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Journal of Virology, October 2001, p. 9613-9622, Vol. 75, No. 20
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.20.9613-9622.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Functional Interaction Map of Lyssavirus Phosphoprotein: Identification of the Minimal Transcription Domains

Yves Jacob,* Eléonore Real, and Noël Tordo

Laboratoire des Lyssavirus, Institut Pasteur, 75724 Paris Cedex 15, France

Received 16 May 2001/Accepted 11 July 2001

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.

* Corresponding author. Mailing address: Laboratoire des Lyssavirus, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. Phone: (33) 1-45 68 87 53. Fax: (33) 1-40 61 32 56. E-mail: yjacob{at}pasteur.fr.

Journal of Virology, October 2001, p. 9613-9622, Vol. 75, No. 20
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.20.9613-9622.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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