Affiliation:
1. Department of Medicine
2. Institute for Molecular Virology
3. Howard Hughes Medical Institute, University of Wisconsin—Madison, Madison, Wisconsin 53706
Abstract
ABSTRACT
Localization of RNA replication to intracellular membranes is a universal feature of positive-strand RNA viruses. Replication complexes of flock house virus (FHV), the best-studied alphanodavirus, are located on outer mitochondrial membranes in infected
Drosophila melanogaster
cells and are associated with the formation of membrane-bound spherules, similar to structures found for many other positive-strand RNA viruses. To further study FHV replication complex formation, we investigated the subcellular localization, membrane association, and membrane topology of protein A, the FHV RNA-dependent RNA polymerase, in the yeast
Saccharomyces cerevisiae
, a host able to support full FHV RNA replication and virion formation. Confocal immunofluorescence revealed that protein A localized to mitochondria in yeast, as in
Drosophila
cells, and that this mitochondrial localization was independent of viral RNA synthesis. Nycodenz gradient flotation and dissociation assays showed that protein A behaved as an integral membrane protein, a finding consistent with a predicted N-proximal transmembrane domain. Protease digestion and selective permeabilization after differential epitope tagging demonstrated that protein A was inserted into the outer mitochondrial membrane with the N terminus in the inner membrane space or matrix and that the C terminus was exposed to the cytoplasm. Flotation and immunofluorescence studies with deletion mutants indicated that the N-proximal region of protein A was important for both membrane association and mitochondrial localization. Gain-of-function studies with green fluorescent protein fusions demonstrated that the N-terminal 46 amino acids of protein A were sufficient for mitochondrial localization and membrane insertion. We conclude that protein A targets and anchors FHV RNA replication complexes to outer mitochondrial membranes, in part through an N-proximal mitochondrial localization signal and transmembrane domain.
Publisher
American Society for Microbiology
Subject
Virology,Insect Science,Immunology,Microbiology
Reference62 articles.
1. Requirements for the self-directed replication of flock house virus RNA 1
2. Ball, L. A., and K. L. Johnson. 1998. Nodaviruses of insects, p. 225-267. In L. K. Miller and L. A. Ball (ed.), The insect viruses. Plenum Publishing, New York, N.Y.
3. Bashiruddin, J. B., and G. F. Cross. 1987. Boolarra virus: ultrastructure of intracytoplasmic virus formation in cultured Drosophila cells. J. Invertebr. Pathol. 49 : 303-315.
4. Bonifacino, J. S. 2000. Characterization of cellular proteins, p. 5.0.1-5.5.11. In K. S. Morgan (ed.), Current protocols in cell biology. John Wiley & Sons, Inc., New York, N.Y.
5. Burgyan, J., L. Rubino, and M. Russo. 1996. The 5′-terminal region of a tombusvirus genome determines the origin of multivesicular bodies. J. Gen. Virol. 77 : 1967-1974.
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