A Fluorescent Cell-Based System for Imaging Zika Virus Infection in Real-Time

McFadden, Michael J.; Mitchell-Dick, Aaron; Vazquez, Christine; Roder, Allison; Labagnara, Kevin; McMahon, John J.; Silver, Debra L.; Horner, Stacy M.

doi:10.3390/v10020095

Cited by 17 publications

(21 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Currently, the role of AXL in mediating viral entry remains controversial, with some studies showing its requirement for the infection of a human fibroblast cell line (HT1080) (Persaud et al 2018) and others showing that AXL is not a viral entry receptor but rather enhances infection by suppressing ZIKV-induced activation of type 1 interferon genes (Chen et al 2018). A role for AXL in the host immune response would help explain and reconcile earlier studies showing it to be dispensable in Axl KO mouse models and the effectiveness of interferon alpha receptor blocking antibody treatments (McFadden et al 2018).…”

Section: Mechanisms Underlying Zikv Infection and Pathogenesis In Thementioning

confidence: 95%

Pathophysiology and Mechanisms of Zika Virus Infection in the Nervous System

Christian

Song

Ming

2019

Annu. Rev. Neurosci.

View full text Add to dashboard Cite

In 2015, public awareness of Zika virus (ZIKV) rose in response to alarming statistics of infants with microcephaly being born to women who were infected with the virus during pregnancy, triggering global concern over these potentially devastating consequences. Although we have discovered a great deal about the genome and pathogenesis of this reemergent flavivirus since this recent outbreak, we still have much more to learn, including the nature of the virus-host interactions and mechanisms that determine its tropism and pathogenicity in the nervous system, which are in turn shaped by the continual evolution of the virus. Inevitably, we will find out more about the potential long-term effects of ZIKV exposure on the nervous system from ongoing longitudinal studies. Integrating clinical and epidemiological data with a wider range of animal and human cell culture models will be critical to understanding the pathogenetic mechanisms and developing more specific antiviral compounds and vaccines.

show abstract

Section: Mechanisms Underlying Zikv Infection and Pathogenesis In Thementioning

confidence: 95%

Pathophysiology and Mechanisms of Zika Virus Infection in the Nervous System

Christian

Song

Ming

2019

Annu. Rev. Neurosci.

View full text Add to dashboard Cite

show abstract

“…To monitor enterovirus infection in real-time, we adapted cell-based reporter methodologies previously used for flaviviruses and hepatitis C virus that rely on viral protease cleavage-dependent translocation of a membrane-anchored cytoplasmic fluorescent proteins to the nucleus [ 27 , 28 , 29 ]. However, given that these systems do not allow for the visualization of virus-induced manipulation of host cell organelles, we sought to design reporter plasmids expressing a fluorescent protein to indicate infection in addition to fluorescent protein-targeted organelle markers.…”

Section: Resultsmentioning

confidence: 99%

“…However, the insertion of a fluorescent protein open reading frame in small positive-strand RNA viruses, including enteroviruses and flaviviruses, can dramatically attenuate viral progeny [ 14 , 25 , 26 ], thereby limiting the scope of assays for which these viruses can provide accurate information. Previous reports have overcome these limitations by developing plasmid-based reporters to detect hepaci- and flavivirus infections [ 27 , 28 , 29 ]. These reporters rely on viral protease-dependent cleavage to allow for the translocation of a fluorescent protein to the nucleus from the mitochondria [ 27 ] or the ER [ 28 , 29 ].…”

Section: Introductionmentioning

confidence: 99%

“…Previous reports have overcome these limitations by developing plasmid-based reporters to detect hepaci- and flavivirus infections [ 27 , 28 , 29 ]. These reporters rely on viral protease-dependent cleavage to allow for the translocation of a fluorescent protein to the nucleus from the mitochondria [ 27 ] or the ER [ 28 , 29 ]. However, these reporters only detect virus infection and do not allow for the characterization of virus-induced remodeling of intracellular host cell structures without the introduction of multiple fluorescent protein marker expression vectors.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Lennemann

Evans

Coyne

2020

Viruses

View full text Add to dashboard Cite

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.

show abstract

“…An alternative approach is the use of engineered fluorescent reporter proteins stably expressed in cells and altering their subcellular distribution upon viral infection (21)(22)(23). Building on this idea, here we established a reporter system based on an ERanchored green fluorescent protein (GFP) that upon cleavage by a viral protease is released from the ER and translocated into the nucleus.…”

Section: Introductionmentioning

confidence: 99%

A versatile reporter system to monitor virus infected cells and its application to dengue virus and SARS-CoV-2

Pahmeier

Neufeldt

Cerikan

et al. 2020

Preprint

View full text Add to dashboard Cite

Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of that are flaviviruses, such as dengue virus and Zika virus that cause millions of yearly infections and spread around the globe, and coronaviruses, such as SARS-CoV-2, which is the cause of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of virology research in determining mechanisms to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective interventions. Here, we describe the generation and characterization of a reporter system to visualize dengue virus and SARS-CoV-2 replication in live cells. The system is based on viral protease activity causing cleavage and nuclear translocation of an engineered fluorescent protein that is expressed in the infected cells. We show the suitability of the system for live cell imaging and visualization of single infected cells as well as for screening and testing of antiviral compounds. Given the modular building blocks, the system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCEReporter systems are useful tools for fast and quantitative visualization of viral replication and spread within a host cell population. Here we describe a reporter system that takes advantage of virus-encoded proteases that are expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the fluorescent protein translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.

show abstract

A Fluorescent Cell-Based System for Imaging Zika Virus Infection in Real-Time

Cited by 17 publications

References 45 publications

Pathophysiology and Mechanisms of Zika Virus Infection in the Nervous System

Pathophysiology and Mechanisms of Zika Virus Infection in the Nervous System

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

A versatile reporter system to monitor virus infected cells and its application to dengue virus and SARS-CoV-2

Contact Info

Product

Resources

About