scholarly journals Fusion of a fluorescent protein to the pUL25 minor capsid protein of pseudorabies virus allows live-cell capsid imaging with negligible impact on infection

2012 ◽  
Vol 93 (1) ◽  
pp. 124-129 ◽  
Author(s):  
Kevin P. Bohannon ◽  
Patricia J. Sollars ◽  
Gary E. Pickard ◽  
Gregory A. Smith
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Nuclear Dynamics ◽  
Infection Models ◽  
Simplex Virus

In order to resolve the location and activity of submicroscopic viruses in living cells, viral proteins are often fused to fluorescent proteins (FPs) and visualized by microscopy. In this study, we describe the fusion of FPs to three proteins of pseudorabies virus (PRV) that allowed imaging of capsids in living cells. Included in this study are the first recombinant PRV strains expressing FP–pUL25 fusions based on a design applied to herpes simplex virus type 1 by Homa and colleagues. The properties of each reporter virus were compared in both in vitro and in vivo infection models. PRV strains expressing FP–pUL25 and FP–pUL36 preserved wild-type properties better than traditional FP–pUL35 isolates in assays of plaque size and virulence in mice. The utility of these strains in studies of axon transport, nuclear dynamics and viral particle composition are documented.

scholarly journals Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

2016 ◽  
Vol 198 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Na Ke ◽  
Dirk Landgraf ◽  
Johan Paulsson ◽  
Mehmet Berkmen
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Functional Protein ◽  
Content Type ◽  
Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

scholarly journals Visualizing Herpesvirus Procapsids in Living Cells

2016 ◽  
Vol 90 (22) ◽  
pp. 10182-10192 ◽  
Author(s):  
Oana Maier ◽  
Patricia J. Sollars ◽  
Gary E. Pickard ◽  
Gregory A. Smith
Keyword(s):  
Fluorescent Protein ◽  
Living Cells ◽  
Capsid Assembly ◽  
Wild Type ◽  
Capsid Shell ◽  
Wide Range ◽  
Green Fluorescent ◽  
Simplex Virus

ABSTRACTA complete understanding of herpesvirus morphogenesis requires studies of capsid assembly dynamics in living cells. Although fluorescent tags fused to the VP26 and pUL25 capsid proteins are available, neither of these components is present on the initial capsid assembly, the procapsid. To make procapsids accessible to live-cell imaging, we made a series of recombinant pseudorabies viruses that encoded green fluorescent protein (GFP) fused in frame to the internal capsid scaffold and maturation protease. One recombinant, a GFP-VP24 fusion, maintained wild-type propagation kineticsin vitroand approximated wild-type virulencein vivo. The fusion also proved to be well tolerated in herpes simplex virus. Viruses encoding GFP-VP24, along with a traditional capsid reporter fusion (pUL25/mCherry), demonstrated that GFP-VP24 was a reliable capsid marker and revealed that the protein remained capsid associated following entry into cells and upon nuclear docking. These dual-fluorescent viruses made possible the discrimination of procapsids during infection and monitoring of capsid shell maturation kinetics. The results demonstrate the feasibility of imaging herpesvirus procapsids and their morphogenesis in living cells and indicate that the encapsidation machinery does not substantially help coordinate capsid shell maturation.IMPORTANCEThe familyHerpesviridaeconsists of human and veterinary pathogens that cause a wide range of diseases in their respective hosts. These viruses share structurally related icosahedral capsids that encase the double-stranded DNA (dsDNA) viral genome. The dynamics of capsid assembly and maturation have been inaccessible to examination in living cells. This study has overcome this technical hurdle and provides new insights into this fundamental stage of herpesvirus infection.

scholarly journals CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Peng-Fei Fu ◽  
Xuan Cheng ◽  
Bing-Qian Su ◽  
Li-Fang Duan ◽  
Cong-Rong Wang ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Antiviral Agents ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Economic Losses ◽  
Green Fluorescent ◽  
Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

scholarly journals Construction of an Excisable Bacterial Artificial Chromosome Containing a Full-Length Infectious Clone of Herpes Simplex Virus Type 1: Viruses Reconstituted from the Clone Exhibit Wild-Type Properties In Vitro and In Vivo

2003 ◽  
Vol 77 (2) ◽  
pp. 1382-1391 ◽  
Author(s):  
Michiko Tanaka ◽  
Hiroyuki Kagawa ◽  
Yuji Yamanashi ◽  
Tetsutaro Sata ◽  
Yasushi Kawaguchi
Keyword(s):  
Vero Cells ◽  
Infectious Molecular Clone ◽  
Wild Type ◽  
Molecular Clone ◽  
Viral Genes ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT In recent years, several laboratories have reported on the cloning of herpes simplex virus type 1 (HSV-1) genomes as bacterial artificial chromosomes (BACs) in Escherichia coli and on procedures to manipulate these genomes by using the bacterial recombination machinery. However, the HSV-BACs reported so far are either replication incompetent or infectious, with a deletion of one or more viral genes due to the BAC vector insertion. For use as a multipurpose clone in research on HSV-1, we attempted to generate infectious HSV-BACs containing the full genome of HSV-1 without any loss of viral genes. Our results were as follows. (i) E. coli (YEbac102) harboring the full-length HSV-1 genome (pYEbac102) in which a BAC flanked by loxP sites was inserted into the intergenic region between UL3 and UL4 was constructed. (ii) pYEbac102 was an infectious molecular clone, given that its transfection into rabbit skin cells resulted in production of infectious virus (YK304). (iii) The BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus YK304 by coinfection of Vero cells with YK304 and a recombinant adenovirus, AxCANCre, expressing Cre recombinase. (iv) As far as was examined, the reconstituted viruses from pYEbac102 could not be phenotypically differentiated from wild-type viruses in vitro and in vivo. Thus, the viruses grew as well in Vero cells as did the wild-type virus and exhibited wild-type virulence in mice on intracerebral inoculation. (v) The infectious molecular clone pYEbac102 is in fact useful for mutagenesis of the HSV-1 genome by bacterial genetics, and a recombinant virus carrying amino acid substitutions in both copies of the α0 gene was generated. pYEbac102 will have multiple applications to the rapid generation of genetically engineered HSV-1 recombinants in basic research into HSV-1 and in the development of HSV vectors in human therapy.

scholarly journals In vivo and in vitro reactivation impairment of a herpes simplex virus type 1 latency-associated transcript variant in a rabbit eye model.

1991 ◽  
Vol 65 (12) ◽  
pp. 6989-6993 ◽  
Author(s):  
M D Trousdale ◽  
I Steiner ◽  
J G Spivack ◽  
S L Deshmane ◽  
S M Brown ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Eye Model ◽  
Rabbit Eye ◽  
Simplex Virus

scholarly journals Antiviral effect of oryzacystatin, a proteinase inhibitor in rice, against herpes simplex virus type 1 in vitro and in vivo.

1995 ◽  
Vol 39 (4) ◽  
pp. 846-849 ◽  
Author(s):  
H Aoki ◽  
T Akaike ◽  
K Abe ◽  
M Kuroda ◽  
S Arai ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Rice Seed ◽  
Simplex Virus ◽  
Hsv 1

Oryzacystatin (OC) is the first-described cystatin originating from rice seed; it consists of two molecular species, OC-I and OC-II, which have antiviral action against poliovirus in vitro (H. Kondo, S. Ijiri, K. Abe, H. Maeda, and S. Arai, FEBS Lett. 299:48-50, 1992). In the experiments reported here, we investigated the effects of OC-I and OC-II on the replication of herpes simplex virus type 1 (HSV-1) in vitro and in vivo. HSV-1 was inoculated onto monolayers of monkey kidney epithelial cells (CV-1 cells) at a multiplicity of infection of 0.1 PFU per cell. After adsorption of the virus onto cells, the cultures were incubated in the presence of either OC-I or OC-II in the concentration range of 1.0 to 300 microM, and the supernatant virus yield was quantitated at 24 h. The effective concentration for 90% inhibition of HSV-1 was 14.8 microM, while a cytotoxic effect on CV-1 cells without infection of HSV-1 was not observed below 500 microM OC-I. Therefore, the apparent in vitro chemotherapeutic index was estimated to be more than 33. In the mouse model of HSV-1-induced keratitis and encephalopathy, topical administration of OC-I to the mouse cornea produced a significant decrease in virus production in the cornea (mean virus yields: 3.11 log10 PFU in the treated group and 4.37 log10 PFU in the control group) and significant improvement in survival rates (P = 0.01). The in vivo antiherpetic effect of OC-I was comparable to that of acyclovir, indicating that topical treatment of HSV-1 infection in humans with OC-I might be possible. Our data also suggest the importance of some thiol proteinases, which may be derived from either the host's cells or HSV-1, during the replication process of HSV-1.

scholarly journals Properties of Fluorescent Far-Red Anti-TNF Nanobodies

Antibodies ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 43 ◽  
Author(s):  
Ekaterina Gorshkova ◽  
Grigory Efimov ◽  
Ksenia Ermakova ◽  
Ekaterina Vasilenko ◽  
Diana Yuzhakova ◽  
...  
Keyword(s):  
Autoimmune Diseases ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Humanized Mice ◽  
Whole Body ◽  
Whole Body Imaging ◽  
Theranostic Agent ◽  
Variable Heavy

Upregulation of the expression of tumor necrosis factor (TNF-α, TNF) has a significant role in the development of autoimmune diseases. The fluorescent antibodies binding TNF may be used for personalized therapy of TNF-dependent diseases as a tool to predict the response to anti-TNF treatment. We generated recombinant fluorescent proteins consisting of the anti-TNF module based on the variable heavy chain (VHH) of camelid antibodies fused with the far-red fluorescent protein Katushka (Kat). Two types of anti-TNF VHH were developed: one (BTN-Kat) that was bound both human or mouse TNF, but did not neutralize their activity, and a second (ITN-Kat) that was binding and neutralizing human TNF. BTN-Kat does not interfere with TNF biological functions and can be used for whole-body imaging. ITN-Kat can be evaluated in humanized mice or in cells isolated from humanized mice. It is able to block human TNF (hTNF) activities both in vitro and in vivo and may be considered as a prototype of a theranostic agent for autoimmune diseases.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 200010
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Emir Bora Akmeriç ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

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