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Imaging-Based Reporter Systems to Define CVB-Induced Membrane Remodeling in Living Cells.
Abstract
Enteroviruses manipulate host membranes to form replication organelles, which concentrate
viral and host factors to allow for efficient replication. However, this process has
not been well-studied in living cells throughout the course of infection. To define
the dynamic process of enterovirus membrane remodeling of major secretory pathway
organelles, we have developed plasmid-based reporter systems that utilize viral protease-dependent
release of a nuclear-localized fluorescent protein from the endoplasmic reticulum
(ER) membrane during infection, while retaining organelle-specific fluorescent protein
markers such as the ER and Golgi. This system thus allows for the monitoring of organelle-specific
changes induced by infection in real-time. Using long-term time-lapse imaging of living
cells infected with coxsackievirus B3 (CVB), we detected reporter translocation to
the nucleus beginning ~4 h post-infection, which correlated with a loss of Golgi integrity
and a collapse of the peripheral ER. Lastly, we applied our system to study the effects
of a calcium channel inhibitor, 2APB, on virus-induced manipulation of host membranes.
We found that 2APB treatment had no effect on the kinetics of infection or the percentage
of infected cells. However, we observed aberrant ER structures in CVB-infected cells
treated with 2APB and a significant decrease in viral-dependent cell lysis, which
corresponded with a decrease in extracellular virus titers. Thus, our system provides
a tractable platform to monitor the effects of inhibitors, gene silencing, and/or
gene editing on viral manipulation of host membranes, which can help determine the
mechanism of action for antivirals.
Type
Journal articleSubject
Cell Line, TumorIntracellular Membranes
Endoplasmic Reticulum
Golgi Apparatus
Humans
Enterovirus B, Human
Calcium Channel Blockers
Virus Replication
Kinetics
Genes, Reporter
Plasmids
Host-Pathogen Interactions
Secretory Pathway
Optical Imaging
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https://hdl.handle.net/10161/22574Published Version (Please cite this version)
10.3390/v12101074Publication Info
Lennemann, Nicholas J; Evans, Azia S; & Coyne, Carolyn B (2020). Imaging-Based Reporter Systems to Define CVB-Induced Membrane Remodeling in Living
Cells. Viruses, 12(10). pp. 1074-1074. 10.3390/v12101074. Retrieved from https://hdl.handle.net/10161/22574.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
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Show full item recordScholars@Duke
Carolyn Coyne
George Barth Geller Distinguished Professor of Immunology
We study the pathways by which microorganisms cross cellular barriers and the mechanisms
by which these barriers restrict microbial infections. Our studies primarily focus
on the epithelium that lines the gastrointestinal tract and on placental trophoblasts,
the cells that comprise a key cellular barrier of the human placenta. Our work is
highly multidisciplinary and encompasses aspects of cell biology, immunology, and
microbiology. Our long-term goals are to identify pathogen- and host-spe

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