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Journal of Virology, January 2002, p. 68-77, Vol. 76, No. 1
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.1.68-77.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

The C-Terminal 88 Amino Acids of the Sendai Virus P Protein Have Multiple Functions Separable by Mutation

Jeffery Tuckis, Sherin Smallwood, Joyce A. Feller, and Sue A. Moyer^*

Department of Molecular Genetics and Microbiology University of Florida College of Medicine, Gainesville, Florida 32610

Received 2 August 2001/ Accepted 27 September 2001

The Sendai virus P-L polymerase complex binds the NP-encapsidated nucleocapsid (NC) template through a P-NP interaction. To identify P amino acids responsible for binding we performed site-directed mutagenesis on the C-terminal 88 amino acids in the NC binding domain. The mutant P proteins expressed from plasmids were assayed for viral RNA synthesis and for various protein-protein interactions. All the mutants formed P oligomers and bound to L protein. While two mutants, JT3 and JT8, retained all P functions at or near the levels of wild-type (wt) P, three others—JT4, JT6, and JT9—were completely defective for both transcription and genome replication in vitro. Each of the inactive mutants retained significant NC binding but had a different spectrum of other binding interactions and activities, suggesting that the NC binding domain also affects the catalytic function of the polymerase. NC binding was inhibited by combinations of the inactive mutations. The remaining P mutants were active in transcription but defective in various aspects of genome replication. Some P mutants were defective in NP₀ binding and abolished the reconstitution of replication from separate P-L and NP₀-P complexes. In some of these cases the coexpression of the wt polymerase with the mutant NP₀-P complex could rescue the defect in replication, suggesting an interaction between these complexes. For some P mutants replication occurred in vivo, but not in vitro, suggesting that the intact cell is providing an unknown function that cannot be reproduced in extracts of cells. Thus, the C-terminal region of P is complex and possesses multiple functions besides NC binding that can be separated by mutation.

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* Corresponding author. Mailing address: University of Florida College of Medicine, Department of Molecular Genetics and Microbiology, 1600 S.W. Archer Rd., P.O. Box 100266, Gainesville, FL 32610-0266. Phone: (352) 392-3131. Fax: (352) 846-2042. E-mail: smoyer{at}ufl.edu.

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Journal of Virology, January 2002, p. 68-77, Vol. 76, No. 1
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.1.68-77.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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