scholarly journals Development of a Novel High-Throughput Surrogate Assay to Measure HIV Envelope/CCR5/CD4-Mediated Viral/Cell Fusion Using BacMam Baculovirus Technology

2003 ◽  
Vol 8 (4) ◽  
pp. 463-470 ◽  
Author(s):  
Stephen Jenkinson ◽  
David C. Mc Coy ◽  
Sandy A. Kerner ◽  
Robert G. Ferris ◽  
Wendell K. Lawrence ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
High Throughput ◽  
Viral Envelope ◽  
Host Cells ◽  
Luciferase Reporter ◽  
Viral Fusion ◽  
Fusion Event ◽  
Surrogate Assay ◽  
Cell Cell

The initial event by which M-tropic HIV strains gain access to cells is via interaction of the viral envelope protein gp120 with the host cell CCR5 coreceptor and CD4. Inhibition of this event reduces viral fusion and entry into cells in vitro. The authors have employed BacMam baculovirus-mediated gene transduction to develop a cell/cell fusion assay that mimics the HIV viral/cell fusion process and allows high-throughput quantification of this fusion event. The assay design uses human osteosarcoma (HOS) cells stably transfected with cDNAs expressing CCR5, CD4, and long terminal repeat (LTR)-luciferase as the recipient host cell. An HEK-293 cell line transduced with BacMam viral constructs to express the viral proteins gp120, gp41, tat, and rev represents the virus. Interaction of gp120 with CCR5/CD4 results in the fusion of the 2 cells and transfer of tat to the HOS cell cytosol; tat, in turn, binds to the LTR region on the luciferase reporter and activates transcription, resulting in an increase in cellular luciferase activity. In conclusion, the cell/cell fusion assay developed has been demonstrated to be a robust and reproducible high-throughput surrogate assay that can be used to assess the effects of compounds on gp120/CCR5/CD4-mediated viral fusion into host cells.

scholarly journals HIV Cell Fusion Assay

2013 ◽  
Vol 19 (1) ◽  
pp. 108-118 ◽  
Author(s):  
Elizabeth B. Smith ◽  
Robert A. Ogert ◽  
David Pechter ◽  
Artjohn Villafania ◽  
Susan J. Abbondanzo ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
Chemokine Receptor ◽  
Viral Entry ◽  
Envelope Glycoprotein ◽  
Laser Scanning ◽  
Viral Envelope ◽  
Fusion Event ◽  
Cxcr4 Signaling ◽  
Hiv Entry

The health and disease-related biology of the CXCR4 chemokine receptor presents the challenge of finding a small molecule that can bind CXCR4 and block T-cell tropic human immunodeficiency virus type 1 (HIV-1) cell entry, while preserving the ability of CXCR4 to respond to its native ligand, CXCL12. HIV entry into the host cell involves the interaction of the viral envelope glycoprotein gp120 binding to CD4, followed by a rearrangement in gp120, and subsequent interaction with the chemokine receptor CXCR4 or CCR5. These initial events can be re-created in a cell fusion assay that represents a surrogate system, mimicking the early stages of viral entry via these host cell receptors. In the current study, a T-tropic HIV cell fusion assay was established using U2OS cells expressing the envelope glycoprotein gp160 from the T-tropic HIV NL4-3 and HeLa cells expressing CD4 and CXCR4. Detection of the cell fusion event was based on a Gal4/VP16-activated β-lactamase signal and was measured by automated microscopy or laser scanning plate cytometry. Changes in morphology associated with cell fusion were combined with β-lactamase activity to generate results with robust assay statistics in both 384-well and 1536-well plates. Compounds were subsequently characterized by CXCR4 signaling assays to eliminate functional antagonists and allow the identification of a function-sparing HIV entry inhibitor.

scholarly journals Suppression of a Fusion Defect by Second Site Mutations in the Ecotropic Murine Leukemia Virus Surface Protein

1999 ◽  
Vol 73 (6) ◽  
pp. 5034-5042 ◽  
Author(s):  
Tatiana Zavorotinskaya ◽  
Lorraine M. Albritton
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Surface Protein ◽  
Host Cells ◽  
Virus Surface ◽  
Fusion Defect ◽  
Murine Leukemia ◽  
Cell Cell

ABSTRACT Entry of ecotropic murine leukemia virus initiates when the envelope surface protein recognizes and binds to the virus receptor on host cells. The envelope transmembrane protein then mediates fusion of viral and host cell membranes and penetration into the cytoplasm. Using a genetic selection, we isolated an infectious retrovirus variant containing three changes in the surface protein—histidine 8 to arginine, glutamine 227 to arginine, and aspartate 243 to tyrosine. Single replacement of histidine 8 with arginine (H8R) resulted in almost complete loss of infectivity, even though the mutant envelope proteins were stable and efficiently incorporated into virions. Virions carrying H8R envelope were proficient at binding cells expressing receptor but failed to induce cell-cell fusion of XC cells, indicating that the histidine at position 8 plays an essential role in fusion during penetration of the host cell membrane. Thus, there is at least one domain in SU that is involved in fusion; the fusion functions do not reside exclusively in TM. In contrast, envelope with all three changes induced cell-cell fusion of XC cells and produced virions that were 10,000-fold more infectious than those containing only the H8R substitution, indicating that changes at positions 227 and 243 can suppress a fusion defect caused by loss of histidine 8 function. Moreover, the other two changes acted synergistically, indicating that both compensate for the loss of the same essential function of histidine 8. The ability of these changes to suppress this fusion defect might provide a means for overcoming postbinding defects found in targeted retroviral vectors for use in human gene therapy.

scholarly journals pH and Receptor Induced Conformational Changes-Implications Towards S1 Dissociation of SARS-CoV2 Spike Glycoprotein

2020 ◽  
Author(s):  
Jesu E. Castin ◽  
Daniel A. Gideon ◽  
Karthik S. Sudarsha ◽  
Sherlin A. Rosita
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
Fusion Proteins ◽  
Proteolytic Enzymes ◽  
Host Cells ◽  
Viral Fusion ◽  
Health Crisis ◽  
S Protein ◽  
Viral Attachment ◽  
Cell Receptors

AbstractViruses, being obligate intracellular parasites, must first attach themselves and gain entry into host cells. Viral fusion machinery is the central player in the viral attachment process in almost every viral disease. Viruses have incorporated an array of efficient fusion proteins on their surfaces to bind efficiently to host cell receptors. They make use of the host proteolytic enzymes to rearrange their surface protein(s) into the form which facilitates their binding to host-cell membrane proteins and subsequently, fusion. This stage of viral entry is very critical and has many therapeutic implications. The current global pandemic of COVID-19 has sparked severe health crisis and economic shutdowns. SARS-CoV2, the etiological agent of the disease has led to millions of deaths and brought the scientific community together in an attempt to understand the mechanisms of SARS-CoV2 pathogenesis and mortality. Like other viral fusion machinery, CoV2 spike (S) glycoprotein- ‘The Demogorgon’ poses the same questions about viral-host cell fusion. The intermediate stages of S protein-mediated viral fusion are unclear owing to the lack of structural insights and concrete biochemical evidence. The mechanism of conformational transition is still unclear. S protein binding and fusion with host cell receptors, Eg., angiotensin-converting enzyme-2 (ACE2) is accompanied by cleavage of S1/S2 subunits. To track the key events of viral-host cell fusion, we have identified (in silico) that low pH-induced conformational change and ACE-2 binding events promote S1 dissociation. Deciphering key mechanistic insights of SARS-CoV2 fusion will further our understanding of other class-I fusion proteins

scholarly journals Reciprocal Regulation of AKT and MAP Kinase Dictates Virus-Host Cell Fusion

2010 ◽  
Vol 84 (9) ◽  
pp. 4366-4382 ◽  
Author(s):  
Nishi R. Sharma ◽  
Prashant Mani ◽  
Neha Nandwani ◽  
Rajakishore Mishra ◽  
Ajay Rana ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
Sendai Virus ◽  
Host Cells ◽  
Mitogen Activated Protein Kinase ◽  
Viral Fusion ◽  
Reciprocal Regulation ◽  
Mapk Cascades ◽  
Mitogen Activated Protein ◽  
Syncytial Virus

ABSTRACT Viruses of the Paramyxoviridae family bind to their host cells by using hemagglutinin-neuraminidase (HN), which enhances fusion protein (F)-mediated membrane fusion. Although respiratory syncytial virus and parainfluenza virus 5 of this family are suggested to trigger host cell signaling during infection, the virus-induced intracellular signals dictating virus-cell fusion await elucidation. Using an F- or HN-F-containing reconstituted envelope of Sendai virus, another paramyxovirus, we revealed the role and regulation of AKT1 and Raf/MEK/ERK cascades during viral fusion with liver cells. Our observation that extracellular signal-regulated kinase (ERK) activation promotes viral fusion via ezrin-mediated cytoskeletal rearrangements, whereas AKT1 attenuates fusion by promoting phosphorylation of F protein, indicates a counteractive regulation of viral fusion by reciprocal activation of AKT1 and mitogen-activated protein kinase (MAPK) cascades, establishing a novel conceptual framework for a therapeutic strategy.

Herpes simplex virus glycoprotein B (gB) mutations define structural sites in domain I, the membrane proximal region, and the cytodomain that regulate entry.

2021 ◽  
Author(s):  
Qing Fan ◽  
Richard Longnecker ◽  
Sarah A. Connolly
Keyword(s):  
Cell Fusion ◽  
Virus Entry ◽  
Proximal Region ◽  
Glycoprotein B ◽  
Viral Fusion ◽  
Viral Fusion Protein ◽  
Membrane Proximal Region ◽  
Domain V ◽  
Domain I ◽  
Cell Cell

The viral fusion protein glycoprotein B (gB) is conserved in all herpesviruses and is essential for virus entry. During entry, gB fuses viral and host cell membranes by refolding from a prefusion to a postfusion form. We previously introduced three structure-based mutations (gB-I671A/H681A/F683A) into the domain V arm of the gB ectodomain that resulted in reduced cell-cell fusion. A virus carrying these three mutations (called gB3A) displayed a small plaque phenotype and remarkably delayed entry into cells. To identify mutations that could counteract this phenotype, we serially passaged the gB3A virus and selected for revertant viruses with increased plaque size. Genomic sequencing revealed that the revertant viruses had second-site mutations in gB, including E187A, M742T, and S383F/G645R/V705I/V880G. Using expression constructs encoding these mutations, only gB-V880G was shown to enhance cell-cell fusion. In contrast, all of the revertant viruses showed enhanced entry kinetics, underscoring the fact that cell-cell fusion and virus-cell fusion are different. The results indicate that mutations in three different regions of gB (domain I, the membrane proximal region, and the cytoplasmic tail domain) can counteract the slow entry phenotype of gB3A virus. Mapping these compensatory mutations to prefusion and postfusion structural models suggests sites of intramolecular functional interactions with the gB domain V arm that may contribute to the gB fusion function. Importance The nine human herpesviruses are ubiquitous and cause a range of disease in humans. Glycoprotein B (gB) is an essential viral fusion protein that is conserved in all herpesviruses. During host cell entry, gB mediates virus-cell membrane fusion by undergoing a conformational change. Structural models for the prefusion and postfusion form of gB exist, but the details of how the protein converts from one to the other are unclear. We previously introduced structure-based mutations into gB that inhibited virus entry and fusion. By passaging this entry-deficient virus over time, we selected second-site mutations that partially restore virus entry. The location of these mutations suggest regulatory sites that contribute to fusion and gB refolding during entry. gB is a target of neutralizing antibodies and defining how gB refolds during entry could provide a basis for the development of fusion inhibitors for future research or clinical use.

Studies of membrane fusion. VI. Mechanism of the membrane fusion and cell swelling stages of Sendai virus-mediated cell fusion

1980 ◽  
Vol 43 (1) ◽  
pp. 103-118
Author(s):  
S. Knutton
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Sendai Virus ◽  
Freeze Fracture ◽  
Viral Envelope ◽  
Cell Swelling ◽  
Fusion Event ◽  
Virus Envelope ◽  
Membrane Rupture ◽  
Membrane Tubules

The membrane fusion and cell swelling stages of Sendai virus-mediated cell-cell fusion have been studied by thin-section and freeze-fracture electron microscopy. Sites of membrane fusion have been detected in human erythrocytes arrested at the membrane fusion stage of cell fusion and in virtually all cases a fused viral envelope or envelope components has been identified thus providing further direct evidence that cell-viral envelope-cell bridge formation is the membrane fusion event in Sendai virus-induced cell fusion. Radial expansion of a single virus bridge connecting 2 cells is sufficient to produce a fused cell. Membrane redistribution which occurs during this cell swelling stage of the fusion process is often accompanied by the formation of a system of membrane tubules in the plane of expansion of the virus bridge. The tubules originate from points of fusion between the bridging virus envelope and the erythrocyte membrane and also expand radially as cells swell. Ultimately membrane rupture occurs and the tubules appear to break down as small vesicles. When previously observed in cross-sectioned cells these membrane tubules were interpreted as sites of direct membrane fusion. The present study indicates that this interpretation is incorrect and shows that the tubules are generated subsequent to membrane fusion when 2 cells connected by a virus bridge are induced to swell. A mechanism to explain the formation of this system of membrane tubules is proposed.

The role of cell swelling and haemolysis in Sendai virus-induced cell fusion and in the diffusion of incorporated viral antigens

1980 ◽  
Vol 42 (1) ◽  
pp. 153-167
Author(s):  
S. Knutton ◽  
T. Bachi
Keyword(s):  
Cell Fusion ◽  
Erythrocyte Membrane ◽  
Sendai Virus ◽  
Viral Envelope ◽  
Cell Swelling ◽  
Haemolytic Activity ◽  
Erythrocyte Ghosts ◽  
Viral Antigens ◽  
Cell Cell

The role of the haemolytic activity of Sendai virus in cell-cell fusion has been examined in monolayers of human erythrocytes and erythrocyte ghosts fused with either haemolytic or non-haemolytic virus. Morphological observations indicate that cell swelling and haemolysis is a distinct event in cell-cell fusion irrespective of whether it is virally induced or, in the case of non-haemolytic virus, experimentally induced. Osmotic swelling appears to be the driving force by which cells which have established sites of membrane fusion expand such sites to form poly-erythrocytes. Immunofluorescent labelling of viral antigens incorporated into the erythrocyte membrane as a result of viral envelope-cell fusion indicates that diffusion of antigens in the plane of the membrane is restricted in intact erythrocytes and resealed erythrocyte ghosts but not in haemolysed erythrocytes or unsealed ghosts. A perturbation of the erythrocyte membrane resulting from osmotic lysis appears to form a prerequisite for the lateral diffusion of viral elements.

Semliki Forest virus induced cell-cell fusion at neutral extracellular pH

1990 ◽  
Vol 10 (4) ◽  
pp. 363-374 ◽  
Author(s):  
Christoph Kempf ◽  
Marcel R. Michel ◽  
Adames Omar ◽  
Pia Jentsch ◽  
Andreas Morell
Keyword(s):  
Cell Fusion ◽  
Semliki Forest Virus ◽  
Covalent Modification ◽  
Surface Proteins ◽  
Acidic Ph ◽  
Fusion Event ◽  
Cell Surface Proteins ◽  
Infected Cells ◽  
Peptide Segments ◽  
Cell Cell

Semliki Forest virus-induced cell-cell fusion from within was considered to exclusively occur at mildly acidic pH (<6.2). Data of this study show that such cell fusion can also be triggered by transient acidification of the cytoplasm of infected cells at an extracellular, neutral pH. Results were obtained by utilizing NH4Cl pulses combined with covalent modification of cell surface proteins. The observation implies a revision of the current consensus regarding the mechanism of Semliki Forest virus induced cell-cell fusion. We propose a model in which at least two peptide segments of the viral spike protein E1 may be involved in triggering the fusion event.

scholarly journals Development of a Novel Dual CCR5-Dependent and CXCR4-Dependent Cell-Cell Fusion Assay System with Inducible gp160 Expression

2005 ◽  
Vol 11 (1) ◽  
pp. 65-74 ◽  
Author(s):  
Changhua Ji ◽  
Jun Zhang ◽  
Nick Cammack ◽  
Surya Sankuratri
Keyword(s):  
Cell Fusion ◽  
Fold Increase ◽  
Assay System ◽  
Luciferase Reporter ◽  
Specific Cell ◽  
Entry Inhibitors ◽  
Fusion Inhibitors ◽  
Dependent Cell ◽  
Hiv Entry ◽  
Cell Cell

In the current study, a novel coreceptor-specific cell-cell fusion (CCF) assay system is reported. The system possesses the following features: dual CCR5-dependent and CXCR4-dependent CCF assays, all stable cell lines, inducible expression of gp160 to minimize cytotoxicity, robust luciferase reporter, and 384-well format. These assays have been validated using various known HIV entry inhibitors targeting various stages of the HIV entry/fusion process, including fusion inhibitors, gp120 inhibitors, CCR5 antagonists, CCR5 antibodies, and CXCR4 antagonists. IC 50data generated from this assay system were well correlated to that from the antiviral assays. The effects of DMSOon this assay systemwere assessed, and a 2-to 3-fold increase in luciferase activitywas observed in the presence of 0.05% to2% DMSO. Although cell-cell fusion efficiencywas enhanced, no changes in drug response kinetics for entry inhibitors were found in the presence of 0.1% or 0.5% DMSO. This assay system has been successfully used for the identification and characterization of thousands of CCR5 inhibitors.

scholarly journals Ehrlichia chaffeensis TRP120 Binds a G+C-Rich Motif in Host Cell DNA and Exhibits Eukaryotic Transcriptional Activator Function

2011 ◽  
Vol 79 (11) ◽  
pp. 4370-4381 ◽  
Author(s):  
Bing Zhu ◽  
Jeeba A. Kuriakose ◽  
Tian Luo ◽  
Efren Ballesteros ◽  
Sharu Gupta ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Dna Binding ◽  
Host Cell ◽  
High Throughput ◽  
Tandem Repeat ◽  
Binding Sites ◽  
Host Gene ◽  
Host Cells ◽  
Ehrlichia Chaffeensis ◽  
Content Type

ABSTRACTEhrlichia chaffeensisis an obligately intracellular bacterium that modulates host cell gene transcription in the mononuclear phagocyte, but the host gene targets and mechanisms involved in transcriptional modulation are not well-defined. In this study, we identified a novel tandem repeat DNA-binding domain in theE. chaffeensis120-kDa tandem repeat protein (TRP120) that directly binds host cell DNA. TRP120 was observed by immunofluorescent microscopy in the nucleus ofE. chaffeensis-infected host cells and was detected in nuclear extracts by Western immunoblotting with TRP120-specific antibody. The TRP120 binding sites and associated host cell target genes were identified using high-throughput deep sequencing (Illumina) of immunoprecipitated DNA (chromatin immunoprecipitation and high-throughput DNA sequencing). Multiple em motif elicitation (MEME) analysis of the most highly enriched TRP120-bound sequences revealed a G+C-rich DNA motif, and recombinant TRP120 specifically bound synthetic oligonucleotides containing the motif. TRP120 target gene binding sites were mapped most frequently to intersecting regions (intron/exon; 49%) but were also identified in upstream regulatory regions (25%) and downstream locations (26%). Genes targeted by TRP120 were most frequently associated with transcriptional regulation, signal transduction, and apoptosis. TRP120 targeted inflammatory chemokine genes, CCL2, CCL20, and CXCL11, which were strongly upregulated duringE. chaffeensisinfection and were also upregulated by direct transfection with recombinant TRP120. This study reveals that TRP120 is a novel DNA-binding protein that is involved in a host gene transcriptional regulation strategy.

Sign in / Sign up

Export Citation Format

Share Document