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Journal of Virology, November 2009, p. 11429-11439, Vol. 83, No. 22
0022-538X/09/$08.00+0     doi:10.1128/JVI.00813-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mutations in the C-Terminal Loop of the Nucleocapsid Protein Affect Vesicular Stomatitis Virus RNA Replication and Transcription Differentially{triangledown}

Djamila Harouaka1,2,3 and Gail W. Wertz1,2*

Department of Pathology, University of Virginia, Charlottesville, Virginia,1 Department of Microbiology, University of Virginia, Charlottesville, Virginia,2 Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama3

Received 21 April 2009/ Accepted 21 August 2009

The 2.9-Å structure of the vesicular stomatitis virus nucleocapsid (N) protein bound to RNA shows the RNA to be tightly sequestered between the two lobes of the N protein. Domain movement of the lobes of the N protein has been postulated to facilitate polymerase access to the RNA template. We investigated the roles of individual amino acid residues in the C-terminal loop, involved in long-range interactions between N protein monomers, in forming functional ribonucleoprotein (RNP) templates. The effects of specific N protein mutations on its expression, interaction with the phosphoprotein, and formation of RNP templates that supported viral RNA replication and transcription were examined. Mutations introduced into the C-terminal loop, predicted to break contact with other residues in the loop, caused up to 10-fold increases in RNA replication without an equivalent stimulation of transcription. Mutation F348A, predicted to break contact between the C-terminal loop and the N-terminal arm, formed templates that supported wild-type levels of RNA replication but almost no transcription. These data show that mutations in the C-terminal loop of the N protein can disparately affect RNA replication and transcription, indicating that the N protein plays a role in modulating RNP template function beyond its structural role in RNA encapsidation.

* Corresponding author. Mailing address: Department of Pathology, University of Virginia, MR5 Building, P.O. Box 800904, Charlottesville, VA 22908-0904. Phone: (434) 982-6039. Fax: (434) 982-2151. E-mail: gww4f{at}virginia.edu

{triangledown} Published ahead of print on 2 September 2009.

Journal of Virology, November 2009, p. 11429-11439, Vol. 83, No. 22
0022-538X/09/$08.00+0     doi:10.1128/JVI.00813-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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