Real-time Fluorescence Measurement of Enterovirus Uncoating
肠道病毒脱壳的实时荧光检测
发布: 2020年04月05日第10卷第7期 DOI: 10.21769/BioProtoc.3582 浏览次数: 3838
评审: David PaulMoona HuttunenAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2019
Abstract
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.
Picornavirus (肠道病毒)
Uncoating (脱壳)
Genome (基因组)
RNA (RNA)
RNase (RNA酶)
SYBR Green II (SYBR Green II)
Fluorescence spectroscopy (荧光光谱)
Background
In our previous study, we found that infectious intermediate echovirus 1 particle allows SYBR Green II, a RNA intercalating dye, to enter the virus particle (Myllynen et al., 2016). This can be observed as an increase of fluorescence and the recorded fluorescence is not susceptible to RNase digestion (Myllynen et al., 2016). Using this information, we developed a real-time method to monitor virus opening using the SYBR Green II dye and RNase in fluorescence spectroscopy. We could follow the fenestration of the particles in real-time at +37 °C, or other temperature of interest, in a 96-well plate format by adding SYBR Green II and factors triggering the uncoating, and observing the increase of SYBR Green II fluorescence. Addition of RNase into parallel wells allowed us to monitor the extent of RNA release from the virions, as RNase readily degrades RNA from the solution, but not from inside of the virion (RNAse cannot enter through the small fenestrations inside to the virus particle, Myllynen et al., 2016). In case of intact virus particles, only very low amount of fluorescence was observed. As an example, in our previous study, a DPBS solution supplemented with 0.01% fatty acid free BSA produced high amounts of intermediate echovirus 1 particles. For more details see the original publication (Ruokolainen et al., 2019).
Materials and Reagents
- Pipette tips
- Sarstedt 96-well plate (Sarstedt, catalog number: 83.3924 ) (or similar)
- 1.5 ml tubes
- Purified virus stock (1 μg of virus per measured well; stored at -80 °C)
Purification of enteroviruses can be done using either 5-20% or 10-40% sucrose gradient and is described in detail in the original publication (Ruokolainen et al., 2019). Also, CsCl purification may be used but it was observed by us to have more variation from batch-to-batch than sucrose purified virus. The protocol has been tested and observed to work with echovirus 1 and coxsackieviruses A9 and B3 suggesting its wide applicability for enteroviruses and probably for picornaviruses in general. - Buffer/solution of your interest
In our paper we used a wide spectrum of concentrations of different ions and albumin to study the virus priming and opening. As an example, DPBS solution supplemented with 0.01% BSA resulted in high amount of intermediate virus particles. - SYBR Green II RNA gel stain (Invitrogen; ThermoFisher Scientific, catalog number S7564 ; stored at -20 °C)
- RNase A, 10 mg/ml (ThermoFisher Scientific catalog number EN0531 ; stored at -20 °C)
- 150 mM NaCl solution
- Ice
Equipment
- Pipette with a volume range including 100 µl
- 8-channel multipipette with a volume range including 50 µl, or similar (optional)
- Perkin Elmer 2030 Multilabel Reader Victor X4 (or similar fluorescence plate reader with suitable filter options)
Software
- Perkin Elmer 2030 Manager
- Microsoft Excel
Procedure
文章信息
版权信息
© 2020 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Ruokolainen, V., Laajala, M. and Marjomäki, V. (2020). Real-time Fluorescence Measurement of Enterovirus Uncoating. Bio-protocol 10(7): e3582. DOI: 10.21769/BioProtoc.3582.
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分类
分子生物学 > RNA > RNA 检测
生物化学 > RNA > RNA-蛋白质相互作用
生物化学 > RNA > RNA结构
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