Combining single RNA sensitive probes with subdiffraction-limited and live-cell imaging enables the characterization of virus dynamics in cells

ACS Nano
Eric AlonasPhilip J Santangelo

Abstract

The creation of fluorescently labeled viruses is currently limited by the length of imaging observation time (e.g., labeling an envelope protein) and the rescue of viral infectivity (e.g., encoding a GFP protein). Using single molecule sensitive RNA hybridization probes delivered to the cytoplasm of infected cells, we were able to isolate individual, infectious, fluorescently labeled human respiratory syncytial virus virions. This was achieved without affecting viral mRNA expression, viral protein expression, or infectivity. Measurements included the characterization of viral proteins and genomic RNA in a single virion using dSTORM, the development of a GFP fusion assay, and the development of a pulse-chase assay for viral RNA production that allowed for the detection of both initial viral RNA and nascent RNA production at designated times postinfection. Live-cell measurements included imaging and characterization of filamentous virion fusion and the quantification of virus replication within the same cell over an eight-hour period. Using probe-labeled viruses, individual viral particles can be characterized at subdiffraction-limited resolution, and viral infections can be quantified in single cells over an entire cycle of repl...Continue Reading

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Citations

Feb 16, 2016·Methods : a Companion to Methods in Enzymology·Eric AlonasPhilip J Santangelo
Dec 9, 2014·RNA Biology·J Matthew TaliaferroChristopher B Burge
Jul 6, 2014·The Anatomical Record : Advances in Integrative Anatomy and Evolutionary Biology·Ellen Jensen, David J Crossman
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Sep 25, 2017·Nature Communications·Daryll VanoverPhilip J Santangelo
May 16, 2020·Materials Science & Engineering. C, Materials for Biological Applications·Lu Chen, Jiangong Liang
Aug 2, 2018·Bioconjugate Chemistry·Kristin H LoomisPhilip J Santangelo
May 8, 2017··Dayong JinYujia Liu

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Methods Mentioned

BETA
scraping
transmission electron microscopy
ELISA
fluorescence microscopy
Fluorescence
super-resolution microscopy

Software Mentioned

Image Studio
Sigma Plot
custom
dSTORM
Applied
Volocity
Excel
Biosystems
CytoViva
ND

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