New Reporter Cell Lines to Study Macrophage-Tropic HIV Envelope Protein-Mediated Cell-Cell Fusion

ABSTRACT Human immunodeficiency virus (HIV) entry is triggered by interactions between a pair of heptad repeats in the gp41 ectodomain, which convert a prehairpin gp41 trimer into a fusogenic three-hairpin bundle. Here we examined the disposition and antigenic nature of these structures during the HIV-mediated fusion of HeLa cells expressing either HIVHXB2 envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various lengths of time and then arrested. Fusion intermediates were then examined for reactivity with various monoclonal antibodies (MAbs) against immunogenic cluster I and cluster II epitopes in the gp41 ectodomain. All of these MAbs produced similar staining patterns indicative of reactivity with prehairpin gp41 intermediates or related structures. MAb staining was seen on Env cells only upon exposure to soluble CD4, CD4-positive, coreceptor-negative cells, or stromal cell-derived factor-treated target cells. In the fusion system, the MAbs reacted with the interfaces of attached Env and target cells within 10 min of coculture. MAb reactivity colocalized with the formation of gp120-CD4-coreceptor tricomplexes after longer periods of coculture, although reactivity was absent on cells exhibiting cytoplasmic dye transfer. Notably, the MAbs were unable to inhibit fusion even when allowed to react with soluble-CD4-triggered or temperature-arrested antigens prior to initiation of the fusion process. In comparison, a broadly neutralizing antibody, 2F5, which recognizes gp41 antigens in the HIV envelope spike, was immunoreactive with free Env cells and Env-target cell clusters but not with fused cells. Notably, exposure of the 2F5 epitope required temperature-dependent elements of the HIV envelope structure, as MAb binding occurred only above 19°C. Overall, these results demonstrate that immunogenic epitopes, both neutralizing and nonneutralizing, are accessible on gp41 antigens prior to membrane fusion. The 2F5 epitope appears to depend on temperature-dependent elements on prefusion antigens, whereas cluster I and cluster II epitopes are displayed by transient gp41 structures. Such findings have important implications for HIV vaccine approaches based on gp41 intermediates.

Download Full-text

Deletion of ER-retention motif on SARS-CoV-2 spike protein reduces cell hybrid during cell–cell fusion

Cell & Bioscience ◽

10.1186/s13578-021-00626-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Xuening Wang ◽

Chih-Hsiung Chen ◽

Saiaditya Badeti ◽

Jong Hyun Cho ◽

Alireza Naghizadeh ◽

...

Keyword(s):

Cell Fusion ◽

Cell Lines ◽

Cell Hybrid ◽

Host Cells ◽

Spike Protein ◽

Expression Vectors ◽

S Protein ◽

Er Retention ◽

Syncytia Formation ◽

Cell Cell

Abstract Background The novel SARS-CoV-2 has quickly become a global pandemic since the first reported case in December 2019, with the virus infecting millions of people to date. The spike (S) protein of the SARS-CoV-2 virus plays a key role in binding to angiotensin-converting enzyme 2 (ACE2), a host cell receptor for SARS-CoV-2. S proteins that are expressed on the cell membrane can initiate receptor-dependent syncytia formation that is associated with extensive tissue damage. Formation of syncytia have been previously observed in cells infected with various other viruses (e.g., HIV, Ebola, Influenza, and Herpesviruses). However, this phenomenon is not well documented and the mechanisms regulating the formation of the syncytia by SARS-CoV-2 are not fully understood. Results In this study, we investigated the possibility that cell fusion events mediated by the S protein of SARS-CoV-2 and ACE2 interaction can occur in different human cell lines that mimic different tissue origins. These cell lines were transduced with either wild-type (WT-S) S protein or a mutated variant where the ER-retention motif was removed (Δ19-S), as well as human ACE2 expression vectors. Different co-culture combinations of spike-expressing 293T, A549, K562, and SK-Hep1 cells with hACE2-expressing cells revealed cell hybrid fusion. However, only certain cells expressing S protein can form syncytial structures as this phenomenon cannot be observed in all co-culture combinations. Thus, SARS-CoV-2 mediated cell–cell fusion represents a cell type-dependent process which might rely on a different set of parameters. Recently, the Δ19-S variant is being widely used to increase SARS-CoV-2 pseudovirus production for in vitro assays. Comparison of cell fusion occurring via Δ19-S expressing cells shows defective nuclear fusion and syncytia formation compared to WT-S. Conclusions This distinction between the Δ19-S variant and WT-S protein may have downstream implications for studies that utilize pseudovirus-based entry assays. Additionally, this study suggest that spike protein expressed by vaccines may affect different ACE2-expressing host cells after SARS-CoV-2 vaccine administration. The long-term effects of these vaccines should be monitored carefully. Δ19-S mRNA may represent a safer mRNA vaccine design in the future.

Download Full-text

Deletion of ER-retention Motif on SARS-CoV-2 Spike Protein Reduces Cell Hybrid During Cell-cell Fusion

10.21203/rs.3.rs-380389/v1 ◽

2021 ◽

Author(s):

Chih-Hsiung Chen ◽

Saiaditya Badeti ◽

Jong Hyun Cho ◽

Alireza Naghizadeh ◽

Xuening Wang ◽

...

Keyword(s):

Cell Fusion ◽

Cell Lines ◽

Cell Hybrid ◽

Host Cells ◽

Spike Protein ◽

In Vitro Assays ◽

S Protein ◽

Er Retention ◽

Syncytia Formation ◽

Cell Cell

Abstract The novel SARS-CoV-2 has quickly become a global pandemic since the first reported case in December 2019, with the virus infecting millions of people to date. The spike (S) protein of the SARS-CoV-2 virus plays a key role in binding to angiotensin-converting enzyme 2 (ACE2), a host cell receptor for SARS-CoV-2. S proteins that are expressed on the cell membrane can initiate receptor-dependent syncytia formation that is associated with extensive tissue damage. Formation of syncytia have been previously observed in cells infected with various other viruses (e.g., HIV, Ebola, Influenza, and Herpesviruses). However, this phenomenon is not well documented and the mechanisms regulating the formation of these syncytia by SARS-CoV-2 are not fully understood. In this study, we investigated the possibility that cell fusion events mediated by the S protein of SARS-CoV-2 and ACE2 interaction can occur in different human cell lines that mimic different tissue origins. These cell lines were stably transduced with either wild-type (WT-S) S protein or a mutated variant where the ER-retention motif was removed (Δ19-S), or human ACE2 vectors. Different co-culture combinations of spike-expressing 293T, A549, K562, and SK-Hep1 cells with hACE2-expressing cells revealed cell hybrid fusion. However, only certain cells expressing S protein can form syncytial structures as this phenomenon cannot be observed in all co-culture combinations. Thus, SARS-CoV-2 mediated cell-cell fusion represents a cell type-dependent process which might rely on a different set of parameters. Recently, the Δ19-S variant is being widely used to increase SARS-CoV-2 pseudovirus production for in vitro assays. Comparison of cell fusion occurring via Δ19-S expressing cells shows defective nuclear fusion and syncytia formation compared to WT-S. This distinction between the Δ19-S variant and WT-S protein may have downstream implications for studies that utilize pseudovirus-based entry assays. Additionally, this study suggest that spike protein expressed by vaccines may affect different ACE2-expressing host cells after SARS-CoV-2 vaccine administration. The long-term effects of these vaccines should be monitored carefully.

Download Full-text

Generation of Recombinant Antibody Manufacturing Cell Lines Using Cell-Cell Fusion

ESACT Proceedings - Animal Cell Technology Meets Genomics ◽

10.1007/1-4020-3103-3_93 ◽

2005 ◽

pp. 483-488

Author(s):

Wenlin Zeng ◽

Ela Puchacz ◽

Katy Heineken ◽

Isaac Raymond ◽

Conway Chang ◽

...

Keyword(s):

Cell Fusion ◽

Cell Lines ◽

Recombinant Antibody ◽

Manufacturing Cell ◽

Cell Cell

Download Full-text

Role of the Mutation Q252R in Activating Membrane Fusion in the Murine Leukemia Virus Surface Envelope Protein

Journal of Virology ◽

10.1128/jvi.77.20.10841-10849.2003 ◽

2003 ◽

Vol 77 (20) ◽

pp. 10841-10849 ◽

Cited By ~ 8

Author(s):

Chi-Wei Lu ◽

Monica J. Roth

Keyword(s):

Cell Fusion ◽

Receptor Binding ◽

Envelope Protein ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Host Cells ◽

Cell Binding ◽

Surface Envelope ◽

Murine Leukemia ◽

Cell Cell

ABSTRACT Entry of retroviruses into host cells requires the fusion between the viral and cellular membranes. It is unclear how receptor binding induces conformational changes within the surface envelope protein (SU) that activate the fusion machinery residing in the transmembrane envelope protein (TM). In this report, we have isolated a point mutation, Q252R, within the proline-rich region of the 4070A murine leukemia virus SU that altered the virus-cell binding characteristics and induced cell-cell fusion. Q252R displays a SU shedding-sensitive phenotype. Cell-cell fusion is receptor dependent and is observed only in the presence of MuLV Gag-Pol. Both cellular binding and fusion by Q252R are greatly enhanced in conjunction of G100R, a mutation within the SU variable region A which increases viral binding through an independent mechanism. Deletion of a conserved histidine (His36) at the SU N terminus abolished cell-cell fusion by G100R/Q252R Env without compromising virus-cell binding. Although G100R/Q252R virus has no detectable titer, replacement of the N-terminal nine 4070A SU amino acids with the equivalent ecotropic MuLV sequence restored viral infectivity. These studies provide insights into the functional cooperation between multiple elements of SU required to signal receptor binding and activate the fusion machinery.

Download Full-text

Characterisation of the NES2Y cell line and its use in the production of human glucose-responsive insulin producing (hGRIP) cell lines by cell-cell fusion

Islets ◽

10.4161/isl.1.2.9432 ◽

2009 ◽

Vol 1 (2) ◽

pp. 117-123 ◽

Cited By ~ 4

Author(s):

Gary G. Adams ◽

A Uddin ◽

M Vives-Pi ◽

R Pujol-Borrell

Keyword(s):

Cell Line ◽

Cell Fusion ◽

Cell Lines ◽

Cell Cell

Download Full-text

HIV-Envelope–Dependent Cell-Cell Fusion: Quantitative Studies

The Scientific World JOURNAL ◽

10.1100/tsw.2009.90 ◽

2009 ◽

Vol 9 ◽

pp. 746-763 ◽

Cited By ~ 14

Author(s):

Leonor Huerta ◽

Nayali López-Balderas ◽

Evelyn Rivera-Toledo ◽

Guadalupe Sandoval ◽

Guillermo Gómez-Icazbalceta ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Fusion ◽

Relative Size ◽

Resonance Energy ◽

Biological Properties ◽

Target Cells ◽

Multinucleated Cells ◽

Hiv Envelope ◽

Cell Cell

Interactionin vitrobetween cells infected with human immunodeficiency virus (HIV) and surrounding, uninfected, target cells often leads to cell fusion and the formation of multinucleated cells, called syncytia. The presence in HIV-infected individuals of virus strains able to induce syncytia in cultures of T cells is associated with disease progression and AIDS. Even in the asymptomatic stage of infection, multinucleated cells have been observed in different organs, indicating that fused cells may be generated and remain viable in the tissues of patients. We used lymphocytic cells transfected for the expression of the HIV-envelope (Env) glycoproteins to develop a method for the direct quantification of fusion events by flow cytometry (Huerta et al., 2006,J. Virol. Methods138, 17–23; López-Balderas et al., 2007,Virus Res.123, 138–146). The method involves the staining of fusion partners with lipophilic probes and the use of fluorescence resonance energy transfer (FRET) to distinguish between fused and aggregated cells. We have shown that such a flow-cytometry assay is appropriate for the screening of compounds that have the potential to modulate HIV-Env–mediated cell fusion. Even those syncytia that are small or few in numbers can be detected. Quantitative analysis of the fusion products was performed with this technique; the results indicated that the time of reaction and initial proportion of fusion partners determine the number, relative size, and average cellular composition of syncytia. Heterogeneity of syncytia generated by HIV-Env–mediated cell-cell fusion may result in a variety of possible outcomes that, in turn, may influence the biological properties of the syncytia and surrounding cells, as well as replication of virus. Given the myriad immune abnormalities leading to AIDS, the full understanding of the extent, diverse composition, and role of fused cells in the pathogenesis of, and immune response to, HIV infection is an important, pending issue.

Download Full-text

Use of Anti-HIV Immunotoxins as Probes of the Biology of HIV-Infected Cells

Canadian Journal of Infectious Diseases ◽

10.1155/1994/576154 ◽

1994 ◽

Vol 5 (suppl a) ◽

pp. 23A-27A ◽

Cited By ~ 3

Author(s):

SETH H Pincus ◽

Jan McClure ◽

Hua Fang

Keyword(s):

Infected Cell ◽

Cell Lines ◽

Envelope Protein ◽

Protein S ◽

Viral Envelope ◽

Soluble Form ◽

Target Antigen ◽

Viral Receptor ◽

Infected Cells ◽

Hiv Envelope

OBJECTIVE: Anti-human immunodeficiency virus (HIV) immunotoxins are potential treatments for HIV infection. but they may also be used as probes to study the relationship between HIV and the cell it infects. Data from the present study indicate the complexity of this relationship.DESIGN: A panel of monoclonal antibodies directed against different epitopes on the HIV envelope protein(s) gp l20 and gp 41 was conjugated to ricin A chain. The activity of these immunotoxins on HIV-infected cell lines was studied.RESULTS: The data demonstrate that HIV-infected cell lines may be killed by some, but not all, of these immunotoxins. The killing is not directly proportional to the binding of the antibody to the infected cell and is influenced by the viral strain. The immunotoxins were used to select persistently infected cell lines for immunotoxin-resistant variants: these demonstrate several different viral or cellular defects. The incubation of infected cells with a soluble form of the viral receptor increases the sensitivity of the cells to anti-gp41, but not anti-gpl20, immunotoxins by altering both the levels of expression and internalization of the viral envelope. Drugs that inhibit lysosomal degradation (ammonium chloride, monensin. chloroquine) enhance the efficacy of these immunotoxins.CONCLUSIONS: Because immunotoxins must be internalized to function, they may be used to study the intracellular trafficking of the target antigens. In the present study, this was done using the HIV-envelope protein as expressed in infected cells as the target antigen

Download Full-text