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Journal of Virology, April 2009, p. 3770-3777, Vol. 83, No. 8
0022-538X/09/$08.00+0     doi:10.1128/JVI.00265-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Site of Human Rhinovirus RNA Uncoating Revealed by Fluorescent In Situ Hybridization{triangledown}

Marianne Brabec-Zaruba,1 Beatrix Pfanzagl,1 Dieter Blaas,2 and Renate Fuchs1*

Department of Pathophysiology, Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090, Vienna, Austria,1 Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, Medical University of Vienna, Dr. Bohr Gasse 9/3, A-1030 Vienna, Austria2

Received 5 February 2008/ Accepted 9 January 2009

By using fluorescent in situ hybridization (FISH), we visualized viral RNA of human rhinovirus type 2 (HRV2) during its entry into HeLa cells. RNA uncoating of HRV2 is entirely dependent on low endosomal pH (≤5.6). When internalized into cells treated with bafilomycin, which results in neutralization of the endosomal pH, no FISH signal was recorded, whereas in the absence of the drug, fluorescent dots were seen. Therefore, FISH detects the genomic viral RNA only upon its release from the capsid. Free viral RNA was first seen at 10 min postinfection (p.i.) in the perinuclear area of the cell, which is indicative of RNA release in/from late endosomal compartments. Pulse-chase experiments and observation of HRV2 RNA and capsid proteins via microscopy, Western blotting, and reverse transcription-PCR revealed that the RNA signal persisted whereas the protein signal disappeared. This demonstrates transport of capsids to lysosomes and degradation. In contrast, viral RNA that had already been transferred into the cytoplasm escaped lysosomal breakdown as indicated by a persistent FISH signal. Taken together, our results demonstrate by direct means RNA arrival in the cytosol within 10 min p.i. Based on persistence of the FISH signal and productive infection in the presence of the microtubule-depolymerizing drug nocodazole, we localized this process to endosomal carrier vesicles/late endosomes.

* Corresponding author. Mailing address: Department of Pathophysiology, Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090, Vienna, Austria. Phone: 431 40400 5127. Fax: 431 404005130. E-mail: renate.fuchs{at}meduniwien.ac.at

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

Journal of Virology, April 2009, p. 3770-3777, Vol. 83, No. 8
0022-538X/09/$08.00+0     doi:10.1128/JVI.00265-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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