Real-time Fluorescence Measurement of Enterovirus Uncoating
Ruokolainen, V., Laajala, M., & Marjomäki, V. (2020). Real-time Fluorescence Measurement of Enterovirus Uncoating. Bio-protocol, 10(7), Article e3582. https://doi.org/10.21769/BioProtoc.3582
Viruses need to open, i.e., uncoat, in order to release their genomes for efficient replication and translation. Especially for non-enveloped viruses, such as enteroviruses, the cues leading to uncoating are less well known. The status of the virus has previously been observed mainly by transmission electron microscopy using negative staining, cryo electron microscopy, X-ray crystallography or gradient separation (reviewed in Tuthill et al., 2010, Myllynen et al., 2016, Ruokolainen et al., 2019). However, monitoring of uncoating has been limited by the lack of methods detecting dynamic changes of the virions. Here, we present a real-time fluorescence based protocol, which detects the viral genome (RNA) during various stages of uncoating in vitro, while RNA is still inside the particle that has been expanded before the actual RNA release, and when the RNA has been totally released from the viral particle. Our method allows to explore how various molecular factors may promote or inhibit virus uncoating. ...
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Additional information about fundingWe acknowledge Jane and Aatos Erkko foundation and doctoral school of University of Jyväskylä for funding and the authors of the original paper (Ruokolainen et al., 2019) for contributing to the work where the protocol was introduced.
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