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 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 Lassa Fever Virus Glycoprotein Mediates LAMP1- and Low pH-Dependent Cell-Cell Fusion through a Stalk-Pore Mechanism

2018 ◽  
Vol 114 (3) ◽  
pp. 605a
Author(s):  
Ruben M. Markosyan ◽  
Mariana Marin ◽  
Fredric S. Cohen ◽  
Gregory B. Melikyan
Keyword(s):  
Cell Fusion ◽  
Lassa Fever ◽  
Low Ph ◽  
Fever Virus ◽  
Virus Glycoprotein ◽  
Lassa Fever Virus ◽  
Dependent Cell ◽  
Ph Dependent ◽  
Cell Cell

scholarly journals Micro-fusion inhibition tests: quantifying antibody neutralization of virus-mediated cell–cell fusion

2020 ◽  
Author(s):  
Nazia Thakur ◽  
Carina Conceicao ◽  
Ariel Isaacs ◽  
Stacey Human ◽  
Naphak Modhiran ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Cell Fusion ◽  
Bimolecular Fluorescence Complementation ◽  
Luciferase Reporter ◽  
Live Virus ◽  
Viral Spread ◽  
Fusion Inhibition ◽  
Recent Emergence ◽  
Inhibitory Antibodies ◽  
Cell Cell

Although enveloped viruses canonically mediate particle entry through virus–cell fusion, certain viruses can spread by cell–cell fusion, brought about by receptor engagement and triggering of membrane-bound, viral-encoded fusion proteins on the surface of cells. The formation of pathogenic syncytia or multinucleated cells is seen in vivo, but their contribution to viral pathogenesis is poorly understood. For the negative-strand paramyxoviruses respiratory syncytial virus (RSV) and Nipah virus (NiV), cell–cell spread is highly efficient because their oligomeric fusion protein complexes are active at neutral pH. The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has also been reported to induce syncytia formation in infected cells, with the spike protein initiating cell–cell fusion. Whilst it is well established that fusion protein-specific antibodies can block particle attachment and/or entry into the cell (canonical virus neutralization), their capacity to inhibit cell–cell fusion and the consequences of this neutralization for the control of infection are not well characterized, in part because of the lack of specific tools to assay and quantify this activity. Using an adapted bimolecular fluorescence complementation assay, based on a split GFP–Renilla luciferase reporter, we have established a micro-fusion inhibition test (mFIT) that allows the identification and quantification of these neutralizing antibodies. This assay has been optimized for high-throughput use and its applicability has been demonstrated by screening monoclonal antibody (mAb)-mediated inhibition of RSV and NiV fusion and, separately, the development of fusion-inhibitory antibodies following NiV vaccine immunization in pigs. In light of the recent emergence of coronavirus disease 2019 (COVID-19), a similar assay was developed for SARS-CoV-2 and used to screen mAbs and convalescent patient plasma for fusion-inhibitory antibodies. Using mFITs to assess antibody responses following natural infection or vaccination is favourable, as this assay can be performed entirely at low biocontainment, without the need for live virus. In addition, the repertoire of antibodies that inhibit cell–cell fusion may be different to those that inhibit particle entry, shedding light on the mechanisms underpinning antibody-mediated neutralization of viral spread.

scholarly journals Design of Potent Membrane Fusion Inhibitors against SARS-CoV-2, an Emerging Coronavirus with High Fusogenic Activity

2020 ◽  
Vol 94 (14) ◽  
Author(s):  
Yuanmei Zhu ◽  
Danwei Yu ◽  
Hongxia Yan ◽  
Huihui Chong ◽  
Yuxian He
Keyword(s):  
Public Health ◽  
Severe Acute Respiratory Syndrome ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Heptad Repeat ◽  
Global Public Health ◽  
S Protein ◽  
Fusion Inhibitors ◽  
Cell Cell

ABSTRACT The 2019 coronavirus disease (COVID-19), caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed serious threats to global public health and economic and social stabilities, calling for the prompt development of therapeutics and prophylactics. In this study, we first verified that SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as a cell receptor and that its spike (S) protein mediates high membrane fusion activity. The heptad repeat 1 (HR1) sequence in the S2 fusion protein of SARS-CoV-2 possesses markedly increased α-helicity and thermostability, as well as a higher binding affinity with its corresponding heptad repeat 2 (HR2) site, than the HR1 sequence in S2 of severe acute respiratory syndrome coronavirus (SARS-CoV). Then, we designed an HR2 sequence-based lipopeptide fusion inhibitor, termed IPB02, which showed highly potent activities in inhibiting SARS-CoV-2 S protein-mediated cell-cell fusion and pseudovirus transduction. IPB02 also inhibited the SARS-CoV pseudovirus efficiently. Moreover, the structure-activity relationship (SAR) of IPB02 was characterized with a panel of truncated lipopeptides, revealing the amino acid motifs critical for its binding and antiviral capacities. Therefore, the results presented here provide important information for understanding the entry pathway of SARS-CoV-2 and the design of antivirals that target the membrane fusion step. IMPORTANCE The COVID-19 pandemic, caused by SARS-CoV-2, presents a serious global public health emergency in urgent need of prophylactic and therapeutic interventions. The S protein of coronaviruses mediates viral receptor binding and membrane fusion, thus being considered a critical target for antivirals. Herein, we report that the SARS-CoV-2 S protein has evolved a high level of activity to mediate cell-cell fusion, significantly differing from the S protein of SARS-CoV that emerged previously. The HR1 sequence in the fusion protein of SARS-CoV-2 adopts a much higher helical stability than the HR1 sequence in the fusion protein of SARS-CoV and can interact with the HR2 site to form a six-helical bundle structure more efficiently, underlying the mechanism of the enhanced fusion capacity. Also, importantly, the design of membrane fusion inhibitors with high potencies against both SARS-CoV-2 and SARS-CoV has provided potential arsenals to combat the pandemic and tools to exploit the fusion mechanism.

scholarly journals EWI-2 Inhibits Cell–Cell Fusion at the HIV-1 Virological Presynapse

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1082
Author(s):  
Emily E. Whitaker ◽  
Nicholas J. Matheson ◽  
Sarah Perlee ◽  
Phillip B. Munson ◽  
Menelaos Symeonides ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Virological Synapse ◽  
Virus Particles ◽  
Fusion Inhibitors ◽  
Producer Cell ◽  
Cellular Components ◽  
Cell To Cell Transfer ◽  
Hiv 1 ◽  
Cell Cell

Cell-to-cell transfer of virus particles at the Env-dependent virological synapse (VS) is a highly efficient mode of HIV-1 transmission. While cell–cell fusion could be triggered at the VS, leading to the formation of syncytia and preventing exponential growth of the infected cell population, this is strongly inhibited by both viral (Gag) and host (ezrin and tetraspanins) proteins. Here, we identify EWI-2, a protein that was previously shown to associate with ezrin and tetraspanins, as a host factor that contributes to the inhibition of Env-mediated cell–cell fusion. Using quantitative fluorescence microscopy, shRNA knockdowns, and cell–cell fusion assays, we show that EWI-2 accumulates at the presynaptic terminal (i.e., the producer cell side of the VS), where it contributes to the fusion-preventing activities of the other viral and cellular components. We also find that EWI-2, like tetraspanins, is downregulated upon HIV-1 infection, most likely by Vpu. Despite the strong inhibition of fusion at the VS, T cell-based syncytia do form in vivo and in physiologically relevant culture systems, but they remain small. In regard to that, we demonstrate that EWI-2 and CD81 levels are restored on the surface of syncytia, where they (presumably) continue to act as fusion inhibitors. This study documents a new role for EWI-2 as an inhibitor of HIV-1-induced cell–cell fusion and provides novel insight into how syncytia are prevented from fusing indefinitely.

scholarly journals The Combination of gQ1 and gQ2 in Human Herpesvirus 6A and 6B Regulates the Viral Tetramer Function for Their Receptor Recognition

2020 ◽  
Author(s):  
Aika Wakata ◽  
Lidya Handayani Tjan ◽  
Mitsuhiro Nishimura ◽  
Akiko Kawabata ◽  
Anna Lystia Poetranto ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Essential Gene ◽  
Critical Role ◽  
Specific Interaction ◽  
Human Herpesvirus ◽  
Assay System ◽  
Virus Propagation ◽  
Receptor Recognition ◽  
A Cell ◽  
Cell Cell

Human herpesvirus 6A (HHV-6A) and HHV-6B use different cellular receptors, human CD46 and CD134, respectively and have different cell tropisms although they have 90% similarity at the nucleotide level. An important feature that characterizes HHV-6A/6B is the glycoprotein H (gH)/gL/gQ1/gQ2 complex (a tetramer) that each virus has specifically on its envelope. Here, to determine which molecules in the tetramer contribute to the specificity for each receptor, we developed a cell-cell fusion assay system for HHV-6A and HHV-6B that uses the cells expressing CD46 or CD134. With this system, when we replaced the gQ1 or gQ2 of HHV-6A with that of HHV-6B in the tetramer, the cell fusion activity mediated by glycoproteins via CD46 was lower than that done with the original-type tetramer. When we replaced the gQ1 or the gQ2 of HHV-6A with that of HHV-6B in the tetramer, the cell fusion mediated by glycoproteins via CD134 was not seen. In addition, we generated two types of C-terminal truncation mutants of HHV-6A gQ2 (AgQ2) to examine the interaction domains of HHV-6A gQ1 (AgQ1) and AgQ2. We found that amino acid residues 163 to 185 in AgQ2 are important for interaction of AgQ1 and AgQ2. Finally, to investigate whether HHV-6B gQ2 (BgQ2) can complement AgQ2, an HHV-6A genome harboring BgQ2 was constructed. The mutant could not produce an infectious virus, indicating that BgQ2 cannot work for the propagation of HHV-6A. These results suggest that gQ2 supports the tetramer's function, and the combination of gQ1 and gQ2 is critical for virus propagation. IMPORTANCE Glycoprotein Q2 (gQ2), an essential gene for virus propagation, forms a heterodimer with gQ1. The gQ1/gQ2 complex has a critical role in receptor recognition in the gH/gL/gQ1/gQ2 complex (a tetramer). We investigated whether gQ2 regulates the specific interaction between the HHV-6A or -6B tetramer and CD46 or CD134. We established a cell-cell fusion assay system for HHV-6A/6B and switched the gQ1 or gQ2 of HHV-6A with that of HHV-6B in the tetramer. Although cell fusion was induced via CD46 when gQ1 or gQ2 was switched between HHV-6A and -6B, the activity was lower than that of the original combination. When gQ1 or gQ2 was switched in HHV-6A and -6B, no cell fusion was observed via CD134. HHV-6B gQ2 could not complement the function of HHV-6A's gQ2 in HHV-6A propagation, suggesting that the combination of gQ1 and gQ2 is critical to regulate the specificity of the tetramer's function for virus entry.

scholarly journals Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion

2020 ◽  
Author(s):  
Guido Papa ◽  
Donna L. Mallery ◽  
Anna Albecka ◽  
Lawrence Welch ◽  
Jérôme Cattin-Ortolá ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Cell Receptor ◽  
Furin Cleavage ◽  
S Protein ◽  
Viral Spread ◽  
Host Cell Receptor ◽  
Host Membrane ◽  
Dependent Cell ◽  
Syncytia Formation ◽  
Cell Cell

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor Ace2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors will not prevent viral spread.

scholarly journals Human Immunodeficiency Virus Type 1 Entry Inhibitors PRO 542 and T‐20 Are Potently Synergistic in Blocking Virus‐Cell and Cell‐Cell Fusion

2001 ◽  
Vol 183 (7) ◽  
pp. 1121-1125 ◽  
Author(s):  
Kirsten A. Nagashima ◽  
Daniah A. D. Thompson ◽  
Soraya I. Rosenfield ◽  
Paul J. Maddon ◽  
Tatjana Dragic ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Fusion ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Entry Inhibitors ◽  
Immunodeficiency Virus ◽  
Cell Cell

scholarly journals Potent Strategy To Inhibit HIV-1 by Binding both gp120 and gp41

2010 ◽  
Vol 55 (1) ◽  
pp. 264-275 ◽  
Author(s):  
Ioannis Kagiampakis ◽  
Arbi Gharibi ◽  
Marie K. Mankowski ◽  
Beth A. Snyder ◽  
Roger G. Ptak ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Hiv Replication ◽  
Nmr Spectrum ◽  
Binding Peptide ◽  
Hiv Entry ◽  
Covalently Linked ◽  
Hiv Gp120 ◽  
Hiv Microbicide ◽  
Anti Hiv ◽  
Cell Cell

ABSTRACTThe development of an anti-HIV microbicide is critical in the fight against the spread of HIV. It is shown here that the covalent linking of compounds that bind gp120 with compounds that bind gp41 can inhibit HIV entry even more potently than individual inhibitors or noncovalent combinations. The most striking example involves griffithsin, a potent HIV inhibitor that binds to the surface of HIV gp120. While griffithsin inhibits HIV Env-mediated fusion in a CCR5-tropic cell-cell fusion assay with a 50% inhibitory concentration (IC50) of 1.31 ± 0.87 nM and the gp41-binding peptide C37 shows an IC50of 18.2 ± 7.6 nM, the covalently linked combination of griffithsin with C37 (Griff37) has an IC50of 0.15 ± 0.05 nM, exhibiting a potency 8.7-fold greater than that of griffithsin alone. Similarly, in CXCR4-tropic cell-cell fusion assays, Griff37 is 5.2-fold more potent than griffithsin alone. In viral assays, both griffithsin and Griff37 inhibit HIV replication at midpicomolar levels, but the linked compound Griff37 is severalfold more potent than griffithsin alone against both CCR5- and CXCR4-tropic virus strains. Another example of this strategy is the covalently linked combination of peptide C37 with a variant of the gp120-binding peptide CD4M33 (L. Martin et al., Nat. Biotechnol. 21:71-76, 2003). Also, nuclear magnetic resonance (NMR) spectra for several of these compounds are shown, including, to our knowledge, the first published NMR spectrum for griffithsin.

Sign in / Sign up

Export Citation Format

Share Document