Establishment of S-WT-EGFP, S-Δ19-mGFP effector cell lines and hACE2-mCherry target cell lines.
Various cell lines were transduced with the indicated plasmids. Then, cells were sorted based on high expression and verified by either enhanced GFP or mCherry fluorescence using a widefield fluorescence microscope and filter combinations optimized for the appropriate fluorescent proteins (Fig. 1A). To confirm the intensities of GFP of either wild-type S protein or Δ19 S protein in 293T cells, the images were processed using the multi-scale cell instance segmentation framework method. Then the intensities of membrane-bound S protein or cytosol-bound S protein were plotted with mean intensities of all cells. The WT-S protein were mainly located on the cell membrane and ER region surrounding the nucleus. The S-Δ19 bar chart demonstrated less intensities compared to wild-type S protein (Fig. 1B). Thus, we successfully established different types of cell lines with S-WT-EGFP, S-Δ19-mGFP, and hACE2-mCherry expression.
Co-culture of S-WT-EGFP 293T cells and hACE2-mCherry 293T cells forms hybrid cell fusion.
To establish a method for visualizing Spike protein-mediated cell-cell fusion, we first designated 293T cells expressing wild-type S or S-Δ19 conjugated to enhanced green fluorescent protein (EGFP) as effector cells and 293T cells expressing the human ACE2 conjugated to mCherry as target cells. Both cells were co-cultured together in glass chamber slides. In the co-culture combinations performed, we could observe cell fusion events and larger-than-normal cells (the controls). Using confocal microscopy, we could visualize fused cells in detail with more than ten S-EGFP and hACE2-mCherry cells per sample fused with intact cell membranes and containing multiple lysed nuclei (Fig. 2). However, in 293T-S-Δ19-GFP with 293T-ACE2-mCherry co-cultures, we observed fused cells that had individual, non-fused nucleus. This phenomenon may be explained by the deletion of the ER retention motif in S-Δ19 cells. Therefore, in S-Δ19 cells, translated S proteins are not retained in the ER, but instead trafficked to the cell surface or secreted as viral particles. Because there is no Spike protein retained in the ER, we hypothesize that there will be minimal Spike protein and hACE2 interaction juxtaposed to the ER of contacting cells and therefore no fusion of captured nuclei. However, Spike WT expressing cells have normal ER signaling retention of S proteins. Therefore, when ACE2 and S proteins are both present in a fused cell, it may lead to ER fusion which can lead to clumped or fused nuclei due to the close proximity between ER and nucleus.
Cell fusion in Spike or hACE2 expressed A549 cells.
Next, we performed the same experiment with A549 cells, a human alveolar basal epithelial adenocarcinoma cell line , expressing either S proteins or ACE2, and confirmed the expression of either protein by confocal imaging (Fig. 3). The co-culture of hACE2-A549 with S-WT-293T demonstrated hybrid cell fusion formation, while mixed culture of S-Δ19-293T with hACE2-A549 only manifested few smaller cell fusion events after 24 hours (Fig. 4B). Co-culture of S-WT-A549 or S-Δ19-A549 cells with hACE2-293T cells showed similar results (Fig. 4C). These results were confirmed with high resolution confocal microscopy (Fig. 5). However, A549-S-WT or S-Δ19-GFP cells co-cultured with A549-ACE2-mCherry exhibited few cell fusion formations (Fig. 4C). The number of fused cells we could observe were much less compared to other cell co-culture combinations which may indicate that other signaling checkpoints are necessary for Spike protein-mediated cell fusion to occur.
Cell fusion in Spike or hACE2 expressed K562 cells.
To investigate SARS-CoV-2’s impact on the hematopoietic system, we transduced the S protein and hACE2 onto K562 cells. K-562 is a human erythroleukemia line derived from a 53-year-old female chronic myelogenous leukemia patient in blast crisis . K562 cells can develop characteristics similar to early-stage erythrocytes .
Co-culture of S-WT or S-Δ19 K562 with hACE2 K562 cells showed cell fusion in a few cells (Fig. 6). However, fluorescence images of other co-cultures using A549 and K562 cells showed that while there were indeed cell fusions, albeit with different patterns, fused hACE2 cells expressed a combination of both fused and unfused nuclei when co-cultured with S-WT and S-Δ19 cells (Fig. 7). We speculate that nuclear fusion in fused cells happens in a time-dependent manner where individual nuclei present in fused cells at the beginning of the process eventually fuse together into one large nucleus as the intact individual nuclei begin to disintegrate. Interestingly, we also observed that S-Δ19 cells seem to express the S protein more strongly in the cytosol than on the cell membrane. Further studies would have to look more into the expression level and trafficking of the S-Δ19 protein, as it compares to the original S-WT protein.
Interaction of Spike and hACE2 mediated-cell fusion events between different Cell types.
Studies showed that chronic liver disease might predispose to poorer outcomes following SARS-CoV-2 infection due to an altered immune profile and systemic inflammation . We investigated whether similar cell fusion events could take place in liver cancer cells by co-culturing S-WT/S-Δ19 K562 cells with hACE2-SK-Hep1 cells. S-WT-K562 induced syncytia formation when combined with hACE2-SK-Hep1 cells, and co-culture of S-Δ19-K562 with hACE2-SK-Hep1 demonstrated different fusion patterns to the wild-type S protein (Fig. 8A &B).
Furthermore, to demonstrate that the observed giant cells are indeed fused cells and not just clumps of individual cells, we performed flow cytometry on 293T cells following 24 hour co-cultures (S-WT-GFP/S-Δ19-GFP with hACE2-mCherry) and observed that there were some cell populations that were double positive for S-WT-GFP/S-Δ19-GFP and hACE2-mCherry (data not shown). Cell fusion upon S protein and ACE2 contact may be of interest to the scientific community as there could be a potential possibility that efficacious COVID-19 vaccines induce transient or permanent cell fusion events within vaccinated individuals. Lysis of fused cells may damage the affected tissues in the long run.