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Journal of Virology, November 1998, p. 8578-8585, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Intracellular Localization of Poliovirus Plus- and Minus-Strand RNA Visualized by Strand-Specific Fluorescent In Situ Hybridization

Roger Bolten,1,dagger Denise Egger,1 Rainer Gosert,1 Gabriela Schaub,1 Lukas Landmann,2 and Kurt Bienz1,*

Institute for Medical Microbiology1 and Department of Anatomy,2 University of Basel, Basel, Switzerland

Received 28 April 1998/Accepted 21 July 1998

The time courses of poliovirus plus- and minus-strand RNA synthesis in infected HEp-2 cells were monitored separately, using a quantitative RNase assay. In parallel, viral RNA and proteins were located in situ by confocal microscopy within cells fixed by a protocol determined to retain their native size and shape. Plus- and minus-strand RNAs were visualized by fluorescent in situ hybridization (FISH) with strand-specific riboprobes. The probes were labelled with different fluorochromes to allow for the simultaneous detection of plus- and minus-strand RNA. The FISH experiments showed minus-strand RNA to be present in distinct, regularly sized, round structures throughout the viral replication cycle. Plus-strand RNA was found in the same structures and also in smaller clusters of vesicles. Association of viral RNA with membranes was demonstrated by combining FISH with immunofluorescence (IF) detection of the viral 2B- and 2C-containing P2 proteins, which are known to be markers for virus-induced membranes. At early times postinfection, the virus-induced membranous structures were distributed through most of the cytoplasm, whereas around peak RNA synthesis, both RNA-associated membranous structures migrated to the center of the cell. During this process, the plus- and minus-strand-containing larger structures stayed as recognizable entities, whereas the plus-strand-containing granules coalesced into a juxtanuclear area of membranous vesicles. An involvement of Golgi-derived membranes in the formation of virus-induced vesicles and RNA synthesis early in infection was investigated by IF with 2C- and Golgi-specific antibodies.

* Corresponding author. Mailing address: Institute for Medical Microbiology, University of Basel, Petersplatz 10, CH-4003 Basel, Switzerland. Phone: 41 61 267 3290. Fax: 41 61 267 3298. E-mail: Bienz{at}ubaclu.unibas.ch.

dagger Present address: Drossapharm, Arlesheim, Switzerland.

Journal of Virology, November 1998, p. 8578-8585, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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