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ID 116524
タイトル別表記
Quantitative Virion Maturation Fluorescence Microscopy
著者
Sarca, Anamaria D. Kyoto University
Sardo, Luca University of the Sciences, Philadelphia|Merck & Co.
Fukuda, Hirofumi Kyoto University
Matsui, Hiroyuki Kyoto University
Shirakawa, Kotaro Kyoto University
Takaori-Kondo, Akifumi Kyoto University
泉, 泰輔 Kyoto University|Henry M. Jackson Foundation for the Advancement of Military Medicine KAKEN研究者をさがす
キーワード
HIV-1 Gag maturation
Förster Resonance Energy Transfer
single virion imaging
protease inhibitor
fluorescence microscopy
資料タイプ
学術雑誌論文
抄録
HIV-1 infectivity is achieved through virion maturation. Virus particles undergo structural changes via cleavage of the Gag polyprotein mediated by the viral protease, causing the transition from an uninfectious to an infectious status. The majority of proviruses in people living with HIV-1 treated with combination antiretroviral therapy are defective with large internal deletions. Defective proviral DNA frequently preserves intact sequences capable of expressing viral structural proteins to form virus-like particles whose maturation status is an important factor for chronic antigen-mediated immune stimulation and inflammation. Thus, novel methods to study the maturation capability of defective virus particles are needed to characterize their immunogenicity. To build a quantitative tool to study virion maturation in vitro, we developed a novel single virion visualization technique based on fluorescence resonance energy transfer (FRET). We inserted an optimized intramolecular CFP-YPF FRET donor-acceptor pair bridged with an HIV-1 protease cleavage sequence between the Gag MA-CA domains. This system allowed us to microscopically distinguish mature and immature virions via their FRET signal when the FRET donor and acceptor proteins were separated by the viral protease during maturation. We found that approximately 80% of the FRET labeled virus particles were mature with equivalent infectivity to wild type. The proportion of immature virions was increased by treatment of virus producer cells with a protease inhibitor in a dose-dependent manner, which corresponded to a relative decrease in infectivity. Potential areas of application for this tool are assessing maturation efficiency in different cell type settings of intact or deficient proviral DNA integrated cells. We believe that this FRET-based single-virion imaging platform will facilitate estimating the impact on the immune system of both extracellular intact and defective viruses by quantifying the Gag maturation status.
掲載誌名
Frontiers in Microbiology
ISSN
1664302X
出版者
Frontiers Media S.A.
12
開始ページ
647452
発行日
2021-03-09
権利情報
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) (https://creativecommons.org/licenses/by/4.0/). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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言語
eng
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部局
医学系