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ID 116524
Title Alternative
Quantitative Virion Maturation Fluorescence Microscopy
Author
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
Izumi, Taisuke Kyoto University|Henry M. Jackson Foundation for the Advancement of Military Medicine KAKEN Search Researchers
Keywords
HIV-1 Gag maturation
Förster Resonance Energy Transfer
single virion imaging
protease inhibitor
fluorescence microscopy
Content Type
Journal Article
Description
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.
Journal Title
Frontiers in Microbiology
ISSN
1664302X
Publisher
Frontiers Media S.A.
Volume
12
Start Page
647452
Published Date
2021-03-09
Rights
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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language
eng
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departments
Medical Sciences