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Journal of Virology, July 2005, p. 8909-8919, Vol. 79, No. 14
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.14.8909-8919.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

In Vivo Self-Interaction of Nodavirus RNA Replicase Protein A Revealed by Fluorescence Resonance Energy Transfer

Billy T. Dye,1,2 David J. Miller,2,{dagger} and Paul Ahlquist1,2*

Howard Hughes Medical Institute,1 Institute for Molecular Virology, University of Wisconsin, Madison, Wisconsin 537062

Received 21 January 2005/ Accepted 29 March 2005

Flock house virus (FHV) is the best-characterized member of the Nodaviridae, a family of small, positive-strand RNA viruses. Unlike most RNA viruses, FHV encodes only a single polypeptide, protein A, that is required for RNA replication. Protein A contains a C-proximal RNA-dependent RNA polymerase domain and localizes via an N-terminal transmembrane domain to the outer mitochondrial membrane, where FHV RNA replication takes place in association with invaginations referred to as spherules. We demonstrate here that protein A self-interacts in vivo by using flow cytometric analysis of fluorescence resonance energy transfer (FRET), spectrofluorometric analysis of bioluminescence resonance energy transfer, and coimmunoprecipitation. Several nonoverlapping protein A sequences were able to independently direct protein-protein interaction, including an N-terminal region previously shown to be sufficient for localization to the outer mitochondrial membrane (D. J. Miller and P. Ahlquist, J. Virol. 76:9856-9867, 2000). Mutations in protein A that diminished FRET also diminished FHV RNA replication, a finding consistent with an important role for protein A self-interaction in FHV RNA synthesis. Thus, the results imply that FHV protein A functions as a multimer rather than as a monomer at one or more steps in RNA replication.

* Corresponding author. Mailing address: Institute for Molecular Virology, University of Wisconsin-Madison, 1525 Linden Dr., Madison, WI 53706. Phone: (608) 263-5916. Fax: (608) 265-9214. E-mail: ahlquist{at}facstaff.wisc.edu

{dagger} Present address: Division of Infectious Diseases, Department of Medicine, University of Michigan Medical School, Ann Arbor, Michigan.

Journal of Virology, July 2005, p. 8909-8919, Vol. 79, No. 14
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.14.8909-8919.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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