scholarly journals A Versatile Reporter System to Monitor Virus Infected Cells and Its Application to Dengue Virus and SARS-CoV-2

2020 ◽  
Author(s):  
Felix Pahmeier ◽  
Christopher J. Neufeldt ◽  
Berati Cerikan ◽  
Vibhu Prasad ◽  
Costantin Pape ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Nuclear Localization Sequence ◽  
Reporter System ◽  
Infected Cells ◽  
Positive Strand Rna

Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of this include flaviviruses, such as dengue virus and Zika virus that cause millions of yearly infections around the globe, and coronaviruses, such as SARS-CoV-2, the source of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of research aimed at determining methods to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. Here, we describe the generation and characterization of a reporter system that can be used to visualize and identify cells infected with dengue virus or SARS-CoV-2. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. We show the suitability of this system for live cell imaging, for visualization of single infected cells, and for screening and testing of antiviral compounds. With the integrated modular building blocks, this system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility. IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of virus infected cells 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 GFP moiety 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.

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

2020 ◽  
Author(s):  
Felix Pahmeier ◽  
Christoper J Neufeldt ◽  
Berati Cerikan ◽  
Vibhu Prasad ◽  
Costantin Pape ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Viral Replication ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Reporter System ◽  
Infected Cells ◽  
Positive Strand Rna

ABSTRACTPositive-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.

scholarly journals A plasmid-based reporter system for live cell imaging of dengue virus infected cells

2015 ◽  
Vol 211 ◽  
pp. 55-62 ◽  
Author(s):  
Carey L. Medin ◽  
Sierra Valois ◽  
Chinmay G. Patkar ◽  
Alan L. Rothman
Keyword(s):  
Dengue Virus ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Reporter System ◽  
Infected Cells

scholarly journals Combining Single RNA Sensitive Probes with Subdiffraction-Limited and Live-Cell Imaging Enables the Characterization of Virus Dynamics in Cells

ACS Nano ◽  
2013 ◽  
Vol 8 (1) ◽  
pp. 302-315 ◽  
Author(s):  
Eric Alonas ◽  
Aaron W. Lifland ◽  
Manasa Gudheti ◽  
Daryll Vanover ◽  
Jeenah Jung ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Virus Dynamics ◽  
In Cells

scholarly journals Red-Shifted Aminated Derivatives of GFP Chromophore for Live-Cell Protein Labeling with Lipocalins

2018 ◽  
Vol 19 (12) ◽  
pp. 3778 ◽  
Author(s):  
Nina Bozhanova ◽  
Mikhail Baranov ◽  
Nadezhda Baleeva ◽  
Alexey Gavrikov ◽  
Alexander Mishin
Keyword(s):  
Green Fluorescent Protein ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Protein Labeling ◽  
Cell Protein ◽  
Gfp Chromophore ◽  
Green Fluorescent ◽  
Derivatives Of

Fluorogens are an attractive type of dye for imaging applications, eliminating time-consuming washout steps from staining protocols. With just a handful of reported fluorogen-protein pairs, mostly in the green region of spectra, there is a need for the expansion of their spectral range. Still, the origins of solvatochromic and fluorogenic properties of the chromophores suitable for live-cell imaging are poorly understood. Here we report on the synthesis and labeling applications of novel red-shifted fluorogenic cell-permeable green fluorescent protein (GFP) chromophore analogs.

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

scholarly journals A live-cell imaging system for visualizing the transport of Marburg virus nucleocapsid-like structures

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Yuki Takamatsu ◽  
Olga Dolnik ◽  
Takeshi Noda ◽  
Stephan Becker
Keyword(s):  
Live Cell Imaging ◽  
Intracellular Transport ◽  
Actin Polymerization ◽  
Ebola Virus ◽  
Cell Imaging ◽  
Temporal Dynamics ◽  
Imaging System ◽  
Marburg Virus ◽  
Live Cell ◽  
Infected Cells

Abstract Background Live-cell imaging is a powerful tool for visualization of the spatio-temporal dynamics of moving signals in living cells. Although this technique can be utilized to visualize nucleocapsid transport in Marburg virus (MARV)- or Ebola virus-infected cells, the experiments require biosafety level-4 (BSL-4) laboratories, which are restricted to trained and authorized individuals. Methods To overcome this limitation, we developed a live-cell imaging system to visualize MARV nucleocapsid-like structures using fluorescence-conjugated viral proteins, which can be conducted outside BSL-4 laboratories. Results Our experiments revealed that nucleocapsid-like structures have similar transport characteristics to those of nucleocapsids observed in MARV-infected cells, both of which are mediated by actin polymerization. Conclusions We developed a non-infectious live cell imaging system to visualize intracellular transport of MARV nucleocapsid-like structures. This system provides a safe platform to evaluate antiviral drugs that inhibit MARV nucleocapsid transport.

scholarly journals Characterization of a new class of blue-fluorescent lipid droplet markers for live-cell imaging in plants

2015 ◽  
Vol 34 (4) ◽  
pp. 655-665 ◽  
Author(s):  
Soujanya Kuntam ◽  
László G. Puskás ◽  
Ferhan Ayaydin
Keyword(s):  
Lipid Droplet ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
New Class ◽  
Fluorescent Lipid

scholarly journals Detection and Characterization of Protein Interactions In Vivo by a Simple Live-Cell Imaging Method

PLoS ONE ◽  
2013 ◽  
Vol 8 (5) ◽  
pp. e62195 ◽  
Author(s):  
Oriol Gallego ◽  
Tanja Specht ◽  
Thorsten Brach ◽  
Arun Kumar ◽  
Anne-Claude Gavin ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Imaging Method

scholarly journals F-Actin Dynamics in Neurospora crassa

2010 ◽  
Vol 9 (4) ◽  
pp. 547-557 ◽  
Author(s):  
Adokiye Berepiki ◽  
Alexander Lichius ◽  
Jun-Ya Shoji ◽  
Jens Tilsner ◽  
Nick D. Read
Keyword(s):  
Neurospora Crassa ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Content Type ◽  
Actin Cables ◽  
Germ Tubes

ABSTRACT This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.

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