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Genome Replication and Regulation of Viral Gene Expression

Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

James R. Short, Jeffrey A. Speir, Radhika Gopal, Logan M. Pankratz, Jason Lanman, Anette Schneemann
D. S. Lyles, Editor
James R. Short
aDepartment of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA
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Jeffrey A. Speir
aDepartment of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA
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Radhika Gopal
aDepartment of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA
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Logan M. Pankratz
bDepartment of Biological Sciences, Purdue University, West Lafayette, Indiana, USA
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Jason Lanman
bDepartment of Biological Sciences, Purdue University, West Lafayette, Indiana, USA
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Anette Schneemann
aDepartment of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA
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D. S. Lyles
Roles: Editor
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DOI: 10.1128/JVI.03080-15
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ABSTRACT

Viruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infects Drosophila cells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ∼50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infected Drosophila cells. These preparations can be programmed to synthesize both single- and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis.

IMPORTANCE It is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infected Drosophila cells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.

FOOTNOTES

    • Received 9 December 2015.
    • Accepted 14 January 2016.
    • Accepted manuscript posted online 20 January 2016.
  • Address correspondence to Anette Schneemann, aschneem{at}scripps.edu.
  • ↵* Present address: James R. Short, Illumina Inc., San Diego, California, USA; Jeffrey A. Speir, NanoImaging Services, Inc., San Diego, California, USA.

  • Citation Short JR, Speir JA, Gopal R, Pankratz LM, Lanman J, Schneemann A. 2016. Role of mitochondrial membrane spherules in Flock House virus replication. J Virol 90:3676–3683. doi:10.1128/JVI.03080-15.

  • Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Role of Mitochondrial Membrane Spherules in Flock House Virus Replication
James R. Short, Jeffrey A. Speir, Radhika Gopal, Logan M. Pankratz, Jason Lanman, Anette Schneemann
Journal of Virology Mar 2016, 90 (7) 3676-3683; DOI: 10.1128/JVI.03080-15

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Role of Mitochondrial Membrane Spherules in Flock House Virus Replication
James R. Short, Jeffrey A. Speir, Radhika Gopal, Logan M. Pankratz, Jason Lanman, Anette Schneemann
Journal of Virology Mar 2016, 90 (7) 3676-3683; DOI: 10.1128/JVI.03080-15
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