Ge cell fusion user manual: Everything you need to know about the newest cell phone accessory

Several distinct mechanisms for virus cell-to-cell transfer and spreading have been described, but some viruses can use the fusogenic capacity of some viral proteins, usually involved in virus entry, expressed at the surface of infected cells to trigger cell-cell fusion between infected virus-donor cells and neighboring target cells to form enlarged multinucleated cells, often called syncytia. Three main different classes of viral fusion proteins have been structurally described depending the conformational structure and mechanism use for fusion of the viral bilayer envelope with the cell membranes [1]: class I, with a characteristic α-helix trimer (as in HIV-1 transmembrane gp41); class II, with a β-sheet-based elongated ectodomain (as in dengue virus glycoprotein); and class III, composed of an α-helix and β-sheet combined ectodomain (as in rabies virus G glycoprotein). In addition, a fourth class of fusion proteins corresponding to the FAST protein family of some non-enveloped Reoviruses has been also described. Thereby, virus-induced cell-cell fusion and syncytium formation are mainly mediated by specific interactions of certain viral fusion proteins with surface molecules or receptors expressed on neighboring non-infected cells.

Ge cell fusion user manual

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Several viral families, including some human pathogens, have evolved the ability to trigger cell-cell fusion to form syncytia between individual infected cells and neighboring uninfected or infected cells. Even if the specific role of these virus-induced syncytia for pathogenesis during the natural course of some viral infections is still discussed, these infected multinucleated cells can show high capacity of viral production and improved capacities of motility or survival, at least when recapitulated in vitro in tissue-culture assays. With the notable exception of some non-enveloped viruses of the Orthoreovirus and Aquareovirus subfamily of Reoviridae that use this cell-cell membrane fusion process and syncytium formation for virus dissemination, all other animal viruses able to use cell-cell fusion belong to families of enveloped viruses. For example, HIV-1 and SARS-CoV-2, the two major viral pathogens responsible for the global pandemics of AIDS and COVID-19, respectively, can induce cell-cell fusion and syncytium formation as largely evidenced in tissues, such as brain and lungs, of infected patients. Similarly, the presence of infected multinucleated giant cells in skin lesions has long been recognized as the hallmark of infection by some Herpesviridae.

The formation of multinucleated giant cells (MGCs) (or syncytia) following Herpesvirus infection in their natural hosts has been well documented for a long time [2,3,4]. The presence of MGCs in skin lesions has indeed long been recognized as the hallmark of Herpesvirus infection [5] and could be used as diagnostic for Herpes simplex keratitis in eyes [6]. Similarly, MGC formation is also a cytopathologic feature of Herpesvirus infection in the lower respiratory tract [7]. The extent of Herpesvirus-mediated cell-cell fusion leading to MGC formation is related to the identity of the Herpesvirus but also to the infected tissue: VZV infection results in extensive syncytium formation in skin lesions [8], while HSV-2 induces limited syncytia consisting of only a minor population of infected cells in the skin lesions [9]. However, the significance of Herpesvirus-mediated cell-cell fusion for virus replication and spreading in vivo remains unclear. In in vitro tissue culture, the degree of cell-cell fusion mediated by different clinical isolates and laboratory-adapted strains can significantly varies [10,11]. For example, HSV-1 primary isolates cause limited cell-cell fusion [12], whereas viral variants from laboratory stocks induce extensive syncytial formation in tissue culture [13,14].

Herpesviruses enter host cells by enabling membrane fusion of viral envelopes with host cellular membranes, which either occurs at the plasma membrane or in endosomal compartments. This viral entry process is cell-type dependent and depends on the identity of the Herpesvirus. The viral core membrane fusion machinery required for cell-free virus entry but also for cell-cell fusion induced by herpesviruses consists of the viral glycoprotein gB, a type III viral membrane fusion protein that forms homotrimers, and the heterodimer gH/gL, which are conserved envelope proteins among all Herpesviruses [15,16]. gB is a major determinant of Herpesvirus infectivity both in vitro and in vivo [17,18], while the gH/gL heterodimer can interact with the gB and is required for its fusogenic function [19]. The requirement of the gB homotrimers and gH/gL heterodimer for virus entry into target cells is a highly conserved function among all Herpesviruses [20]. The general process for virus cell-fusion of Herpesviruses first involves activation of the gH/gL heterodimer upon binding to the cellular receptors leading to activation and conformational change of gB [20,21] for insertion of its fusion loops into the host cell membrane, followed by the refolding of gB to drive merge of the viral envelope with the cell membrane [16]. The core fusion machinery is required both for entry of cell-free Herpesviruses into target cells and Herpesvirus-induced cell-cell fusion [15,16,17,18,19,20,21,22]. However, the mechanism of Herpesvirus-induced cell-cell fusion is still poorly understood and is highly cell type-dependent, suggesting that specific cellular cofactors may play important roles in this process. For example, VZV induces extensive syncytial formation of primary keratinocytes but poorly causes cell-cell fusion of primary fibroblasts [8].

In addition, the membrane fusion process sometimes also involved non-conserved membrane glycoproteins specific to each Herpesvirus, which can bind to host cell-specific receptors. For example, HSV-1 and HSV-2 express a non-conserved glycoprotein gD that binds to different host cell receptors depending of the target cell [23]: nectin-1 and nectin-2, cell adhesion molecules expressed on neurons and epithelial cells [24]; the Herpesvirus entry mediator (HVEM), also called Herpesvirus entry mediator A (HveA) or tumor necrosis factor receptor superfamily member 14 (TNFRSF14), expressed on activated lymphocytes [25]; and a non-protein receptor called soluble 3-O-sulfated heparan sulfate [26]. HCMV expresses a glycoprotein gO, as well as 3 small glycoproteins encoded by viral UL128, UL130, and UL131 genes, to mediate receptor binding and regulate cellular tropism, but the respective host cell receptors for these additional glycoproteins remain unclear [27,28]. HHV-6A and HHV-6B also express a glycoprotein gO and additional gQ1/gQ2 proteins to engage the CD46 cell surface receptor on human target cells [29,30], but some observations indicated that HHV-6 type B can also use human CD134 as an alternative receptor for viral entry [31]. EBV expresses a soluble gp42 to bind MHC class II receptor HLA-DR1 on B lymphocytes but uses gH/gL heterodimer to directly bind cell surface integrins, such as αvβ5, αvβ6, or αvβ8, for viral membrane fusion [32,33].

Whereas the gD receptors (HVEM, and nectin-1 or nectin-2) are required for HSV mediated cell-cell fusion [50,51], heparan sulfate appears less important for cell-cell fusion than for viral entry [51,52]. It remains elusive how interaction of gD with its entry receptors leads to cell-cell fusion. One hypothesis is that the binding of gD with entry receptors induces conformational changes of gD, enabling its interaction with the core fusion machinery and activation of its fusogenic activity. Other cellular receptors for gB and/or gH/gL may also exist, and the binding of these glycoproteins to these additional receptors could also trigger fusion activity, and bypass the requirement for gD. The paired immunoglobulin-like type 2 receptor (PILR) has been identified as an entry coreceptor that associates with gB, and interactions between PILR and gB are involved in membrane cell-cell fusion events during HSV-1 infection [53]. Interestingly, the cellular protein tyrosine phosphatase 1B (PTP1B) has been recently reported to be specifically required for the cell-cell fusion by HSV-1 and not for cell-free virus infection [54].

Measles is a highly contagious disease that affects children and young adults, usually causing skin rashes, but severe complications can progress to fatal encephalitis. Measles virus (MV) is an enveloped virus of the Morbillivirus genus that first infects epithelial cells but also dendritic cells and alveolar macrophages of the respiratory tract before spreading by cis-infection to lymphocytes in lymph nodes and finally neurons [72,73,74,75]. Virus replication begins with the attachment of the viral envelope protein Hemagglutinin (H) to different receptors (CD46, Signaling Lymphocyte-Activation Molecule (SLAM), also named SLAMF1 or CD150, and Nectin-4) [76]. The wild-type strain of MV preferentially recognizes SLAM which is expressed on both B and T cells, while attenuated vaccine MV strains uses the CD46 receptor widely expressed on all cells [77]. Finally, nectin-4, a cellular adhesion protein of epithelial cell junctions, acts as a co-receptor for virus entry and can be used by both wild type and attenuated vaccine viruses [78]. The virus entry is mediated by both hemagglutinin and the fusion F protein. Hemagglutinin is organized on tetramers at the surface of virions, and is composed of a hydrophobic fusion peptide, two heptad repeat regions A and B (HRA/HRB, respectively) and a transmembrane domain [79]. After binding to the receptor, hemagglutinin induces a coordinated series of conformational changes of the different domains of the F protein, and the final F conformation triggers insertion of the fusion peptide in the host cell membrane, leading to the fusion between virions and the target cell [80], by a pH-independent process at the plasma membrane [79].

MV, but also other Morbilliviruses able to infect dogs, cats, cattle, seals, and cetaceans, are also characterized by their capacity to induce cell-cell fusion, both in vitro and in vivo. The formation of multinucleated cells is one of the major landmark in the development of measles, and has been observed in lymph nodes, respiratory tract and thymus after MV infection [81,82,83]. In 1973, White and Boy already observed syncytia of infected human thymocytes in lymphoid tissues of infected patients [83]. Similarly, the formation of multinucleated giant cells following MV infection of lymphocytes was initially observed in vivo, in African infected children [84]. Multinucleated giant cells were also initially detected within epidermal cells of Measles skin lesions by electron microscopy [85]. The in vivo MV-infected giant cells are able to produce infectious viral particles and can contribute to viral dissemination in patients [86]. In vitro, MV-induced syncytia have been mainly studied in epithelial cells, but formation of syncytia was also observed in vitro between infected human dendritic cells. Cell-cell fusion was only observed when DCs were activated by lymphocytes through CD40 engagement [87]. More recently, a new study demonstrated that the MV-induced cell-cell fusion leads to an increase of the IFN-I response in infected human epithelial cells and mature dendritic cells, via the nuclear translocation of IRF-3 [70]. In most cell-culture assays used to study MV-induced cell-cell fusion, this process is governed by interaction of viral F and H proteins with the cellular receptors CD46 and SLAM [88]. However, infected epithelial cells, which do not express the SLAM receptor, can form syncytia [89], indicating that the mechanisms by which cell-cell fusion is triggered in MV-infected cells still need better characterization. Interestingly, Kelly et al. [90] have shown in vitro that the formation of syncytia between infected HEK293T cells, a human embryonic kidney cell-line, expressing the cellular receptor SLAM, was drastically reduced when the lipid raft-associated Tetherin/BST2 protein was overexpressed. Tetherin/BST2 is a cellular restriction factor that restricts replication of numerous viruses, including HIV-1, by clustering viral particles at the cell surface and inhibiting virion release [91]. By using truncation mutants, they showed that the modification of the C-terminal glycosyl-phosphatidylinositol (GPI) anchor of BST2 was sufficient for significant decrease of cell-cell fusion and syncytium formation through targeting of the H protein [90]. 2ff7e9595c