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Development of a rapid and quantitative method for the analysis of viral entry and release using a NanoLuc luciferase complementation assay

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Title: Development of a rapid and quantitative method for the analysis of viral entry and release using a NanoLuc luciferase complementation assay
Authors: Sasaki, Michihito Browse this author →KAKEN DB
Anindita, Paulina D Browse this author
Phongphaew, Wallaya Browse this author
Carr, Michael Browse this author →KAKEN DB
Kobayashi, Shintaro Browse this author →KAKEN DB
Orba, Yasuko Browse this author →KAKEN DB
Sawa, Hirofumi Browse this author →KAKEN DB
Keywords: Flavivirus
Virus entry
Virus release
Luciferase complementation assay
Issue Date: 23-Oct-2017
Publisher: Elsevier
Journal Title: Virus research
Volume: 243
Issue: 2
Start Page: 69
End Page: 74
Publisher DOI: 10.1016/j.virusres.2017.10.015
PMID: 29074234
Abstract: Subviral particles (SVPs) self-assemble and are released from cells transfected with expression plasmids encoding flavivirus structural proteins. Flavivirus-like particles (VLPs), consisting of flavivirus structural proteins and a subgenomic replicon, can enter cells and cause single-round infections. Neither SVPs or VLPs possess complete viral RNA genomes, therefore are replication-incompetent systems; however, they retain the capacity to fuse and bud from target cells and follow the same maturation process as whole virions. SVPs and VLPs have been previously employed in studies analyzing entry and release steps of viral life cycles. In this study, we have developed quantitative methods for the detection of cellular entry and release of SVPs and VLPs by applying a luciferase complementation assay based on the high affinity interaction between the split NanoLuc luciferase protein, LgBiT and the small peptide, HiBiT. We introduced HiBiT into the structural protein of West Nile virus and generated SVPs and VLPs harboring HiBiT (SVP-HiBiT and VLP-HiBiT, respectively). As SVP-HiBiT emitted strong luminescence upon exposure to LgBiT and its substrate, the nascently budded SVP-HiBiT in the supernatant was readily quantified by luminometry. Similarly, the cellular entry of VLP-HiBiT generated luminescence when VLP-HiBiT was infected into LgBiT-expressing cells. These methods utilizing SVP-HiBiT and VLP-HiBiT will facilitate research into life cycles of flaviviruses, including WNV.
Rights: ©2017. This manuscript version is made available under the CC-BY-NC-ND 4.0 license
Type: article (author version)
Appears in Collections:人獣共通感染症リサーチセンター (Research Center for Zoonosis Control) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 佐々木 道仁

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