nanoDSF: In vitro Label-Free Method to Monitor Picornavirus Uncoating and Test Compounds Affecting Particle Stability

Frontiers in Microbiology
Antonio Real-HohnHeinrich Kowalski

Abstract

Thermal shift assays measure the stability of macromolecules and macromolecular assemblies as a function of temperature. The Particle Stability Thermal Release Assay (PaSTRy) of picornaviruses is based on probes becoming strongly fluorescent upon binding to hydrophobic patches of the protein capsid (e.g., SYPRO Orange) or to the viral RNA genome (e.g., SYTO-82) that become exposed upon heating virus particles. PaSTRy has been exploited for studying the stability of viral mutants, viral uncoating, and the effect of capsid-stabilizing compounds. While the results were usually robust, the thermal shift assay with SYPRO Orange is sensitive to surfactants and EDTA and failed at least to correctly report the effect of excipients on an inactivated poliovirus 3 vaccine. Furthermore, interactions between the probe and capsid-binding antivirals as well as mutual competition for binding sites cannot be excluded. To overcome these caveats, we assessed differential scanning fluorimetry with a nanoDSF device as a label-free alternative. NanoDSF monitors the changes in the intrinsic tryptophan fluorescence (ITF) resulting from alterations of the 3D-structure of proteins as a function of the temperature. Using rhinovirus A2 as a model, we demo...Continue Reading

References

Aug 1, 1976·Journal of Virology·K Lonberg-HolmE J Shimshick
Nov 1, 1977·The Journal of General Virology·D P FrisbyB M Richards
Jul 1, 1973·Journal of Virology·K Lonberg-Holm, F H Yin
Oct 1, 1996·Journal of Virology·S CurryJ M Hogle
Jun 1, 1997·Archives of Biochemistry and Biophysics·C H ChenR Crainic
Jun 17, 1998·Proceedings of the National Academy of Sciences of the United States of America·J K LewisG Siuzdak
Feb 12, 2000·Journal of Molecular Biology·S K TsangJ M Hogle
Jul 25, 2000·Journal of Molecular Biology·N VerdaguerI Fita
Jul 31, 2002·Molecular Pathology : MP·M S Smyth, J H Martin
Mar 12, 2003·Pharmacotherapy·Naomi R FloreaDavid P Nicolau
Feb 25, 2005·The Journal of Biological Chemistry·Juan RegueraMauricio G Mateu
Oct 30, 2007·Biochimica Et Biophysica Acta·K H StrucksbergJ Fitter
May 1, 2009·Journal of Virology·Umesh KatpallyThomas J Smith
Apr 17, 2010·Current Topics in Microbiology and Immunology·Tobias J TuthillDavid J Rowlands
Oct 29, 2010·Journal of Virology·Mihnea BostinaJames M Hogle
Nov 10, 2011·Assay and Drug Development Technologies·Guillermo SenisterraMasoud Vedadi
Mar 1, 2012·Langmuir : the ACS Journal of Surfaces and Colloids·N Idayu ZahidThorsten Heidelberg
Jun 30, 2012·Journal of Virological Methods·Thomas S WalterElizabeth E Fry
Sep 27, 2013·Journal of Virology·Vamseedhar RayaproluBrian Bothner

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Citations

Feb 3, 2021·Cell Chemical Biology·Christian HentrichFrancisco Ylera
Feb 20, 2021·Archives of Virology·Caio Bidueira DenaniRafael Braga Gonçalves

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Methods Mentioned

BETA
Assay
PCR
Fluorescence
transmission electron microscopy
X-ray
thermal shift
electrophoresis
fluorescence correlation spectroscopy

Software Mentioned

PR
GraphPad Prism
ThermControl
nanoDSF
Chimera
PaSTRy

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