Mapping the interacting domains between the rabies virus polymerase and phosphoprotein.
The 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 vaccinia virus-T7 RNA polymerase recombinant in mammalian cells, they formed a complex as detected by coimmunoprecipitation. Analysis of P and L deletion mutants was performed to identify the regions of both proteins involved in complex formation. The interaction of P with L was not disrupted by large deletions removing the carboxy-terminal half of the P protein. On the contrary, P proteins containing a deletion in the amino terminus were defective in complex formation with L. Moreover, fusion proteins containing the 19 or the 52 first residues of P in frame with green fluorescent protein (GFP) still bound to L. These results indicate that the major L binding site resides within the 19 first residues of the P protein. We also mapped the region of L involved in the interaction with P. Mutant L proteins consisting of the carboxy-terminal 1,656, 956, 690, and 566 amino acids all bound to the P protein, whereas deletion of 789 residues within the terminal region eliminated binding to P protein. This result demonstrates that the carboxy-terminal domain of L is required for the interaction with P.
Chenik
M
M
Laboratoire de Génétique des Virus, CNRS, Gif sur Yvette, France.
Schnell
M
M
Conzelmann
K K
KK
Blondel
D
D
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002874:Chromosome Mapping; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D018832:Molecular Chaperones; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D011820:Rabies virus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics* / Q000378:metabolism
D018832:Molecular Chaperones; C103961:P phosphoprotein, Rabies virus; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C097625:L protein, Rabies virus; D012321:DNA-Directed RNA Polymerases