Functional interaction map of lyssavirus phosphoprotein: Identification ofthe minimal transcription domains

Lyssaviruses, the causative agents of rabies encephalitis, are distributed in seven genotypes. The phylogenetically distant rabies virus (PV strain, g enotype 1) and Mokola virus (genotype 3) were used to develop a strategy to identify functional homologous interactive domains from two proteins (P an d N) which participate in the viral ribonucleoprotein (RNP) transcription-r eplication complex. This strategy combined two-hybrid and green fluorescent protein-reverse two-hybrid assays in Saccharomyces cerevisiae to analyze p rotein-protein interactions and a reverse genetic assay in mammalian cells to study the transcriptional activity of the reconstituted RNP complex. Lys savirus P proteins contain two N-binding domains (N-BDs), a strong one enco mpassing 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 hom omultimerization site. Here we demonstrate that N-P interactions, although weaker, are maintained between proteins of the different genotypes. A minim al transcriptional module of the P protein was obtained by fusing the first 60 N-terminal as containing the L protein binding site to the C-terminal s trong N-BD. Random mutation of the strong N-BD on P protein identified thre e highly conserved K residues crucial for N-P interaction. Their mutagenesi s in full-length P induced a transcriptionally defective RNP. The analysis of homologous interactive domains presented here and previously reported di ssections 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.