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Journal of Virology, October 2001, p. 9613-9622, Vol. 75, No. 20
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.
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
*
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.
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