Generation of a Reporter Cell Line for Detection of Infectious Varicella-Zoster Virus and Its Application to Antiviral Studies

ABSTRACT To simplify the titration of infectious varicella-zoster virus (VZV), we generated a reporter cell line that produced luciferase in a dose-dependent manner upon infection with cell-free VZV. A few VZV-infected cells were detectable by coculturing with the cell line. We demonstrated the usefulness of the cell line for antiviral studies.

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Characterization of Entry Mechanisms of Human Herpesvirus 8 by Using an Rta-Dependent Reporter Cell Line

Journal of Virology ◽

10.1128/jvi.77.14.8147-8152.2003 ◽

2003 ◽

Vol 77 (14) ◽

pp. 8147-8152 ◽

Cited By ~ 49

Author(s):

Naoki Inoue ◽

Jörn Winter ◽

Renu B. Lal ◽

Margaret K. Offermann ◽

Shin Koyano

Keyword(s):

Cell Line ◽

Primary Effusion Lymphoma ◽

Human Herpesvirus ◽

Human Herpesvirus 8 ◽

Dependent Manner ◽

Reporter Cell Line ◽

Reporter Cell ◽

Herpesvirus 8 ◽

Intracellular Expression ◽

Infected Cells

ABSTRACT To analyze the mechanisms of entry of human herpesvirus 8 (HHV-8), we established a reporter cell line T1H6 that contains the lacZ gene under the control of the polyadenylated nuclear RNA promoter, known to be strongly activated by a viral transactivator, Rta. We found that infection with cell-free virus, as well as cocultivation with HHV-8-positive primary effusion lymphoma cell lines, activated the lacZ gene of T1H6 in a sensitive and dose-dependent manner. Addition of Polybrene and centrifugation enhanced, but polysulfonate compounds inhibited, the HHV-8 infectivity. RGD-motif-containing polypeptides and integrins did not decrease the infectivity, suggesting the presence of an additional cellular receptor other than the reported one. The entry was dependent on pH acidification but not on the clathrin pathway. Although conditioned media obtained from human immunodeficiency virus (HIV)-infected cells did not have any effect on the early steps of HHV-8 infection, intracellular expression of a proviral HIV type 1, but not of Tat alone, increased the HHV-8-dependent reporter activation slightly, suggesting a potential of HIV-mediated enhancement of an early step of HHV-8 infection.

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Titration of cell-associated varicella-zoster virus with the MV9G reporter cell line for antiviral studies

Journal of Virological Methods ◽

10.1016/j.jviromet.2018.06.019 ◽

2018 ◽

Vol 260 ◽

pp. 14-20

Author(s):

Xiaojie Li ◽

Xiaoxia Li ◽

Wei Gong ◽

Guanqing Wang ◽

Zhenling Lu ◽

...

Keyword(s):

Varicella Zoster Virus ◽

Cell Line ◽

Reporter Cell Line ◽

Reporter Cell ◽

Varicella Zoster

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A Functional YNKI Motif in the Short Cytoplasmic Tail of Varicella-Zoster Virus Glycoprotein gH Mediates Clathrin-Dependent and Antibody-Independent Endocytosis

Journal of Virology ◽

10.1128/jvi.77.7.4191-4204.2003 ◽

2003 ◽

Vol 77 (7) ◽

pp. 4191-4204 ◽

Cited By ~ 29

Author(s):

Tracy Jo Pasieka ◽

Lucie Maresova ◽

Charles Grose

Keyword(s):

Amino Acids ◽

Varicella Zoster Virus ◽

Cytoplasmic Tail ◽

Dependent Manner ◽

Independent Manner ◽

Varicella Zoster ◽

Cytoplasmic Tails ◽

Infected Cells ◽

Simplex Virus ◽

Alignment Analysis

ABSTRACT The trafficking of varicella-zoster virus (VZV) gH was investigated under both infection and transfection conditions. In initial endocytosis assays performed in infected cells, the three glycoproteins gE, gI, and gB served as positive controls for internalization from the plasma membrane. Subsequently, we discovered that gH in VZV-infected cells was also internalized and followed a similar trafficking pattern. This observation was unexpected because all herpesvirus gH homologues have short endodomains not known to contain trafficking motifs. Further investigation demonstrated that VZV gH, when expressed alone with its chaperone gL, was capable of endocytosis in a clathrin-dependent manner, independent of gE, gI, or gB. Upon inspection of the short gH cytoplasmic tail, we discovered a putative tyrosine-based endocytosis motif (YNKI). When the tyrosine was replaced with an alanine, endocytosis of gH was blocked. Utilizing an endocytosis assay dependent on biotin labeling, we further documented that endocytosis of VZV gH was antibody independent. In control experiments, we showed that gE, gI, and gB also internalized in an antibody-independent manner. Alignment analysis of the VZV gH cytoplasmic tail to other herpesvirus gH homologues revealed two important findings: (i) herpes simplex virus type 1 and 2 homologues lacked an endocytosis motif, while all other alphaherpesvirus gH homologues contained a potential motif, and (ii) the VZV gH and simian varicella virus gH cytoplasmic tails were likely longer in length (18 amino acids) than predicted in the original sequence analyses (12 and 16 amino acids, respectively). The longer tails provided the proper context for a functional endocytosis motif.

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Correction for Shakya et al., “Interferon Gamma Inhibits Varicella-Zoster Virus Replication in a Cell Line-Dependent Manner”

Journal of Virology ◽

10.1128/jvi.02138-19 ◽

2020 ◽

Vol 94 (7) ◽

Author(s):

Akhalesh K. Shakya ◽

Dennis J. O’Callaghan ◽

Seong K. Kim

Keyword(s):

Varicella Zoster Virus ◽

Cell Line ◽

Interferon Gamma ◽

Virus Replication ◽

Dependent Manner ◽

Varicella Zoster ◽

A Cell

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Interferon Gamma Inhibits Varicella-Zoster Virus Replication in a Cell Line-Dependent Manner

Journal of Virology ◽

10.1128/jvi.00257-19 ◽

2019 ◽

Vol 93 (12) ◽

Cited By ~ 5

Author(s):

Akhalesh K. Shakya ◽

Dennis J. O’Callaghan ◽

Seong K. Kim

Keyword(s):

Epithelial Cells ◽

Varicella Zoster Virus ◽

Cell Line ◽

Interferon Gamma ◽

Immediate Early ◽

Dependent Manner ◽

Varicella Zoster ◽

Transactivation Activity ◽

A Cell ◽

Ifn Γ

ABSTRACTThe major immediate early 62 (IE62) protein of varicella-zoster virus (VZV) is delivered to newly infected cell nuclei, where it initiates VZV replication by transactivating viral immediate early (IE), early (E), and late (L) genes. Interferon gamma (IFN-γ) is a potent cytokine produced following primary VZV infection. Furthermore, VZV reactivation correlates with a decline in IFN-γ-producing immune cells. Our results showed that treatment with 20 ng/ml of IFN-γ completely reduced intracellular VZV yield in A549 lung epithelial cells, MRC-5 lung fibroblasts, and ARPE-19 retinal epithelial cells at 4 days post-VZV infection. However, IFN-γ reduced virus yield only 2-fold in MeWo melanoma cells compared to that of untreated cells. IFN-β significantly inhibited VZV replication in both ARPE-19 and MeWo cells. In luciferase assays with VZV open reading frame 61 (ORF61) promoter reporter plasmid, IFN-γ abrogated the transactivation activity of IE62 by 95%, 97%, and 89% in A549, ARPE-19, and MRC-5 cells, respectively. However, IFN-γ abrogated IE62’s transactivation activity by 16% in MeWo cells, indicating that IFN-γ inhibits VZV replication as well as IE62-mediated transactivation in a cell line-dependent manner. The expression of VZV IE62 and ORF63 suppressed by IFN-γ was restored by JAK1 inhibitor treatment, indicating that the inhibition of VZV replication is mediated by JAK/STAT1 signaling. In the presence of IFN-γ, knockdown of interferon response factor 1 (IRF1) increased VZV replication. Ectopic expression of IRF1 reduced VZV yields 4,000-fold in MRC-5 and ARPE-19 cells but 3-fold in MeWo cells. These results suggest that IFN-γ blocks VZV replication by inhibiting IE62 function in a cell line-dependent manner.IMPORTANCEOur results showed that IFN-γ significantly inhibited VZV replication in a cell line-dependent manner. IFN-γ inhibited VZV gene expression after the immediate early stage of infection and abrogated IE62-mediated transactivation. These results suggest that IFN-γ blocks VZV replication by inhibiting IE62 function in a cell line-dependent manner. Understanding the mechanisms by which IFN-γ plays a role in VZV gene programming may be important in determining the tissue restriction of VZV.

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Comparison of cellular assays for TLR activation and development of a species-specific reporter cell line for cattle

Innate Immunity ◽

10.1177/1753425917695445 ◽

2017 ◽

Vol 23 (4) ◽

pp. 329-335 ◽

Cited By ~ 3

Author(s):

Kata Tombácz ◽

Duncan Mwangi ◽

Dirk Werling ◽

Amanda J Gibson

Keyword(s):

Cell Line ◽

Luciferase Assay ◽

Specific Cell ◽

Reporter Cell Line ◽

Reporter Assay ◽

Reporter Cell ◽

Cellular Assays ◽

Ligand Interactions ◽

Species Specific ◽

Dose Dependent

PRRs are sentinels of the innate immune system, with TLRs being the most important. Assays for TLR ligand interactions have been used to gain insights into their function and signaling pathways. As significant differences exist between species with regard to ligand recognition, it is necessary to adapt these tools for TLRs of other species. In the present work, we describe a species-specific cell-based assay adapted for the analysis of single PRRs. Human embryonic kidney 293T cells were stably transfected with the NF-κB-inducible reporter gene secreted embryonic alkaline phosphatase (SEAP) together with bovine TLR2. We compared the SEAP response with an existing luciferase NF-κB reporter assay for correlation with IL-8 production. A dose-dependent response was detected upon stimulation using both methods with good correlation to IL-8 secretion. Lower stimulant concentrations were detected by SEAP assay than IL-8 secretion. The luciferase assay produced high non-specific background for all ligand concentrations. Of all assays tested, we found the bovine-specific SEAP reporter assay to be the most convenient and delivered results in the shortest time. The developed reporter cell line would lend well to rapid, high-throughput TLR ligand screening for cattle.

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Ecological and physiological as well as biochemical properties of representatives of the Genus sedum L.

Ecology and Noospherology ◽

10.15421/031417 ◽

2014 ◽

Vol 25 (3-4) ◽

pp. 24-33

Author(s):

O. I. Dzjuba ◽

M. V. Yatsenko

Keyword(s):

Nitric Oxide ◽

Cell Line ◽

Methanol Extract ◽

Hepatoma Cell ◽

Ethanol Extract ◽

Biochemical Properties ◽

Biologically Active ◽

Hepatoma Cell Line ◽

Dependent Manner ◽

Dose Dependent

The article deals with the history of the study and the current state of research of physiological and biochemical properties of the plant genus Sedum that are useful for human and has been used in folk medicine for many years. It was noticed that antioxidant properties of extracts from plants S. sarmentosum, S. sempervivoides, S. takesimense were caused by the presence of phenolic compounds. Methanol extract of plants S. takesimense exhibited strong scavenging activities against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radicals as well as significant inhibitory effects on lipid peroxidation and low density lipoprotein (LDL) oxidation induced by a metal ion Cu2+. Various immunomodulatory activities of various fractions of plants extracts (S. dendroideum, S. kamtschaticum, S. sarmentosum, S. telephium) are observed. It was shown that the ethanol extract of S. sarmentosum and it’s fractions suppressed specific antibody and cellular responses to ovalbumin in mice. The methanol extract of plants S. sarmentosum reduced the levels of anti-inflammatory markers, such as volume of exudates, number of polymorphonuclear leukocytes, suppressed nitric oxide synthesis in activated macrophages via suppressed induction of inducible nitric oxide synthase (iNOS). Polysaccharides fractions from plants S. telephium inducing productions of tumor necrosis factor alpha (TNF-α), increasing the intensity of phagocytosis in vitro and in vivo. Methanol extract from the whole part of S. kamtschaticum strongly inhibit PGE2 production from lipopolysaccharide-induced RAW 264.7 cells, a mouse macrophage cell line via modulating activity in gene expression of the enzyme cyclooxygenase-2 (COX-2). The methanol extract of plants S. sarmentosum and the major kaempferol glycosides from S. dendroideum have antinociceptive activity. It was noticed that anti-adipogenic activity of extracts from plants S. kamtschaticum were caused by inhibition of peroxisome-proliferator-activated receptor γ (PPARγ) expression and it’s dependent target genes, such as genes encoding adipocyte protein 2 (аР2), lipoprotein lipase (LPL), adiponectin and CD36. Polysaccharides fractions from S. telephium cause inhibition of cell adhesion of human fibroblast (MRC5) to laminin and fibronectin via interfere with integrin-mediated cell behaviour and they contributed to the role of polysaccharides in cell-matrix interaction. The methanol extract of plants S. sarmentosum exhibited a significant inhibitory activity in the chick embryo chorioallantoic membrane angiogenesis in a dose-dependent manner. The crude alkaloid fraction of S. sarmentosum caused a dose-dependent inhibition of cell proliferation on murine hepatoma cell line BNL CL.2 and human hepatoma cell line HepG2 without necrosis or apoptosis. Alkaloids from plants S. sarmentosum may improve survival of hepatoma patients via the inhibition of excessive growth of tumor cells. Plant’s juices have antiviral activity (S. sarmentosum, S. spurium, S. stahlii). Crude ethanol extract S. praealtum have spermicidal activity of the in mice and a relevant inhibitory effect of aqueous extract on human spermatozoa motility as well as an anti-fertilizing activity in rats. Hepatoprotective triterpenes, e.g., δ-amyrone, 3-epi-δ-amyrin, δ-amyrin and sarmentolin were isolated from S. sarmentosum. 2- and 2,6-substituted piperidine alkaloids (e.g., norsedamine, allosedridine, sedamine, allosedamine) are observed in plants S. acre, which in the presence of data on the use of pyridine and piperidine derivatives for treating neurodegenerative diseases (e.g., Alzheimer's disease), points on the promising research in this area. Taking into account that biologically active compounds are accumulated in the aboveground vegetative organs of plants of Sedum, the prospects of further study of the use of Sedum for the purposes of biotechnology and in the pharmaceutical industry becomes apparent. This work extends the existing views regarding the use of plants Sedum.

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