Show simple item record

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

dc.contributor.author Lennemann, Nicholas J
dc.contributor.author Evans, Azia S
dc.contributor.author Coyne, Carolyn B
dc.date.accessioned 2021-04-16T19:51:28Z
dc.date.available 2021-04-16T19:51:28Z
dc.date.issued 2020-09-25
dc.identifier v12101074
dc.identifier.issn 1999-4915
dc.identifier.issn 1999-4915
dc.identifier.uri https://hdl.handle.net/10161/22574
dc.description.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.
dc.language eng
dc.publisher MDPI AG
dc.relation.ispartof Viruses
dc.relation.isversionof 10.3390/v12101074
dc.subject Cell Line, Tumor
dc.subject Intracellular Membranes
dc.subject Endoplasmic Reticulum
dc.subject Golgi Apparatus
dc.subject Humans
dc.subject Enterovirus B, Human
dc.subject Calcium Channel Blockers
dc.subject Virus Replication
dc.subject Kinetics
dc.subject Genes, Reporter
dc.subject Plasmids
dc.subject Host-Pathogen Interactions
dc.subject Secretory Pathway
dc.subject Optical Imaging
dc.title Imaging-Based Reporter Systems to Define CVB-Induced Membrane Remodeling in Living Cells.
dc.type Journal article
duke.contributor.id Coyne, Carolyn B|1086848
dc.date.updated 2021-04-16T19:51:26Z
pubs.begin-page 1074
pubs.end-page 1074
pubs.issue 10
pubs.organisational-group School of Medicine
pubs.organisational-group Molecular Genetics and Microbiology
pubs.organisational-group Duke
pubs.organisational-group Basic Science Departments
pubs.publication-status Published
pubs.volume 12
duke.contributor.orcid Coyne, Carolyn B|0000-0002-1884-6309


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record