title: Functional interaction map of lyssavirus phosphoprotein: identification of the minimal transcription domains.
abstract: Lyssaviruses, the causative agents of rabies encephalitis, are distributed in seven genotypes. The phylogenetically distant rabies virus (PV strain, genotype 1) and Mokola virus (genotype 3) were used to develop a strategy to identify functional homologous interactive domains from two proteins (P and N) which participate in the viral ribonucleoprotein (RNP) transcription-replication complex. This strategy combined two-hybrid and green fluorescent protein-reverse two-hybrid assays in Saccharomyces cerevisiae to analyze protein-protein interactions and a reverse genetic assay in mammalian cells to study the transcriptional activity of the reconstituted RNP complex. Lyssavirus P proteins contain two N-binding domains (N-BDs), a strong one encompassing amino acid (aa) 176 to the C terminus and a weak one in the 189 N-terminal aa. The N-terminal portion of P (aa 52 to 189) also contains a homomultimerization site. Here we demonstrate that N-P interactions, although weaker, are maintained between proteins of the different genotypes. A minimal transcriptional module of the P protein was obtained by fusing the first 60 N-terminal aa containing the L protein binding site to the C-terminal strong N-BD. Random mutation of the strong N-BD on P protein identified three highly conserved K residues crucial for N-P interaction. Their mutagenesis in full-length P induced a transcriptionally defective RNP. The analysis of homologous interactive domains presented here and previously reported dissections of the P protein allowed us to propose a model of the functional interaction network of the lyssavirus P protein. This model underscores the central role of P at the interface between L protein and N-RNA template.
authors.affiliation: Laboratoire des Lyssavirus, Institut Pasteur, 75724 Paris Cedex 15, France. yjacob@pasteur.fr
authors.forename: Y
authors.initials: Y
authors.lastname: Jacob
authors.forename: E
authors.initials: E
authors.lastname: Real
authors.forename: N
authors.initials: N
authors.lastname: Tordo
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D012321:DNA-Directed RNA Polymerases; D005838:Genotype; D018114:Lyssavirus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D019590:Nucleocapsid Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012261:Ribonucleoproteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
chemical_list: D019590:Nucleocapsid Proteins; D010750:Phosphoproteins; D012261:Ribonucleoproteins; D014764:Viral Proteins; C097625:L protein, Rabies virus; D012321:DNA-Directed RNA Polymerases