Journal
VIROLOGY
Volume 485, Issue -, Pages 439-451Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.virol.2015.08.023
Keywords
Live-cell imaging; Double-stranded RNA; RNA binding protein; Fluorescent protein; BiFC; Positive-sense RNA virus; Viral replication complex; SGS3; RDR6; D-bodies
Categories
Funding
- Agriculture and Agri-Food Canada
- Natural Sciences and Engineering Research Council of Canada
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Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. Crown Copyright (C) 2015 Published by Elsevier Inc. All rights reserved.
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