期刊
VIRUS RESEARCH
卷 243, 期 -, 页码 69-74出版社
ELSEVIER
DOI: 10.1016/j.virusres.2017.10.015
关键词
Flavivirus; Virus entry; Virus release; Luciferase complementation assay
类别
资金
- Global Institution for Collaborative Research and Education (GICoRE) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan
- MEXT, Japan [16H06429, 16H06431, 16K21723]
- Grants-in-Aid for Scientific Research [16H06431, 16H06429] Funding Source: KAKEN
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.
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