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 Amino Acid Substitutions within the Leucine Zipper Domain of the Murine Coronavirus Spike Protein Cause Defects in Oligomerization and the Ability To Induce Cell-to-Cell Fusion

1999 ◽  
Vol 73 (10) ◽  
pp. 8152-8159 ◽  
Author(s):  
Zongli Luo ◽  
Avery M. Matthews ◽  
Susan R. Weiss
Keyword(s):  
Cell Surface ◽  
Cell Fusion ◽  
Leucine Zipper ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Fusion Activity ◽  
Alanine Substitution ◽  
Leucine Zipper Domain ◽  
S Proteins ◽  
Murine Coronavirus

ABSTRACT The murine coronavirus spike (S) protein contains a leucine zipper domain which is highly conserved among coronaviruses. To assess the role of this leucine zipper domain in S-induced cell-to-cell fusion, the six heptadic leucine and isoleucine residues were replaced with alanine by site-directed mutagenesis. The mutant S proteins were analyzed for cell-to-cell membrane fusion activity as well as for progress through the glycoprotein maturation process, including intracellular glycosylation, oligomerization, and cell surface expression. Single-alanine-substitution mutations had minimal, if any, effects on S-induced cell-to-cell fusion. Significant reduction in fusion activity was observed, however, when two of the four middle heptadic leucine or isoleucine residues were replaced with alanine. Double alanine substitutions that involved either of the two end heptadic leucine residues did not significantly affect fusion. All double-substitution mutant S proteins displayed levels of endoglycosidase H resistance and cell surface expression similar to those of the wild-type S. However, fusion-defective double-alanine-substitution mutants exhibited defects in S oligomerization. These results indicate that the leucine zipper domain plays a role in S-induced cell-to-cell fusion and that the ability of S to induce fusion may be dependent on the oligomeric structure of S.

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.

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.

scholarly journals Effects of Mutations in the Rubella Virus E1 Glycoprotein on E1-E2 Interaction and Membrane Fusion Activity

1998 ◽  
Vol 72 (11) ◽  
pp. 8747-8755 ◽  
Author(s):  
Decheng Yang ◽  
Dorothy Hwang ◽  
Zhiyong Qiu ◽  
Shirley Gillam
Keyword(s):  
Cell Surface ◽  
Membrane Fusion ◽  
Rubella Virus ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Fusion Activity ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Hydrophobic Domain ◽  
Infected Cells

ABSTRACT Rubella virus (RV) virions contain two glycosylated membrane proteins, E1 and E2, that exist as a heterodimer and form the viral spike complexes on the virion surface. Formation of an E1-E2 heterodimer is required for transport of E1 out of the endoplasmic reticulum lumen to the Golgi apparatus and plasma membrane. To investigate the nature of the E1-E2 interaction, we have introduced mutations in the internal hydrophobic region (residues 81 to 109) of E1. Substitution of serine at Cys82 (mutant C82S) or deletion of this hydrophobic domain (mutant dt) of E1 resulted in a disruption of the E1 conformation that ultimately affected E1-E2 heterodimer formation and cell surface expression of both E1 and E2. Substitution of either aspartic acid at Gly93 (G93D) or glycine at Pro104 (P104G) was found to impair neither E1-E2 heterodimer formation nor the transport of E1 and E2 to the cell surface. Fusion of RV-infected cells is induced by a brief treatment at a pH below 6.0. To test whether this internal hydrophobic domain is involved in the membrane fusion activity of RV, transformed BHK cell lines expressing either wild-type or mutant spike proteins were exposed to an acidic pH and polykaryon formation was measured. No fusion activity was observed in the C82S, dt, and G93D mutants; however, the wild type and the P104G mutant exhibited fusogenic activities, with greater than 60% and 20 to 40% of the cells being fused, respectively, at pH 4.8. These results suggest that it is likely that the region of E1 between amino acids 81 and 109 is involved in the membrane fusion activity of RV and that it may be important for the interaction of that protein with E2 to form the E1-E2 heterodimer.

scholarly journals Comparative Mutagenesis of Pseudorabies Virus and Epstein-Barr Virus gH Identifies a Structural Determinant within Domain III of gH Required for Surface Expression and Entry Function

2015 ◽  
Vol 90 (5) ◽  
pp. 2285-2293 ◽  
Author(s):  
Britta S. Möhl ◽  
Christina Schröter ◽  
Barbara G. Klupp ◽  
Walter Fuchs ◽  
Thomas C. Mettenleiter ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Pseudorabies Virus ◽  
Surface Expression ◽  
Structural Motif ◽  
Cell Surface Expression ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACTHerpesviruses infect cells using the conserved core fusion machinery composed of glycoprotein B (gB) and gH/gL. The gH/gL complex plays an essential but still poorly characterized role in membrane fusion and cell tropism. Our previous studies demonstrated that the conserved disulfide bond (DB) C278/C335 in domain II (D-II) of Epstein-Barr virus (EBV) gH has an epithelial cell-specific function, whereas the interface of D-II/D-III is involved in formation of the B cell entry complex by binding to gp42. To extend these studies, we compared gH of the alphaherpesvirus pseudorabies virus (PrV) with gH of the gammaherpesvirus EBV to identify functionally equivalent regions critical for gH function during entry. We identified several conserved amino acids surrounding the conserved DB that connects three central helices of D-III of PrV and EBV gH. The present study verified that the conserved DB and several contacting amino acids in D-III modulate cell surface expression and thereby contribute to gH function. In line with this finding, we found that DB C404/C439 and T401 are important for cell-to-cell spread and efficient entry of PrV. This parallel comparison between PrV and EBV gH function brings new insights into how gH structure impacts fusion function during herpesvirus entry.IMPORTANCEThe alphaherpesvirus PrV is known for its neuroinvasion, whereas the gammaherpesvirus EBV is associated with cancer of epithelial and B cell origin. Despite low amino acid conservation, PrV gH and EBV gH show strikingly similar structures. Interestingly, both PrV gH and EBV gH contain a structural motif composed of a DB and supporting amino acids which is highly conserved within theHerpesviridae. Our study verified that PrV gH uses a minimal motif with the DB as the core, whereas the DB of EBV gH forms extensive connections through hydrogen bonds to surrounding amino acids, ensuring the cell surface expression of gH/gL. Our study verifies that the comparative analysis of distantly related herpesviruses, such as PrV and EBV, allows the identification of common gH functions. In addition, we provide an understanding of how functional domains can evolve over time, resulting in subtle differences in domain structure and function.

scholarly journals A Single Amino Acid Change in the Newcastle Disease Virus Fusion Protein Alters the Requirement for HN Protein in Fusion

2000 ◽  
Vol 74 (11) ◽  
pp. 5101-5107 ◽  
Author(s):  
Theresa A. Sergel ◽  
Lori W. McGinnes ◽  
Trudy G. Morrison
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Protein Expression ◽  
Newcastle Disease ◽  
Amino Acid Change ◽  
Syncytium Formation ◽  
Single Amino Acid ◽  
Fusion Activity ◽  
Wild Type ◽  
Single Amino Acid Change

ABSTRACT The role of a leucine heptad repeat motif between amino acids 268 and 289 in the structure and function of the Newcastle disease virus (NDV) F protein was explored by introducing single point mutations into the F gene cDNA. The mutations affected either folding of the protein or the fusion activity of the protein. Two mutations, L275A and L282A, likely interfered with folding of the molecule since these proteins were not proteolytically cleaved, were minimally expressed at the cell surface, and formed aggregates. L268A mutant protein was cleaved and expressed at the cell surface although the protein migrated slightly slower than wild type on polyacrylamide gels, suggesting an alteration in conformation or processing. L268A protein was fusion inactive in the presence or absence of HN protein expression. Mutant L289A protein was expressed at the cell surface and proteolytically cleaved at better than wild-type levels. Most importantly, this protein mediated syncytium formation in the absence of HN protein expression although HN protein enhanced fusion activity. These results show that a single amino acid change in the F1 portion of the NDV F protein can alter the stringent requirement for HN protein expression in syncytium formation.

scholarly journals C-terminal region regulates the functional expression of human noradrenaline transporter splice variants

2006 ◽  
Vol 401 (1) ◽  
pp. 185-195 ◽  
Author(s):  
Chiharu Sogawa ◽  
Kei Kumagai ◽  
Norio Sogawa ◽  
Katsuya Morita ◽  
Toshihiro Dohi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Splice Variants ◽  
Functional Expression ◽  
Surface Expression ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
C Terminus ◽  
And Function

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl−-dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

Identification of amino acids in the pore region of Kv1.2 potassium channel that regulate its glycosylation and cell surface expression

2010 ◽  
Vol 112 (4) ◽  
pp. 913-923 ◽  
Author(s):  
Iku Utsunomiya ◽  
Shinya Tanabe ◽  
Tomonori Terashi ◽  
Souichi Ikeno ◽  
Tadashi Miyatake ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Potassium Channel ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Pore Region

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.

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