In vivo self-interaction of nodavirus RNA replicase protein a revealed by fluorescence resonance energy transfer.
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.
Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, WI 53706, USA.
Billy T
BT
Dye
David J
DJ
Miller
Paul
P
Ahlquist
D000818:Animals; D002478:Cells, Cultured; D004331:Drosophila melanogaster; D031541:Fluorescence Resonance Energy Transfer; D047468:Immunoprecipitation; D008163:Luminescent Measurements; D030381:Nodaviridae / Q000235:genetics*; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry*
D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase