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Journal of Virology, April 2009, p. 2872-2882, Vol. 83, No. 7
0022-538X/09/$08.00+0     doi:10.1128/JVI.01780-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Dual Roles for an Arginine-Rich Motif in Specific Genome Recognition and Localization of Viral Coat Protein to RNA Replication Sites in Flock House Virus-Infected Cells{triangledown} ,{dagger}

P. Arno Venter,* Dawn Marshall, and Anette Schneemann*

Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

Received 22 August 2008/ Accepted 13 January 2009

Assembly of many RNA viruses entails the encapsidation of multiple genome segments into a single virion, and underlying mechanisms for this process are still poorly understood. In the case of the nodavirus Flock House virus (FHV), a bipartite positive-strand RNA genome consisting of RNA1 and RNA2 is copackaged into progeny virions. In this study, we investigated whether the specific packaging of FHV RNA is dependent on an arginine-rich motif (ARM) located in the N terminus of the coat protein. Our results demonstrate that the replacement of all arginine residues within this motif with alanines rendered the resultant coat protein unable to package RNA1, suggesting that the ARM represents an important determinant for the encapsidation of this genome segment. In contrast, replacement of all arginines with lysines had no effect on RNA1 packaging. Interestingly, confocal microscopic analysis demonstrated that the RNA1 packaging-deficient mutant did not localize to mitochondrial sites of FHV RNA replication as efficiently as wild-type coat protein. In addition, gain-of-function analyses showed that the ARM by itself was sufficient to target green fluorescent protein to RNA replication sites. These data suggest that the packaging of RNA1 is dependent on trafficking of coat protein to mitochondria, the presumed site of FHV assembly, and that this trafficking requires a high density of positive charge in the N terminus. Our results are compatible with a model in which recognition of RNA1 and RNA2 for encapsidation occurs sequentially and in distinct cellular microenvironments.

* Corresponding author. Mailing address: Department of Molecular Biology, CB262, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Phone for A. Schneemann: (858) 784-8643. Fax: (858) 784-7979. E-mail: aschneem{at}scripps.edu. Phone for P. A. Venter: (858) 784-7123. Fax: (858) 784-7979. E-mail: paventer{at}scripps.edu

{triangledown} Published ahead of print on 21 January 2009.

{dagger} Manuscript 19728 from The Scripps Research Institute.

Journal of Virology, April 2009, p. 2872-2882, Vol. 83, No. 7
0022-538X/09/$08.00+0     doi:10.1128/JVI.01780-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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