Single-Amino-Acid Alterations in a Highly Conserved Central Region of Vesicular Stomatitis Virus N Protein Differentially Affect the Viral Nucleocapsid Template Functions

Authors

Debasis Nayak, University of Nebraska - Lincoln
Debasis Panda, University of Nebraska - LincolnFollow
Subash C. Das, University of Nebraska - LincolnFollow
Ming Lou, University of Alabama, Birmingham
Asit K. Pattnaik, University of Nebraska-LincolnFollow

Document Type

Article

Date of this Version

6-2009

Citation

JOURNAL OF VIROLOGY, June 2009, p. 5525–5534

Comments

Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Abstract

The nucleocapsid protein (N) of vesicular stomatitis virus and other rhabdoviruses plays a central role in the assembly and template functions of the viral N-RNA complex. The crystal structure of the viral N-RNA complex suggests that the central region of the N protein interacts with the viral RNA. Sequence alignment of rhabdovirus N proteins revealed several highly conserved regions, one of which spanned residues 282 to 291 (GLSSKSPYSS) in the central region of the molecule. Alanine-scanning mutagenesis of this region suggested that replacement of the tyrosine residue at position 289 (Y289) with alanine resulted in an N-RNA template that is nonfunctional in viral genome replication and transcription. To establish the molecular basis of this defect, our further studies revealed that the Y289A mutant maintained its interaction with other N protein molecules but that its interactions with the P protein or with the viral RNA were defective. Replacement of Y289 with other aromatic, polar, or large amino acids indicated that the hydrophobic and aromatic nature of this position in the N protein is functionally important and that a larger aromatic residue is less favorable. Interestingly, we have observed that several single-amino-acid substitutions in this highly conserved region of the molecule rendered the nucleocapsid template nonfunctional in transcription without adversely affecting the replication functions. These results suggest that the structure of the N protein and the resulting N-RNA complex, in part, regulate the viral template functions in transcription and replication.