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Journal of Virology, April 2002, p. 3905-3919, Vol. 76, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.8.3905-3919.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Long-Distance Base Pairing in Flock House Virus RNA1 Regulates Subgenomic RNA3 Synthesis and RNA2 Replication

Brett D. Lindenbach,1,2 Jean-Yves Sgro,2,3 and Paul Ahlquist1,2*

Howard Hughes Medical Institute,1 Institute for Molecular Virology,2 Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706-15963

Received 1 November 2001/ Accepted 2 January 2002

Replication of flock house virus (FHV) RNA1 and production of subgenomic RNA3 in the yeast Saccharomyces cerevisiae provide a useful tool for the dissection of FHV molecular biology and host-encoded functions involved in RNA replication. The replication template activity of RNA1 can be separated from its coding potential by supplying the RNA1-encoded replication factor protein A in trans. We constructed a trans-replication system in yeast to examine cis-acting elements in RNA1 that control RNA3 production, as well as RNA1 and RNA2 replication. Two cis elements controlling RNA3 production were found. A proximal subgenomic control element was located just upstream of the RNA3 start site (nucleotides [nt] 2282 to 2777). A short distal element also controlling RNA3 production (distal subgenomic control element) was identified 1.5 kb upstream, at nt 1229 to 1239. Base pairing between these distal and proximal elements was shown to be essential for RNA3 production by covariation analysis and in vivo selection of RNA3-expressing replicons from plasmid libraries containing random sequences in the distal element. Two distinct RNA1 replication elements (RE) were mapped within the 3' quarter of RNA1: the intRE (nt 2322 to 2501) and the 3'RE (nt 2735 to 3011). The 3'RE significantly overlaps the RNA3 region in RNA1, and this information was applied to produce improved RNA3-based vectors for foreign-gene expression. In addition, replication of an RNA2 derivative was dependent on RNA1 templates capable of forming the long-distance interaction that controls RNA3 production.

* Corresponding author. Mailing address: Institute for Molecular Virology and Howard Hughes Medical Institute, 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.

Journal of Virology, April 2002, p. 3905-3919, Vol. 76, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.8.3905-3919.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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