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SARS-CoV-2 requires cholesterol for viral entry and pathological syncytia formation.

ABSTRACT: Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ~6000 drugs and >30 spike variants. Together with quantitative cell biology approaches, the screen reveals an essential role for biophysical aspects of the membrane, particularly cholesterol-rich regions, in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings potentially provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.

SUBMITTER: Sanders DW

PROVIDER: S-EPMC8104966 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Syncytia formation by SARS-CoV-2 infected cells.

Project description:Severe cases of COVID-19 are associated with extensive lung damage and the presence of infected multinucleated syncytial pneumocytes. The viral and cellular mechanisms regulating the formation of these syncytia are not well understood. Here, we show that SARS-CoV-2 infected cells express the Spike protein (S) at their surface and fuse with ACE2-positive neighbouring cells. Expression of S without any other viral proteins triggers syncytia formation. Interferon-induced transmembrane proteins (IFITMs), a family of restriction factors that block the entry of many viruses, inhibit S-mediated fusion, with IFITM1 being more active than IFITM2 and IFITM3. On the contrary, the TMPRSS2 serine protease, which is known to enhance infectivity of cell-free virions, processes both S and ACE2 and increases syncytia formation by accelerating the fusion process. TMPRSS2 thwarts the antiviral effect of IFITMs. Our results show that SARS-CoV-2 pathological effects are modulated by cellular proteins that either inhibit or facilitate syncytia formation.

| S-SCDT-EMBOJ-2020-106267 | biostudies-other

Syncytia formation by SARS-CoV-2-infected cells.

Project description:Severe cases of COVID-19 are associated with extensive lung damage and the presence of infected multinucleated syncytial pneumocytes. The viral and cellular mechanisms regulating the formation of these syncytia are not well understood. Here, we show that SARS-CoV-2-infected cells express the Spike protein (S) at their surface and fuse with ACE2-positive neighboring cells. Expression of S without any other viral proteins triggers syncytia formation. Interferon-induced transmembrane proteins (IFITMs), a family of restriction factors that block the entry of many viruses, inhibit S-mediated fusion, with IFITM1 being more active than IFITM2 and IFITM3. On the contrary, the TMPRSS2 serine protease, which is known to enhance infectivity of cell-free virions, processes both S and ACE2 and increases syncytia formation by accelerating the fusion process. TMPRSS2 thwarts the antiviral effect of IFITMs. Our results show that SARS-CoV-2 pathological effects are modulated by cellular proteins that either inhibit or facilitate syncytia formation.

| S-EPMC7646020 | biostudies-literature

Syncytia formation by SARS-CoV-2-infected cells.

Project description: Not available

| S-EPMC7849166 | biostudies-literature

Palmitoylation of SARS-CoV-2 S protein is critical for S-mediated syncytia formation and virus entry.

Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The S protein is the key viral protein for associating with ACE2, the receptor for SARS-CoV-2. There are many kinds of posttranslational modifications in S protein. However, the detailed mechanism of palmitoylation of SARS-CoV-2 S remains to be elucidated. In our current study, we characterized the palmitoylation of SARS-CoV-2 S. Both the C15 and cytoplasmic tail of SARS-CoV-2 S were palmitoylated. Fatty acid synthase inhibitor C75 and zinc finger DHHC domain-containing palmitoyltransferase (ZDHHC) inhibitor 2-BP reduced the palmitoylation of S. Interestingly, palmitoylation of SARS-CoV-2 S was not required for plasma membrane targeting of S but was critical for S-mediated syncytia formation and SARS-CoV-2 pseudovirus particle entry. Overexpression of ZDHHC2, ZDHHC3, ZDHHC4, ZDHHC5, ZDHHC8, ZDHHC9, ZDHHC11, ZDHHC14, ZDHHC16, ZDHHC19, and ZDHHC20 promoted the palmitoylation of S. Furthermore, those ZDHHCs were identified to associate with SARS-CoV-2 S. Our study not only reveals the mechanism of S palmitoylation but also will shed important light into the role of S palmitoylation in syncytia formation and virus entry.

| S-EPMC8661603 | biostudies-literature

Computationally prioritized drugs inhibit SARS-CoV-2 infection and syncytia formation.

Project description:The pharmacological arsenal against the COVID-19 pandemic is largely based on generic anti-inflammatory strategies or poorly scalable solutions. Moreover, as the ongoing vaccination campaign is rolling slower than wished, affordable and effective therapeutics are needed. To this end, there is increasing attention toward computational methods for drug repositioning and de novo drug design. Here, multiple data-driven computational approaches are systematically integrated to perform a virtual screening and prioritize candidate drugs for the treatment of COVID-19. From the list of prioritized drugs, a subset of representative candidates to test in human cells is selected. Two compounds, 7-hydroxystaurosporine and bafetinib, show synergistic antiviral effects in vitro and strongly inhibit viral-induced syncytia formation. Moreover, since existing drug repositioning methods provide limited usable information for de novo drug design, the relevant chemical substructures of the identified drugs are extracted to provide a chemical vocabulary that may help to design new effective drugs.

| S-EPMC8769897 | biostudies-literature

Syncytia formation during SARS-CoV-2 lung infection: a disastrous unity to eliminate lymphocytes.

Project description: Not available

| S-EPMC8114657 | biostudies-literature

SARS-CoV-2 Alpha, Beta, and Delta variants display enhanced Spike-mediated syncytia formation.

Project description:Severe COVID-19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS-CoV-2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha (B.1.1.7) and Beta (B.1.351) spread and fusion in cell cultures, compared with the ancestral D614G strain. Alpha and Beta replicated similarly to D614G strain in Vero, Caco-2, Calu-3, and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Variant spike proteins displayed higher ACE2 affinity compared with D614G. Alpha, Beta, and D614G fusion was similarly inhibited by interferon-induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes modified fusogenicity, binding to ACE2 or recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS-CoV-2 emerging variants display enhanced syncytia formation.

| S-EPMC8646911 | biostudies-literature

Trypsin enhances SARS-CoV-2 infection by facilitating viral entry.

Project description:Coronaviruses infect cells by cytoplasmic or endosomal membrane fusion, driven by the spike (S) protein, which must be primed by proteolytic cleavage at the S1/S2 furin cleavage site (FCS) and the S2' site by cellular proteases. Exogenous trypsin as a medium additive facilitates isolation and propagation of several coronaviruses in vitro. Here, we show that trypsin enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in cultured cells and that SARS-CoV-2 enters cells via either a non-endosomal or an endosomal fusion pathway, depending on the presence of trypsin. Interestingly, trypsin enabled viral entry at the cell surface and led to more efficient infection than trypsin-independent endosomal entry, suggesting that trypsin production in the target organs may trigger a high level of replication of SARS-CoV-2 and cause severe tissue injury. Extensive syncytium formation and enhanced growth kinetics were observed only in the presence of exogenous trypsin when cell-adapted SARS-CoV-2 strains were tested. During 50 serial passages without the addition of trypsin, a specific R685S mutation occurred in the S1/S2 FCS (681PRRAR685) that was completely conserved but accompanied by several mutations in the S2 fusion subunit in the presence of trypsin. These findings demonstrate that the S1/S2 FCS is essential for proteolytic priming of the S protein and fusion activity for SARS-CoV-2 entry but not for viral replication. Our data can potentially contribute to the improvement of SARS-CoV-2 production for the development of vaccines or antivirals and motivate further investigations into the explicit functions of cell-adaptation-related genetic drift in SARS-CoV-2 pathogenesis.

| S-EPMC8789370 | biostudies-literature

Role of the spike glycoprotein of human Middle East respiratory syndrome coronavirus (MERS-CoV) in virus entry and syncytia formation.

Project description:Little is known about the biology of the emerging human group c betacoronavirus, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Because coronavirus spike glycoproteins (S) mediate virus entry, affect viral host range, and elicit neutralizing antibodies, analyzing the functions of MERS-CoV S protein is a high research priority. MERS-CoV S on lentivirus pseudovirions mediated entry into a variety of cell types including embryo cells from New World Eptesicus fuscus bats. Surprisingly, a polyclonal antibody to the S protein of MHV, a group a murine betacoronavirus, cross-reacted in immunoblots with the S2 domain of group c MERS-CoV spike protein. MERS pseudovirions released from 293T cells contained only uncleaved S, and pseudovirus entry was blocked by lysosomotropic reagents NH4Cl and bafilomycin and inhibitors of cathepsin L. However, when MERS pseudovirions with uncleaved S protein were adsorbed at 4°C to Vero E6 cells, brief trypsin treatment at neutral pH triggered virus entry at the plasma membrane and syncytia formation. When 293T cells producing MERS pseudotypes co-expressed serine proteases TMPRSS-2 or -4, large syncytia formed at neutral pH, and the pseudovirions produced were non-infectious and deficient in S protein. These experiments show that if S protein on MERS pseudovirions is uncleaved, then viruses enter by endocytosis in a cathepsin L-dependent manner, but if MERS-CoV S is cleaved, either during virus maturation by serine proteases or on pseudovirions by trypsin in extracellular fluids, then viruses enter at the plasma membrane at neutral pH and cause massive syncytia formation even in cells that express little or no MERS-CoV receptor. Thus, whether MERS-CoV enters cells within endosomes or at the plasma membrane depends upon the host cell type and tissue, and is determined by the location of host proteases that cleave the viral spike glycoprotein and activate membrane fusion.

| S-EPMC3789674 | biostudies-literature

Impact of Comorbidities on SARS-CoV-2 Viral Entry-Related Genes.

Project description:Viral entry mechanisms for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are an important aspect of virulence. Proposed mechanisms involve host cell membrane-bound angiotensin-converting enzyme 2 (ACE2), type II transmembrane serine proteases (TTSPs), such as transmembrane serine protease isoform 2 (TMPRSS2), lysosomal endopeptidase Cathepsin L (CTSL), subtilisin-like proprotein peptidase furin (FURIN), and even potentially membrane bound heparan sulfate proteoglycans. The distribution and expression of many of these genes across cell types representing multiple organ systems in healthy individuals has recently been demonstrated. However, comorbidities such as diabetes and cardiovascular disease are highly prevalent in patients with Coronavirus Disease 2019 (COVID-19) and are associated with worse outcomes. Whether these conditions contribute directly to SARS-CoV-2 virulence remains unclear. Here, we show that the expression levels of ACE2, TMPRSS2 and other viral entry-related genes, as well as potential downstream effector genes such as bradykinin receptors, are modulated in the target organs of select disease states. In tissues, such as the heart, which normally express ACE2 but minimal TMPRSS2, we found that TMPRSS2 as well as other TTSPs are elevated in individuals with comorbidities compared to healthy individuals. Additionally, we found the increased expression of viral entry-related genes in the settings of hypertension, cancer, or smoking across target organ systems. Our results demonstrate that common comorbidities may contribute directly to SARS-CoV-2 virulence and we suggest new therapeutic targets to improve outcomes in vulnerable patient populations.

| S-EPMC7720121 | biostudies-literature

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