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Journal of Virology, May 2007, p. 4633-4644, Vol. 81, No. 9
0022-538X/07/$08.00+0     doi:10.1128/JVI.02267-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Nodavirus RNA Replication Protein A Induces Membrane Association of Genomic RNA{triangledown}

Priscilla M. Van Wynsberghe,1 Hau-Ren Chen,1,{dagger} and Paul Ahlquist1,2*

Institute for Molecular Virology,1 Howard Hughes Medical Institute, University of Wisconsin—Madison, Madison, Wisconsin 537062

Received 16 October 2006/ Accepted 7 February 2007

Positive-strand RNA virus genome replication occurs in membrane-associated RNA replication complexes, whose assembly remains poorly understood. Here we show that prior to RNA replication, the multifunctional, transmembrane RNA replication protein A of the nodavirus flock house virus (FHV) recruits FHV genomic RNA1 to a membrane-associated state in both Drosophila melanogaster and Saccharomyces cerevisiae cells. Protein A has mitochondrial membrane-targeting, self-interaction, RNA-dependent RNA polymerase (RdRp), and RNA capping domains. In the absence of RdRp activity due to an active site mutation (AD692E), protein A stimulated RNA1 accumulation by increasing RNA1 stability. Protein AD692E stimulated RNA1 accumulation in wild-type cells and in xrn1– yeast defective in decapped RNA decay, showing that increased RNA1 stability was not due to protein A-mediated RNA1 recapping. Increased RNA1 stability was closely linked with protein A-induced membrane association of the stabilized RNA and was highly selective for RNA1. Substantial N- and C-proximal regions of protein A were dispensable for these activities. However, increased RNA1 accumulation was eliminated by deleting protein A amino acids (aa) 1 to 370 but was restored completely by adding back the transmembrane domain (aa 1 to 35) and partially by adding back peripheral membrane association sequences in aa 36 to 370. Moreover, although RNA polymerase activity was not required, even small deletions in or around the RdRp domain abolished increased RNA1 accumulation. These and other results show that prior to negative-strand RNA synthesis, multiple domains of mitochondrially targeted protein A cooperate to selectively recruit FHV genomic RNA to membranes where RNA replication complexes form.

* Corresponding author. Mailing address: Institute for Molecular Virology, University of Wisconsin—Madison, 1525 Linden Dr., Madison, WI 53706-1596. Phone: (608) 263-5916. Fax: (608) 265-9214. E-mail: ahlquist{at}facstaff.wisc.edu

{triangledown} Published ahead of print on 14 February 2007.

{dagger} Present address: Institute of Molecular Biology, National Chung Cheng University, Chia-Yi 621, Taiwan, Republic of China.

Journal of Virology, May 2007, p. 4633-4644, Vol. 81, No. 9
0022-538X/07/$08.00+0     doi:10.1128/JVI.02267-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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