scholarly journals Regulation of Varicella-Zoster Virus-Induced Cell-to-Cell Fusion by the Endocytosis-Competent Glycoproteins gH and gE

2004 ◽  
Vol 78 (6) ◽  
pp. 2884-2896 ◽  
Author(s):  
Tracy Jo Pasieka ◽  
Lucie Maresova ◽  
Kimiyasu Shiraki ◽  
Charles Grose
Keyword(s):  
Cell Surface ◽  
Varicella Zoster Virus ◽  
Cell Fusion ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Wild Type ◽  
Viral Glycoprotein ◽  
Transfected Cells ◽  
Varicella Zoster ◽  
Kolmogorov Smirnov

ABSTRACT The gH glycoprotein of varicella-zoster virus (VZV) is a major fusogen. The realigned short cytoplasmic tail of gH (18 amino acids) harbors a functional endocytosis motif (YNKI) that mediates internalization in both VZV-infected and transfected cells (T. J. Pasieka, L. Maresova, and C. Grose, J. Virol. 77: 4194-4202, 2003). During subsequent confocal microscopy studies of endocytosis-deficient gH mutants, we observed that cells transfected with the gH tail mutants exhibited marked fusion. Therefore, we postulated that VZV gH endocytosis served to regulate cell-to-cell fusion. Subsequent analyses of gH+gL transfection fusion assays by the Kolmogorov-Smirnov statistical test demonstrated that expression of the endocytosis-deficient gH mutants resulted in a statistically significant enhancement of cell-to-cell fusion (P < 0.0001) compared to wild-type gH. On the other hand, coexpression of VZV gE, another endocytosis-competent VZV glycoprotein, was able to temper the fusogenicity of the gH endocytosis mutants by facilitating internalization of the mutant gH protein from the cell surface. When the latter results were similarly analyzed, there was no longer any enhanced fusion by the endocytosis-deficient gH mutant protein. In summary, these studies support a role for gH endocytosis in regulating the cell surface expression of gH and thereby regulating gH-mediated fusion. The data also confirm and extend prior observations of a gE-gH interaction during viral glycoprotein trafficking in a VZV transfection system.

Cell surface expression of the Varicella-zoster virus glycoproteins and Fc receptor

Virology ◽  
1990 ◽  
Vol 178 (1) ◽  
pp. 263-272 ◽  
Author(s):  
Virginia Litwin ◽  
Matyas Sandor ◽  
Charles Grose
Keyword(s):  
Cell Surface ◽  
Varicella Zoster Virus ◽  
Surface Expression ◽  
Fc Receptor ◽  
Cell Surface Expression ◽  
Varicella Zoster

scholarly journals Coexpression of UL20p and gK Inhibits Cell-Cell Fusion Mediated by Herpes Simplex Virus Glycoproteins gD, gH-gL, and Wild-Type gB or an Endocytosis-Defective gB Mutant and Downmodulates Their Cell Surface Expression

2004 ◽  
Vol 78 (15) ◽  
pp. 8015-8025 ◽  
Author(s):  
Elisa Avitabile ◽  
Giulia Lombardi ◽  
Tatiana Gianni ◽  
Miriam Capri ◽  
Gabriella Campadelli-Fiume
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Surface ◽  
Cell Fusion ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Wild Type ◽  
Simplex Virus ◽  
In Cells ◽  
Cell Cell

ABSTRACT Syncytium formation in cells that express herpes simplex virus glycoprotein B (gB), gD, gH, and gL is blocked by gK (E. Avitabile, G. Lombardi, and G. Campadelli-Fiume, J. Virol. 77:6836-6844, 2003). Here, we report the results of two series of experiments. First, UL20 protein (UL20p) expression weakly inhibited cell-cell fusion. Coexpression of UL20p and gK drastically reduced fusion in a cell-line-dependent manner, with the highest inhibition in BHK cells. Singly expressed UL20p and gK localized at the endoplasmic reticulum and nuclear membranes. When they were coexpressed, both proteins relocalized to the Golgi apparatus. Remarkably, in cells that coexpressed UL20p and gK, the antifusion activity correlated with a downmodulation of gD, gB, gH, and gL cell surface expression. Second, gBΔ867 has a partial deletion in the cytoplasmic tail that removed endocytosis motifs. Whereas wild-type (wt) gB was internalized in vesicles lined with the endosomal marker Rab5, gBΔ867 was not internalized, exhibited enhanced cell surface expression, and was more efficient in mediating cell-cell fusion than wt gB. The antifusion activity of UL20p and gK was also exerted when gBΔ867 replaced wt gB in the cell fusion assay. These studies show that the gB C tail carries a functional endocytosis motif(s) and that the removal of the motif correlated with increased gB surface expression and increased fusion activity. We conclude that cell-cell fusion in wt-virus-infected cells is negatively controlled by at least two mechanisms. The novel mechanism described here involves the concerted action of UL20p and gK and correlates with a moderate but consistent reduction in the cell surface expression of the fusion glycoproteins. This mechanism is independent of the one exerted through endocytosis-mediated downmodulation of gB from the plasma membrane.

Acidic amino acids increase fusion activity in the specific fusion domain of Newcastle disease virus fusion protein

2013 ◽  
Vol 59 (9) ◽  
pp. 641-644 ◽  
Author(s):  
Guijie Ren ◽  
Yunlong Zhuang ◽  
Keli Tian ◽  
Huiyu Li ◽  
Xueqin Diao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Cell Fusion ◽  
Newcastle Disease ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Fusion Activity ◽  
Wild Type ◽  
F Protein ◽  
Fusion Domain

To explore the effects of amino acids Gln and Asn within the specific fusion domain of fusion (F) protein on the specific membrane fusion in Newcastle disease virus (NDV), the mutants Q204E–Q205E and N245D were constructed in the specific fusion domain of F protein. The mutant genes were co-expressed with homologous or heterologous hemagglutinin–neuraminidase (HN) in BHK21 cells. Cell fusion functions of mutants were analyzed with Giemsa staining and reporter gene methods. Cell surface expression efficiency was analyzed with immunofluorescence assay and fluorescence-activated cell sorter analysis. Co-immunoprecipitation was performed to analyze the interaction of mutant F proteins with the homotypic HN protein. Both Q204E–Q205E and N245D mutations caused increased cell–cell fusion activity when they were co-expressed with homotypic HN protein. The mutant F proteins had slight changes in cell surface expression compared with that of wild-type F protein. The interactions of Q204E–Q205E or N245D with their homotypic HN increased significantly (P < 0.01) compared with the wild-type F protein. Neither Q204–Q205E nor N245D caused cell fusion in the presence of heterologous HN protein. Our data suggested that the residues Q204, Q205, and N245 play a critical role in the regulation of cell fusion. They may decrease the interaction of wild-type NDV F and NDV HN to suppress the fusion activity for survival of the infected host, which may enable a persistent virus infection and long-term virus reproduction and spread.

scholarly journals Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein–coupled receptor kinase–dependent manner

2013 ◽  
Vol 24 (11) ◽  
pp. 1649-1660 ◽  
Author(s):  
Susumu Hara ◽  
Shigeki Arawaka ◽  
Hiroyasu Sato ◽  
Youhei Machiya ◽  
Can Cui ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Dopamine Transporter ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Receptor Kinase ◽  
Wild Type ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.

scholarly journals ΔF508-CFTR Modulator Screen Based on Cell Surface Targeting of a Chimeric Nucleotide Binding Domain 1 Reporter

2018 ◽  
Vol 23 (8) ◽  
pp. 823-831
Author(s):  
Puay-Wah Phuan ◽  
Guido Veit ◽  
Joseph-Anthony Tan ◽  
Ariel Roldan ◽  
Walter E. Finkbeiner ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cell Surface ◽  
Nucleotide Binding ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Wild Type ◽  
Nucleotide Binding Domain ◽  
Δf508 Cftr ◽  
Nucleotide Binding Domain 1 ◽  
Cftr Modulators

The most common cystic fibrosis–causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine at residue 508 (∆F508). The ∆F508 mutation impairs folding of nucleotide binding domain 1 (NBD1) and interfacial interactions of NBD1 and the membrane spanning domains. Here, we report a domain-targeted screen to identify ∆F508-CFTR modulators that act on NBD1. A biochemical screen for ΔF508-NBD1 cell surface expression was done in Madin–Darby canine kidney cells expressing a chimeric reporter consisting of ΔF508-NBD1, the CD4 transmembrane domain, and an extracellular horseradish peroxidase (HRP) reporter. Using a luminescence readout of HRP activity, the screen was robust with a Z′ factor of 0.7. The screening of ~20,000 synthetic small molecules allowed the identification of compounds from four chemical classes that increased ∆F508-NBD1 cell surface expression by up to 4-fold; for comparison, a 12-fold increased cell surface expression was found for a wild-type NBD1 chimera. While the compounds were inactive as correctors of full-length ΔF508-CFTR, several carboxamide-benzothiophenes had potentiator activity with low micromolar EC50. Interestingly, the potentiators did not activate G551D or wild-type CFTR. Our results provide a proof of concept for a cell-based NBD1 domain screen to identify ∆F508-CFTR modulators that target the NBD1 domain.

scholarly journals Loss of the C Terminus of Melanocortin Receptor 2 (MC2R) Results in Impaired Cell Surface Expression and ACTH Insensitivity

Endocrinology ◽  
2010 ◽  
Vol 151 (12) ◽  
pp. 5971-5971
Author(s):  
Andrea Hirsch ◽  
Eirini Meimaridou ◽  
Monica Fernandez-Cancio ◽  
Amit V. Pandey ◽  
María Clemente ◽  
...  
Keyword(s):  
Cell Surface ◽  
Severe Hypoglycemia ◽  
Surface Expression ◽  
Melanocortin Receptor ◽  
Cell Surface Expression ◽  
Wild Type ◽  
C Terminus ◽  
Exact Function ◽  
Acth Insensitivity ◽  
Glucocorticoid Deficiency

Objective: Mutations in melanocortin receptor 2 (MC2R) and its related melanocortin receptor accessory protein (MRAP) cause familial glucocorticoid deficiency. We identified a novel MC2R mutation, K289fs. This unique mutation in the C terminus of MC2R is located in the intracellular part of the protein for which the exact function is unknown. Setting: A 6-wk-old boy presented with severe hypoglycemia, unmeasurable cortisol, and grossly elevated ACTH but normal electrolytes. Genetic analysis revealed homozygote K289fs mutation in MC2R. His parents and siblings were heterozygous but phenotypically normal. Intervention and Results: The role of the C terminus of MC2R was studied in two cell systems. Because the K289fs mutant changes the last eight amino acids of the protein and leads to protein elongation, wild-type MC2R and C-terminally mutated constructs were tested for activity to respond to ACTH in an OS3 cell-based reporter assay. Wild-type and alanine-substituted constructs responded normally to ACTH. By contrast K289fs and M290X had a total loss of activity. Cell surface assays and confocal localization studies revealed that K289fs and M290X receptors were not found at the cell surface, indicating that their transport from the endoplasmic reticulum to the cell membrane is disrupted. Interestingly, coimmunoprecipitation experiments showed no alteration in the interaction of mutant MC2R with MRAP, suggesting that interaction between these two proteins does not guarantee normal localization. Conclusions: Loss of the C terminus of MC2R impairs cell surface expression and ACTH sensitivity but does not disrupt interaction of MC2R with MRAP. These findings highlight the extreme sensitivity of MC2R to structural disruption.

Headless henipaviral receptor binding glycoproteins reveal key post-receptor binding contributions of the globular head and head/stalk interface in fusion promotion

2021 ◽  
Author(s):  
Yao Yu Yeo ◽  
David W. Buchholz ◽  
Amandine Gamble ◽  
Mason Jager ◽  
Hector C. Aguilar
Keyword(s):  
Cell Fusion ◽  
Receptor Binding ◽  
Viral Entry ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Wild Type ◽  
Emerging Viruses ◽  
Multinucleated Cells ◽  
Head Domain ◽  
Cell Cell

Cedar virus (CedV) is a nonpathogenic member of the Henipavirus (HNV) genus of emerging viruses, which includes the deadly Nipah (NiV) and Hendra (HeV) viruses. CedV forms syncytia, a hallmark of henipaviral and paramyxoviral infections and pathogenicity. However, the intrinsic fusogenic capacity of CedV relative to NiV or HeV remains unquantified. HNV entry is mediated by concerted interactions between the attachment (G) and fusion (F) glycoproteins. Upon receptor binding by the HNV G head domain, a fusion-activating G stalk region is exposed and triggers F to undergo a conformational cascade that leads to viral entry or cell-cell fusion. Here, we first demonstrated quantitatively that CedV is inherently significantly less fusogenic than NiV at equivalent G and F cell surface expression levels. We then generated and tested six headless CedV G mutants of distinct stalk C-terminal lengths, surprisingly revealing highly hyperfusogenic cell-cell fusion phenotypes 3 to 4-fold greater than wild-type CedV levels. Additionally, similarly to NiV, a headless HeV G mutant yielded a less pronounced hyperfusogenic phenotype compared to wild-type HeV. Further, coimmunoprecipitation and cell-cell fusion assays revealed heterotypic NiV/CedV functional G/F bidentate interactions, as well as evidence of HNV G head domain involvement beyond receptor binding or G stalk exposure. All evidence points to the G head/stalk junction being key to modulating HNV fusogenicity, supporting the notion that head domains play several distinct and central roles in modulating stalk domain fusion promotion. Further, this study exemplifies how CedV may help elucidate important mechanistic underpinnings of HNV entry and pathogenicity. IMPORTANCE The Henipavirus genus in the Paramyxoviridae family includes the zoonotic Nipah (NiV) and Hendra (HeV) viruses. NiV and HeV infections often cause fatal encephalitis and pneumonia, but no vaccines or therapeutics are currently approved for human use. Upon viral entry, Henipavirus infections yield the formation of multinucleated cells (syncytia). Viral entry and cell-cell fusion are mediated by the attachment (G) and fusion (F) glycoproteins. Cedar virus (CedV), a nonpathogenic henipavirus, may be a useful tool to gain knowledge on henipaviral pathogenicity. Here, using homotypic and heterotypic full-length and headless CedV, NiV, and HeV G/F combinations, we discovered that CedV G/F are significantly less fusogenic than NiV or HeV G/F, and that the G head/stalk junction is key to modulating cell-cell fusion, refining the mechanism of henipaviral membrane fusion events. Our study exemplifies how CedV may be a useful tool to elucidate broader mechanistic understanding for the important henipaviruses.

Abnormal intracellular sorting of O-linked carbohydrate-deficient interleukin-2 receptors

1988 ◽  
Vol 8 (8) ◽  
pp. 3357-3363
Author(s):  
K F Kozarsky ◽  
S M Call ◽  
S K Dower ◽  
M Krieger
Keyword(s):  
Cell Surface ◽  
Cho Cells ◽  
Interleukin 2 ◽  
Chinese Hamster ◽  
Density Lipoprotein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Structure Stability ◽  
Wild Type ◽  
Intracellular Sorting

The synthesis and intracellular sorting of the interleukin-2 (IL-2) receptor were studied with a line of mutant Chinese hamster ovary (CHO) cells with a reversible defect in protein O glycosylation. Under normal culture conditions the mutant ldlD cannot add N-acetylgalactosamine (Ga1NAc) to proteins. Ga1NAc is the first sugar of mucin-type O-linked oligosaccharides attached to protein. This O-glycosylation defect is rapidly corrected when Ga1NAc is added to the culture mediu. An expression vector for the p55 human IL-2 receptor was transfected into wild-type CHO and ldlD cells and the structure, stability, and cell surface expression of the receptor were examined by immunoprecipitation and antibody-binding assays. Essentially all of the mature form of the normally glycosylated IL-2 receptor in both wild-type CHO cells and ldlD cells incubated with Ga1NAc was expressed on the cell surface. The stability of O-linked carbohydrate-deficient (Od) IL-2 receptors (in ldlD cells without Ga1NAc) was normal; however, missorting of the Od receptors resulted in very little cell surface expression. The sialidase sensitivity and endoglycosidase H resistance of mature Od IL-2 receptors suggest that Od receptor missorting occurred in or beyond the trans Golgi apparatus. The abnormal sorting of the Od IL-2 receptor is compared with the O-glycosylation dependence of the surface expression and stability of the low-density lipoprotein receptor, decay-accelerating factor, and the major antigen envelope protein of Epstein-Barr virus.

Abstract 049: Functional Characterization of KCNQ1 Channel Subunit With an A590T Mutation

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Koshi Kinoshita ◽  
Katsuya Kimoto ◽  
Takuto Komatsu ◽  
Kohki Nishide ◽  
Toshihide Tabata ◽  
...  
Keyword(s):  
Cell Surface ◽  
Functional Characterization ◽  
Coiled Coil ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Delayed Rectifier ◽  
Wild Type ◽  
Cardiac Events ◽  
Channel Function ◽  
Similar Density

Background: KCNQ1 encodes the alpha subunit of the voltage-gated K + channel that mediates the cardiac slow delayed rectifier K + current (I Ks ). A mutation, A590T, in KCNQ1 was incidentally identified in a 40 years old female. She had a mild QTc prolongation in electrocardiogram but has never experienced any cardiac events. A590 is located in the C-terminal domain forming a coiled-coil structure, which has been suggested as a pivotal component for subunit tetramerization and channel trafficking to the cell surface. The previously reported mutations around A590 result in markedly reduced cell surface expression and loss of functional channel. We, for the first time, examined whether and how the A590T mutation affects the I Ks channel function. Methods: To assess the trafficking and channel function of KCNQ1(A590T) mutant subunit, we performed immunostaining, immunoblotting, and voltage-clamp measurements in HEK-293T cells transfected with wild-type or the A590T mutant KCNQ1 or their mixture (WT, A590T, and A590T/WT cells, respectively). Results: The density of a depolarization-activated current in the A590T cells was smaller than that in the WT cells. The threshold, half-maximal activation, and saturating voltages of the depolarization-activated current in the A590T cells were more positive than those in the WT cells. The immunoreactivity against KCNQ1 subunit on the cell surface in the A590T cells is lower than in WT cells. The A590T/WT cells had a similar density of the depolarization-activated current and a similar level of immunoreactivity against the channel subunit to the WT cells. Furthermore, the immunoblotting detected subunit oligomers in the membrane fraction of the A590T cells while their densities were lower than those of the WT cells. Conclusion: Although the A590T mutant subunit can form oligomers for itself, this subunit is not efficiently trafficked to the cell surface without the aid of the WT subunit. Thus, homozygous inheritance of the mutant KCNQ1 might be pathogenic. By contrast, the cells expressing both the mutant and wild-type KCNQ1 subunit had normal I Ks and cell surface expression, indicating that the heterozygous inheritance of the mutant KCNQ1 might not cause severe cardiac diseases.

scholarly journals Abnormal intracellular sorting of O-linked carbohydrate-deficient interleukin-2 receptors.

1988 ◽  
Vol 8 (8) ◽  
pp. 3357-3363 ◽  
Author(s):  
K F Kozarsky ◽  
S M Call ◽  
S K Dower ◽  
M Krieger
Keyword(s):  
Cell Surface ◽  
Cho Cells ◽  
Interleukin 2 ◽  
Chinese Hamster ◽  
Density Lipoprotein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Structure Stability ◽  
Wild Type ◽  
Intracellular Sorting

The synthesis and intracellular sorting of the interleukin-2 (IL-2) receptor were studied with a line of mutant Chinese hamster ovary (CHO) cells with a reversible defect in protein O glycosylation. Under normal culture conditions the mutant ldlD cannot add N-acetylgalactosamine (Ga1NAc) to proteins. Ga1NAc is the first sugar of mucin-type O-linked oligosaccharides attached to protein. This O-glycosylation defect is rapidly corrected when Ga1NAc is added to the culture mediu. An expression vector for the p55 human IL-2 receptor was transfected into wild-type CHO and ldlD cells and the structure, stability, and cell surface expression of the receptor were examined by immunoprecipitation and antibody-binding assays. Essentially all of the mature form of the normally glycosylated IL-2 receptor in both wild-type CHO cells and ldlD cells incubated with Ga1NAc was expressed on the cell surface. The stability of O-linked carbohydrate-deficient (Od) IL-2 receptors (in ldlD cells without Ga1NAc) was normal; however, missorting of the Od receptors resulted in very little cell surface expression. The sialidase sensitivity and endoglycosidase H resistance of mature Od IL-2 receptors suggest that Od receptor missorting occurred in or beyond the trans Golgi apparatus. The abnormal sorting of the Od IL-2 receptor is compared with the O-glycosylation dependence of the surface expression and stability of the low-density lipoprotein receptor, decay-accelerating factor, and the major antigen envelope protein of Epstein-Barr virus.

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