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Journal of Virology, September 2002, p. 8737-8746, Vol. 76, No. 17
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.17.8737-8746.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of a Regulatory Domain on the Carboxyl Terminus of the Measles Virus Nucleocapsid Protein

Xinsheng Zhang,1 Candace Glendening,1 Hawley Linke,1 Christopher L. Parks,2 Charles Brooks,1 Stephen A. Udem,2 and Michael Oglesbee1*

Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio 43210,1 Department of Viral Vaccine Research, Wyeth-Lederle Vaccines and Pediatrics, Pearl River, New York 109652

Received 15 April 2002/ Accepted 23 May 2002

The paramyxovirus template for transcription and genome replication consists of the RNA genome encapsidated by the nucleocapsid protein (N protein). The activity of the complex, consisting of viral polymerase plus template, can be measured with minireplicons in which the genomic coding sequence is replaced by chloramphenical acetyltransferase (CAT) antisense RNA. Using this approach, we showed that the C-terminal 24 amino acids of the measles virus N protein are dispensable for transcription and replication, based upon the truncation of N proteins used to support minireplicon reporter gene expression. Truncation at the C-terminal or penultimate amino acid 524 resulted in no change in CAT expression, whereas larger truncations spanning residues 523 to 502 were accompanied by an approximately twofold increase in basal activity. Reporter gene expression was enhanced by supplementation with the major inducible 70-kDa heat shock protein (Hsp72) for minireplicons with the N protein or the N protein truncated at position 525 or 524 but not in systems with a truncation at position 523 or 522. Naturally occurring sequence variants of the N protein with variations at positions 522 and 523 were also shown to lack Hsp72 responsiveness independent of changes in basal activity. Since these residues lie within a linear sequence predicting a direct Hsp72 interaction, N protein-Hsp72 binding reactions were analyzed by using surface plasmon resonance technology. Truncation of the C-terminal portion of the N protein by protease digestion resulted in a reduced binding affinity between Hsp72 and the N protein. Furthermore, with synthetic peptides, we established a correlation between the functional responsiveness and the binding affinity for Hsp72 of C-terminal N protein sequences. Collectively, these results show that the C-terminal 24 amino acids of the N protein represent a regulatory domain containing a functional motif that mediates a direct interaction with Hsp72.

* Corresponding author. Mailing address: Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210. Phone: (614) 292-9672. Fax: (614) 292-6473. E-mail: oglesbee.1{at}osu.edu.

Journal of Virology, September 2002, p. 8737-8746, Vol. 76, No. 17
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.17.8737-8746.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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