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Imaging-Based Reporter Systems to Define CVB-Induced Membrane Remodeling in Living Cells.

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Date
2020-09-25
Authors
Lennemann, Nicholas J
Evans, Azia S
Coyne, Carolyn B
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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 article
Subject
Cell Line, Tumor
Intracellular 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
Permalink
https://hdl.handle.net/10161/22574
Published Version (Please cite this version)
10.3390/v12101074
Publication 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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Scholars@Duke

Coyne

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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