Quantitative assays reveal cell fusion at minimal levels of SARS-CoV-2
Cell entry of the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by its spike protein S. As a main antigenic determinant, S protein is in focus of various therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here, we present sensitive assay systems with a high dynamic range and high signal-to-noise ratios covering not only particle-cell and cell-cell fusion but also fusion from without (FFWO).
In FFWO, S-containing viral particles induce syncytia independently of de novo synthesis of S. Neutralizing antibodies, as well as sera from convalescent patients, inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring levels of S protein below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as pathological consequence during coronavirus disease 2019 (COVID-19) can proceed at low levels of S protein and may not be effectively prevented by antibodies.
Introduction
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is met with an unprecedented global scientific effort. As the causative agent of the pandemic and the associated coronavirus disease 2019 (COVID-19), SARS-CoV-2 is a typical coronavirus, with a large spike protein (S) inserted in the membrane of the enveloped particle. Being the main surface-exposed antigen and the mediator of cell entry, S protein forms the most important target of current efforts to develop therapeutic drugs and prophylactic vaccines (Gioia et al., 2020; Tang et al., 2020).
As a typical class I viral fusion protein, S protein forms trimers with a globular head domain containing the receptor-binding site which contacts angiotensin-converting enzyme 2 (ACE2) as its entry receptor. Proteolytic cleavage separates the globular head (S1 domain) and the stalk domain (S2), which contains the hydrophobic fusion peptide at its N-terminus and the transmembrane domain toward the C-terminus.
Priming of the fusion-competent state requires processing of two cleavage sites localized in close N-terminal proximity of the fusion peptide. These cleavage sites can be recognized by alternative proteases, thus explaining the high flexibility of the virus to adapt to various tissues. In particular, the S1/S2 site is cleaved by proprotein convertases like furin localized in the trans-Golgi network during trafficking of the newly synthesized S protein to the cell surface.
The S2′ site can be cleaved by the serine protease TMPRSS2 which is exposed on the surface of target cells and contacted when the virus binds to ACE2. In absence of TMPRSS2, virus particles are endocytosed and cleaved by cathepsins (Hasan et al., 2020; Hoffmann et al., 2020a, 2020b; Shang et al., 2020a; Walls et al., 2020). In cell culture, trypsin was described as further alternative protease able to activate membrane fusion (Ou et al., 2020; Xia et al., 2020; Zhang and Kutateladze, 2020).
Thus, two alternative entry routes exist for SARS-CoV-2, notably determined by the availability of activating proteases (Hoffmann et al., 2020b; Tang et al., 2020). For both routes, membrane fusion is pH independent.
Like other fusion proteins that are active pH independently, S protein mediates not only fusion between the viral and the cellular membranes during particle entry but also fusion of infected cells with uninfected cells (Buchrieser et al., 2020). This process is mediated by newly synthesized S protein accumulating at the cell surface. The resulting syncytia are giant cells containing at least three, often many more nuclei.
Cell-cell fusion is used by viruses such as human immunodeficiency virus (HIV) (Compton and Schwartz, 2017; Bracq et al., 2018), measles virus (MV) (Griffin, 2020), or herpesvirus (Cole and Grose, 2003) to spread in a particle-independent way. The resulting syncytia are documented as pathological consequence detectable in various tissues such as the lung (MV), skin (herpesvirus), or lymphoid tissues (HIV).
In the brain, cell-to-cell transmission via hyperfusogenic F proteins constitutes a hallmark of MV-caused encephalitis as a fatal consequence of acute MV infections manifesting years later (Ferren et al., 2019).
Besides particle-cell and cell-cell fusion, a third membrane fusion process mediated by viral fusion proteins has been designated fusion from without (FFWO) (Roller et al., 2008). FFWO results in syncytia in absence of newly expressed fusion protein. In presence of a sufficient concentration of particle-associated fusion protein, adjacent cells are fused, e.g., by bound HIV or herpesvirus particles either directly or after uptake of fusion protein and its presentation at the cell surface (Clavel and Charneau, 1994; Melikyan, 2014).
Here, we investigated the competence of SARS-CoV-2 S protein for these three membrane fusion processes. For each of them, we established quantitative assays relying on expressed S protein, thereby avoiding work with infectious virus at biosafety level 3 (BSL-3). The data reveal a strong membrane fusion activity of the S protein and demonstrate syncytia formation even at undetectable levels of S protein and FFWO.
Examination of sera from convalescent patients with COVID-19 revealed potent neutralizing capacity against particle-cell fusion but only moderate or low activity against cell-cell fusion.
Results
Spike-protein-mediated particle entry
To follow particle entry mediated by S protein, we set out to generate lentiviral vectors (LVs) pseudotyped with S protein. S protein was codon optimized for expression in human cells, and either the full-length protein or the previously described C-terminal truncation variant SΔ19 (Ou et al., 2020) was used for pseudotyping. LVs transferring the β-galactosidase gene lacZ were generated by transient transfection of HEK-293T cells and subsequently purified and concentrated (Figure 1A) (see Transparent Methods for details).
Western blot analysis of LV batches revealed a stronger spike signal relative to p24 for SΔ19, demonstrating its better incorporation into LV particles (Figure 1B). SΔ19-LVs were titrated on cell lines frequently used in coronavirus research, i.e., Vero E6, MRC-5, Calu-3, HEK-293T, and HEK-293T overexpressing ACE2 (293T-ACE2). On all cell lines included, the luminescence signal decreased linearly with increasing dilution of the vector (Figure 1C).
In contrast to LVs pseudotyped with the G protein of vesicular stomatitis virus (VSV-LV), SΔ19-LV did not reach a signal plateau, indicating that only a subsaturating fraction of the cells was transduced. However, transduction rates with SΔ19-LV increased more than 100-fold upon overexpression of hACE2 on HEK-293T cells, reaching a signal-to-noise ratio of more than 2000. VSVG-LV-mediated gene delivery was not affected by overexpression of hACE2 (Figure 1D).
Remarkably, with a saturating dose of SΔ19-LV, a similar luminescence signal was reached on 293T-ACE2 cells as with VSV-LV (Figure 1C).
Figure 1. Spike-mediated particle entry
(A) Generation of pseudotyped lentiviral vectors. Second-generation LVs pseudotyped with S protein were generated by transfection of HEK-293T cells with a packaging plasmid encoding HIV-1 gag/pol, a transfer vector plasmid with a lacZ reporter gene and one of two envelope plasmids encoding codon-optimized SARS-CoV-2 S with or without (SΔ19) the 19 C-terminal amino acids. The C-terminal endoplasmic reticulum retention signal (purple) and the receptor-binding domain (RBD, orange) are indicated.
(B) Incorporation of S protein into LVs determined by Western blotting. S-LV and SΔ19-LV particles (V) and lysates of their producer cells (C) were stained for the presence of S protein (top) and p24 as particle loading control. Top blot was exposed for 30 s and bottom blot for 5 s. Image contrast was adjusted, retaining relative signal strengths.
(C) Gene transfer activities on the indicated cell lines. The indicated dilutions of 5 μL vector stock of SΔ19-LV or VSV-LV were added to the cells. Cell lysates were prepared three days after vector addition, and lacZ reporter activity was quantified as a luminescence readout. Symbols represent means of technical triplicates. Gray shaded area indicates 95 percent confidence interval (CI) of signals from untransduced cells (blanks).
(D) Effect of ACE2 overexpression on reporter transfer. 293T cells transfected with ACE2 expression plasmid or mock plasmid were incubated with 0.2 μL of SΔ19-LV or VSV-LV. Cell lysates were prepared three days after vector addition, and reporter activity was quantified as a luminescence readout. Bars represent geometric means of technical triplicates ±95 percent CIs.
Quantifying cell fusion mediated by SARS-CoV-2 S protein
Upon transfection, SARS-CoV-2 S protein showed a remarkable fusogenic activity. When transfected HEK-293T cells producing SΔ19-LV were detached and cocultured overnight in a 1:1 ratio with Vero E6 cells, plates were covered by large syncytia, each containing at least 10 (and up to 100) nuclei (Figure S1A). To examine this further, the full-length and truncated forms of S were overexpressed in HEK-293T, and cocultures with Vero E6 target cells were imaged by confocal laser scanning microscopy (Figure S1B).
Both, S and SΔ19 protein, induced many large syncytia characterized by cytoskeletal rearrangement, clustering of more than five nuclei and colocalization of the red and green fluorescent protein (RFP and GFP) reporter dye signals (Figure S1C).
To quantify the cell-cell fusion mediated by S protein, an α-complementation assay based on β-galactosidase previously used to evaluate HIV-mediated fusion (Holland et al., 2004) was adapted to the S protein (see Transparent Methods for details): Upon cell-cell fusion, complementation of ω enzyme fragment with the α fragment inside the syncytium results in the formation of active β-galactosidase that is then quantified by a luminescence reaction (Figure 2A).
In a first step, the minimal amount of S protein required to result in a detectable signal was determined. Effector cells were transfected with varying quantities of S protein-encoding plasmid (ranging from 7.5 μg to 7.5 ng per T75 flask) and S protein surface expression was followed by flow cytometry. S protein expression was still clearly detectable with 75 ng of plasmid, while the mean fluorescence intensity (MFI) at 7.5 ng plasmid was indistinguishable from background (Figure 2B).
Notably, this low level of S protein was still sufficient to induce significant cell-cell fusion when the assay was allowed to proceed overnight, highlighting the potent fusogenic activity of S protein (Figure 2C). With higher amounts of plasmid, three hours of incubation were sufficient to obtain signals more than 10-fold above background. Interestingly, when cells were detached prior to coculture with ethylenediaminetetraacetic acid (EDTA) only instead of trypsin-EDTA, the cell fusion activity decreased by at least one order of magnitude, and only overnight incubation yielded signals significantly over background (Figure 2D).
When target cells overexpressed ACE2 and were detached with trypsin, the maximum extent of the fusion signal increased by another order of magnitude (Figure 2D). Now, the signal-to-noise ratio reached two orders of magnitude already after one-hour incubation including the samples expressing the lowest amounts of S protein. This highlights the dependence of S protein activity on the presence of ACE2. Under optimal conditions and with ACE2 overexpressing cells, a signal-to-noise ratio of 2.9 orders of magnitude was reached.
Figure 2. Quantifying S protein-mediated cell-cell fusion
(A) Experimental setup for the quantification of cell-cell fusion. Active β-galactosidase is formed when effector cells expressing S protein and the α-fragment fuse with target cells expressing ACE2 and the ω-fragment.
(B–D) Effector cells transfected with different amounts of S-protein expression plasmid (ranging from 7500 ng to 7.5 ng per T75 flask) were assessed for S protein expression by flow cytometry (B) and then used in the fusion assay (C-D). (B) Mean fluorescence intensity (MFI, orange bars) and the percentage of S-positive cells (yellow bars) were determined in flow cytometry. Bars represent (geometric) means ±95 percent confidence intervals (CIs), n = 3. p values are from one-way analysis of variance (ANOVA).
(C) Activities of β-galactosidase in cocultures of effector and target cells in absence of ACE2 overexpression. Effector cells were transfected with the indicated amounts of S protein encoding plasmid, detached with trypsin and cultivated for the indicated time periods (o/n: overnight) with target cells which were transfected with the ω-fragment encoding plasmid only. Bars represent geometric means ±95 percent CIs, n = 3. p values are from two-way ANOVA.
(D) Influence of proteolytic processing and ACE2 overexpression on cell fusion activity. Left panel: Effector and target cells were detached without trypsin using 5 mM EDTA in PBS. Right panel: Target cells were co-transfected with the ω-fragment and ACE2 encoding plasmids. Bars represent geometric means of technical triplicates ±95 percent CIs.
This is taken from a very long article. Read the rest here: sciencedirect.com
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Hubert
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Again I ask, is COVID a real virus? Has it been isolated or not because stories like this make me wonder
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CodexCoder
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You might want to read Dr. David Martin’s document about the patents governing all the various pieces of the COVID problem that existed before its “discovery” and “leak” into the world.
https://www.davidmartin.world/wp-content/uploads/2021/01/The_Fauci_COVID-19_Dossier.pdf
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Wojt
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Too much for perusal…what is your point?
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Wojt
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‘Ripley’s Believe it or Not’…
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Tom
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I don’t think even my expert doctor would understand much of this.
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barry paul robinson
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This is from way back, 19 march 2021, nothing to see here or we would have known about from the likes of Stefan Lanka and Co.
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tom0mason
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Later research shows some difference …
From https://www.nature.com/articles/s41418-021-00795-y
“Syncytia formation during SARS-CoV-2 lung infection: a disastrous unity to eliminate lymphocytes” by Liangyu Lin, Qing Li, Ying Wang & Yufang Shi, Published: 12 May 2021
This paper states this in it’s first paragraph …
“Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has become a major threat to the global public health and economy [1, 2]. While the majorities of SARS-CoV-2 infected individuals only suffer mild or moderate symptoms, unfortunately, some patients develop severe chronic respiratory diseases (CRDs) and have to be admitted to intensive care units [3, 4]. …”
[my bold]
On Explaining Syncytia —
“…Syncytia are evolutionarily conserved cellular structures form by the multiple cell fusions of uninuclear cells. In mammals, the best example of physiological syncytia is muscle fibers, which contain thousands of fused muscle cells to allows their rapid coordinated contraction [7]. …”
“…Syncytia can also be induced by certain types of infections by viruses, such as human immunodeficiency virus, respiratory syncytial virus, and herpes simplex virus [9]. It could be envisioned that virus-induced cell fusion facilitates the transfer of viral genomes to the neighboring cells. However, the viral and cellular mechanisms regulating the formation of syncytia during SARS-CoV-2 infection remains largely elusive. …”
However it also has these little nuggets in it —
“…the syncytia blocking drugs, niclosamide, an oral anti-helminthic agent, was found to be effective at a very low dose (IC50 = 0.34 μM) and could prevent cell from virus-induced cell death.”
[note niclosamide is used as as a treatment for tapeworm in humans and many animals. -TM]
And
“Despite the observation of multinucleate pneumocytes in autopsy, it is still not known whether such syncytia play a critical role in the pathogenesis of CRDs of severe COVID-19 patients. Recently, an antidepressant drug, fluvoxamine, was shown to lower the likelihood of clinical deterioration of severe COVID-19 patients in a randomized clinical trial [12]. Interestingly, fluvoxamine could facilitate TMEMF activation and phosphatidylserine exposure [13]. It is imperative to examine whether fluvoxamine affects syncytia formation. It is also worthy to evaluate the whether the combine uses of anti-syncytia drugs with other COVID-19 targets would yield better clinical outcomes [14, 15].”
Makes me wonder, if COVID does cause some fusing of cells (Syncytia), what other syncytia blocking drugs (or combinations of drugs) are there that could help with treating COVID, helping to reduced the mortality threat perhaps?
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Hubert
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Simple question… does covid exist yes or no, if so, please point me to the relevant source that can present covid in isolate form, thank you kindly
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Herb Rose
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Hi Hubert,
Yes covid exists. It is a causative agent for diseases people get but I do not think the present identification of a virus as that agent is correct. I believe the pathogen is a protein that can link with DNA so that it produces RNA to make more copies of the protein. This is why both the mRNA vaccines and the Johnson and Johnson spike protein vaccine all cause bleeding, blot clots, and the disease.
Herb
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Mark Tapley
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Hello Herb:
You have been baffled by the baloney. Did you not watch the PSI video of Brendon D. Murphy in which he exposes the Zionist plan going back over a decade? They even had Wuhan figured in. This medical fraud is just. a revamped version of the AIDS “epidemic” that PCR inventor Kary Mullis exposed back in the eighties. That was followed up by several more trail run virus frauds, including H1N1, Bird flu, Swine flu, Ebola, Zika and Anthrax. All of these medical hoaxes made billions for the insiders as they turned out debilitating and deadly vaccines.
We have a clear trail of the Current Covid 19 fraud. In 2014 Journalist Harry Vox exposed this latest eugenicist scam explicitly laid out by the Rockefeller Documents of 2010, video below. We know that all the Zionist controlled countries has their fake PCR tests ordered back in 2018, long before the springing of the fake virus. We know that eugenicist Gates held his pre pandemic 201 meeting on 9-18-19 where all those present were instructed on how to push the virus narrative. We know that shortly after this meeting our shabbas goy senators held their closed door meeting from which all their big donors were tipped off. We know that subsequently several of these same sensors were caught selling millions of dollars of stock at the top of the market right before the announcement of the fake virus and then tanking of the market.
How obvious can it get, with the announcement of the fake virus we had the usual. Jew MSM spin and scare tactics from all the puppet actor insiders just like you get with every fake event the government pulls such as a 911, Sandy Hook, Los Vegas, fake Floyd or WND’s. The only difference is that this time they incorporated a fake test to go with the rest of the subterfuge.
Naturally the insiders are going to obfuscate the issue and baffle everyone with scientific jargon as the billions continue to roll in for the many insiders. This is just part of the medical fear porn. That is standard procedure. Only by the presentation of an elaborate hoax with constant re enforcement by the MSM whores, Rockefeller CDC, big Pharma and their captive medical establishment and the controlled politicians, can this imaginary Insilco computer generated nonsense continue. The conspirators have no sample of any pathogen. There is no excess death rate. The spike protein hypothesis has never been proven and indeed viruses do not exist as proven conclusively by microbiologist Stephan Lanka and others. The main ingredient in all four blood toxin injections is graphene oxide (over 99% of contents). The Bible refers to people like you: Romans 1: 19-22
19Because that which may be known of God is manifest in them; for God hath shewed it unto them. 20For the invisible things of him from the creation of the world are clearly seen, being understood by the things that are made, even his eternal power and Godhead; so that they are without excuse: 21Because that, when they knew God, they glorified him not as God, neither were thankful; but became vain in their imaginations, and their foolish heart was darkened. 22Professing themselves to be wise, they became fools
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Mark Tapley
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Got in too big of a hurry. Forgot to post video referred to in previous comment.
https://www.youtube.com/watch?v=wuOxG-rnj30
https://www.bitchute.com/video/lgFmeUIb0vJ5/
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