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Journal of Virology, May 2009, p. 4498-4507, Vol. 83, No. 9
0022-538X/09/$08.00+0     doi:10.1128/JVI.00040-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mitochondrion-Enriched Anionic Phospholipids Facilitate Flock House Virus RNA Polymerase Membrane Association{triangledown}

Kenneth A. Stapleford,1 Doron Rapaport,2 and David J. Miller1,3,4*

Program in Cellular and Molecular Biology,1 Departments of Internal Medicine,3 Microbiology & Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109,4 Interfaculty Institute for Biochemistry, Hoppe-Seyler-Strasse 4, University of Tübingen, 72076 Tübingen, Germany2

Received 8 January 2009/ Accepted 13 February 2009

One characteristic of all positive-strand RNA viruses is the necessity to assemble viral RNA replication complexes on host intracellular membranes, a process whose molecular details are poorly understood. To study viral replication complex assembly we use the established model system of Flock House virus (FHV), which assembles its replication complexes on the mitochondrial outer membrane. The FHV RNA-dependent RNA polymerase, protein A, is the only viral protein necessary for genome replication in the budding yeast Saccharomyces cerevisiae. To examine the host components involved in protein A-membrane interactions, an initial step of FHV RNA replication complex assembly, we established an in vitro protein A membrane association assay. Protein A translated in vitro rapidly and specifically associated with mitochondria isolated from yeast, insect, and mammalian cells. This process was temperature dependent but independent of protease-sensitive mitochondrial outer membrane components or the host mitochondrial import machinery. Furthermore, lipid-binding studies revealed that protein A preferentially bound to specific anionic phospholipids, in particular the mitochondrion-specific phospholipid cardiolipin. These studies implicate membrane phospholipids as important host determinants for FHV RNA polymerase membrane association and provide evidence for the involvement of host phospholipids in positive-strand RNA virus membrane-specific targeting.

* Corresponding author. Mailing address: 3560B MSRB II, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-5688. Phone: (734) 763-0565. Fax: (734) 764-0101. E-mail: milldavi{at}umich.edu

{triangledown} Published ahead of print on 25 February 2009.

Journal of Virology, May 2009, p. 4498-4507, Vol. 83, No. 9
0022-538X/09/$08.00+0     doi:10.1128/JVI.00040-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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