisting of the carboxy-terminal 1,656, 956, 690, and 566 aminoacids all bound to the P protein, whereas deletion of789 residues within the terminal region eliminated bindingto P protein. This result demonstrates that the carboxy-terminal domain of L is required for the interactionwith P.Rhabdoviruses contain a single-stranded negative-senseRNA genome (11 to 15 kb) which is tightly encapsidated withthe viral nucleoprotein (N) to form an RNP (nucleocapsid)template for transcription and replication. During transcrip-tion, a 47-nucleotide-long leader RNA and five capped andpolyadenylated mRNAs are synthesized (32). The replicationprocess yields nucleocapsids containing full-length anti-genome-sense RNA which in turn serve as templates for thesynthesis of genome-sense RNA. The active virus-encodedRNA polymerase complex is composed of the large protein (L)and its cofactor, the phosphoprotein (P) (14). The L protein isa multifunctional enzyme and is the RNA-dependent RNApolymerase. This protein may carry out all enzymatic steps oftranscription, including initiation and elongation of transcriptsas well as cotranscriptional modifications of RNAs such ascapping, methylation, and polyadenylation (1). The sequencesof rhabdovirus L-protein amino acids have been comparedwith those of other negative-strand RNA viruses (9, 27, 33).Four motifs (A to D) constitute the so-called polymerase mod-ule and are conserved in all viral RNA-dependent DNA andRNA polymerases (24). Part of the highly conserved motif Cwhich is located within the amino-terminal half of the rabiesvirus L protein has recently been shown to be involved in theformation of the catalytic center of the protein (26). Functionsof the P protein are not well defined. Studies with vesicularstomatitis virus (VSV), the best-characterized rhabdovirus,have shown that the P protein is a noncatalytic cofactor and aregulatory protein: it associates with the L protein in the poly-merase complex and interacts with both soluble and genome-associated N protein (13, 21, 30). The P protein has differentphosphorylation states and is believed to bind with differentaffinities to the RNP template and to have different transcrip-tion activities (2, 3, 17). Furthermore, the VSV P protein hasbeen shown to form multimers, and multimerization seems tobe necessary for binding both to the L protein and to thetemplate (12, 17). Rabies virus and VSV are structurally sim-ilar. Thus, by analogy, their RNA polymerase complexes mayhave similar properties. In vitro and in vivo studies have shownthat rabies virus P protein forms specific complexes with Nproteins (8, 15). We have previously demonstrated the exis-tence of two N-protein binding sites on the P protein; one islocated between amino acids 69 and 177, and another requiresthe carboxy-terminal region comprising the amino acids 268 to297 (8). The rabies virus P protein has at least two differentlyphosphorylated forms (34). Four additional proteins (P2, P3,P4, and P5) translated from the P mRNA have been found inpurified virus, in infected cells, and in cells transfected with aplasmid encoding the complete P protein. Translation of theseproteins is initiated from internal in-frame AUG initiationcodons by a leaky scanning mechanism (7).To characterize functional domains of the rabies virus RNApolymerase, we have expressed P and L proteins from plasmidsin cultured cells, and we show that they form a complex thatcan be immunoprecipitated. Analyses of P-protein deletionmutants reveal that the amino-terminal residues of the P pro-tein are involved in the interaction with the L protein and thatthey are sufficient to mediate binding to L of a green fluores-cent protein (GFP) fusion protein. Analysis ofthe P-L complexformation with truncated L proteins shows that binding to theP protein is mediated by the carboxy-terminal part of the Lprotein.MATERIALS AND METHODSCells and virus. BSR cells, cloned from BHK-21 (baby hamster kidney) cells,were grown in Eagle’s minimal essential medium supplemented with 10% calfserum. The CVS strain of rabies virus was cultivated and purified as previouslydescribed (18).Recombinant vaccinia virus vTF7-3, containing the T7 RNA polymerase gene,was kindly provided by B. Moss, National Institutes of Health, Bethesda, Md.(16).* Corresponding author. Mailing address: Laboratoire de Ge ´ne ´t-ique des Virus, CNRS, 91198 Gif sur Yvette Cedex, France. Phone: 331 69 82 38 37. Fax: 33 1 69 82 43 08. E-mail: danielle blondel@cnrs-gif.fr.1925
