# SARS-CoV-2 entry and fusion are independent of ACE2 localization to lipid rafts

William Bolland, Inès Marechal, Chloé Petiot, Françoise Porrot, Florence Guivel-Benhassine, Anne Brelot, Nicoletta Casartelli, Olivier Schwartz, Julian Buchrieser

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William Bolland, Inès Marechal, Chloé Petiot, Françoise Porrot, Florence Guivel-Benhassine, et al. SARS-CoV-2 entry and fusion are independent of ACE2 localization to lipid rafts. Journal of Virology, 2025, 99 (1), pp.e0182324. 10.1128/jvi.01823-24. pasteur-05109813

HAL Id: pasteur-05109813

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# SARS-CoV-2 entry and fusion are independent of ACE2 localization to lipid rafts

William Bolland,¹,² Inès Marechal,¹ Chloé Petiot,¹ Françoise Porrot,¹ Florence Guivel-Benhassine,¹ Anne Brelot,³ Nicoletta Casartelli,¹ Olivier Schwartz,¹,⁴ Julian Buchrieser¹

**AUTHOR AFFILIATIONS** See affiliation list on p. 14.

**ABSTRACT** Membrane fusion occurs at the early stages of SARS-CoV-2 replication, during entry of the virus, and later during the formation of multinucleated cells called syncytia. Fusion is mediated by the binding of the viral Spike protein to its receptor ACE2. Lipid rafts are dynamic nanodomains enriched in cholesterol and sphingolipids. Rafts can act as platforms for entry of different viruses by localizing virus receptors, and attachment factors to the same membrane domains. Here, we first demonstrate that cholesterol depletion by methyl-beta-cyclodextrin inhibits Spike-mediated fusion and entry. To further study the role of ACE2 lipid raft localization in SARS-CoV-2 fusion and entry, we designed a GPI-anchored ACE2 construct. Both ACE2 and ACE2-GPI proteins were similarly expressed at the plasma membrane. Through membrane flotation assays, we show that in different cell lines, ACE2-GPI localizes predominantly to raft domains of the plasma membrane while ACE2 is non-raft associated. We then compare the ability of ACE2 and ACE2-GPI to permit SARS-CoV-2 entry, replication, and syncytia formation of different viral variants. We find little difference in the two proteins. Our results demonstrate that SARS-CoV-2 entry and fusion are cholesterol-dependent and raft-independent processes.

**IMPORTANCE** Rafts are often exploited by viruses and used as platforms to enhance their entry into the cell or spread from cell to cell. The membrane localization of ACE2 and the role of lipid rafts in SARS-CoV-2 entry and cell-to-cell spread are poorly understood. The function of lipid rafts in viral fusion is often studied through their disruption by cholesterol-depleting agents. However, this process may have off-target impacts on viral fusion independently of lipid-raft disruption. Therefore, we created an ACE2 construct that localizes to lipid rafts using a GPI anchor. Conversely, wild-type ACE2 was non-raft associated. We find that the localization of ACE2 to lipid rafts does not modify the fusion dynamics of SARS-CoV-2.

**KEYWORDS** SARS-CoV-2, fusion, lipid rafts, cholesterol, syncytia

Enveloped viruses, such as coronaviruses, use fusogens to facilitate entry and infection at the cell surface or within the endocytic pathway (1, 2). SARS-CoV-2 Spike protein binds to the angiotensin-converting enzyme 2 (ACE2) receptor, leading to conformational changes in Spike to allow the release of the fusion peptide through cleavage of the S2' site by cell surface proteases, such as TMPRSS2, or endosomal proteases such as cathepsins (3, 4). During the late stages of viral replication, Spike leaks from the viral packing site in the endoplasmic reticulum and Golgi network to the cell surface due to a suboptimal COPI-binding motif (5). There, Spike interacts with neighboring ACE2-expressing cells, leading to cell-to-cell fusion and the formation of multinucleated cells, known as syncytia. Syncytia formation by SARS-CoV-2 has been well documented in *in vitro* studies and from analyses of lung samples of COVID-19

**Editor** Stacey Schultz-Cherry, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

Address correspondence to Olivier Schwartz, olivier.schwartz@pasteur.fr, or Julian Buchrieser, julian.buchrieser@pasteur.fr.

Olivier Schwartz and Julian Buchrieser contributed equally to this article.

The authors declare no conflict of interest.

**Received** 21 October 2024

**Accepted** 24 October 2024

**Published** 21 November 2024

Copyright © 2024 Bolland et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
patients (6–9). We and others have shown that the SARS-CoV-2 variants possess different syncytia-forming capacities due to mutations in the Spike protein (10–14) and that high fusogenic activity is linked to increased cytopathy (13). The role of syncytia during SARS-CoV-2 infection includes dissemination of the virus between cells while escaping the humoral immunity (15, 16). However, questions remain surrounding the role of syncytia in pathogenesis and the mechanisms regulating syncytia formation.

Lipid rafts are highly dynamic nanodomains (<200 nm) present within the plasma membranes of all eukaryotic cells that are enriched in cholesterol and sphingolipids. Post-translational modifications of proteins, such as the addition of a glycosylphosphatidylinositol (GPI) anchor, localize the proteins to lipid rafts (17). Rafts perform cellular functions such as signal transduction by concentrating and crosslinking signaling receptors, and exocytosis by organizing SNAP REceptor (SNARE) proteins at the plasma membrane (18, 19).

Lipid rafts have been proposed to act as platforms for the entry of diverse enveloped and nonenveloped viruses through localization of the virus receptor, co-receptors, and attachment factors (20). For example, human herpesvirus-6 recruits its receptor CD46 to rafts to permit infection (21), likewise with the attachment factors of hepatitis C virus (22), and the CCR5 and CXCR4 co-receptors of human immunodeficiency virus-1 (23, 24). Both SARS-CoV and SARS-CoV-2 have been proposed to employ ACE2 localization into rafts to facilitate host cell entry through the caveolin-dependent pathway (25). However, the exact localization of ACE2 in the plasma membrane is disputed with reports suggesting it is a raft-associated protein and others suggesting the contrary (26–29). Therefore, clarification of the membrane localization of ACE2 and the importance of its localization in SARS-CoV-2 entry is needed.

The classical approach in studying the role of lipid rafts during viral replication is by their disruption by cholesterol-depleting agents such as cyclodextrins and statins (30, 31). Disruption of rafts in ACE2-expressing cells reduces entry of SARS-CoV and SARS-CoV-2, indicating that cholesterol plays an important role and suggesting that lipid rafts may be involved in this process (26, 27, 32). Nevertheless, cholesterol depletion is not specific for rafts (33) and may have off-target impacts on cell homeostasis, such as glucose transport and membrane elasticity (34, 35). Investigating the role of lipid rafts in SARS-CoV-2 fusion and entry through ACE2 modifications has not been explored.

Here, we confirm the importance of cholesterol in the entry and fusion of SARS-CoV-2 by depletion using Methyl β cyclodextrin (MβCD). We then show that wild-type ACE2 localizes to non-raft regions of the membrane and subsequently design a GPI-anchored ACE2 construct that localizes to lipid rafts. Through reporter assays for syncytia formation and pseudovirus entry, we find that ACE2 localization to lipid rafts has no significant impact on SARS-CoV-2 entry and fusion, except in HEK293T cells where raft localization slightly increases syncytia formation and pseudovirus entry. We then corroborate these findings using the SARS-CoV-2 virus by measuring viral replication and syncytia formation.

# MATERIALS AND METHODS

## Cell lines

HEK293T (Herein referred to as 293T cells), MRC5, U2OS, and HeLa cells and their derivatives were purchased from the ATCC or generously provided by fellow staff at Institut Pasteur. All cell lines were cultured at 37°C, 5% CO₂ in Dulbecco's modified Eagle medium media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin antibiotics. For fusion assays, GFP-split stable cell lines were generated by lentivector transduction containing pQCXIP-derived plasmids for GFP1-10 or GFP11 subunits. GFP1-10- or GFP11-expressing cells were selected through puromycin resistance and cultured by supplementation of the media with puromycin (293T, U2OS, HeLa—1 µg/mL; MRC5—10 µg/mL). 293T and MRC5 ACE2 and ACE2-GPI stable cell lines were generated by lentivector transduction containing pLV-derived plasmids for ACE2
or ACE2-GPI. ACE-2 and ACE2-GPI-expressing cells were selected through hygromycin B resistance and cultured by supplementation of the media with 100 µg/mL (293T) or 200 µg/mL (MRC5) hygromycin B. All cells used in this study were routinely checked and found negative for mycoplasma.

## SARS-CoV-2 isolates

The D614G and XBB.1.5 isolates were previously described (12). Briefly, the D614G and XBB.1.5 strains were isolated and cultured on VeroE6 or IGROV-1 cell lines, respectively. The D614G strain (hCoV-19/France/GE1973/2020) was obtained from the National Reference Centre for Respiratory Viruses at Institut Pasteur. The XBB.1.5 strain was isolated from a nasopharyngeal swab of an anonymous individual at the Hopital European Georges Pompidou (HEGP; Assistance Publique, Hôpitaux de Paris). Virus stock titers were calculated by TCID₅₀ measurements as previously described. All work using the SARS-CoV-2 virus was carried out in a BSL-3 laboratory, under the guidelines of the risk prevention service at Institut Pasteur.

## GFP-split fusion assay

SARS-CoV-2 Spike-induced cell-to-cell fusion was measured using the GFP-split system. All transfections were carried out using lipofectamine 2000 reagent (ThermoFisher) and incubated on a shaking incubator at 1,000 rpm 37°C for 2 h. For 293T, U2OS, and HeLa GFP-split-expressing cell lines, the respective GFP1-10 cell lines were transfected with plasmids encoding ACE2 (pLV) or ACE2-GPI (pLV) plasmids in the presence or absence of TMPRSS2 (pcDEST). The GFP11 cell lines were transfected with plasmids encoding D614G Spik or XBB.1.5 Spike (pVAX1, Invitrogen). Control conditions were performed using a pQCXIP-Empty plasmid. Following transfection, cells were centrifuged at 500 g to pellet cells, and the transfection mix was discarded. Cells were resuspended and GFP1-10 and GFP11 cells were co-cultured in µClear black 96-well plates (Greiner Bio-One) for 18 h. Cells were cultured at the following densities (50:50 ratio of GFP1-10 and GFP11 cells): 293T—6.0 × 10⁴ cells per well; U2OS—3.0 × 10⁴ cells per well; and HeLa—5.0 × 10⁴ cells per well. For MRC5 fusion assays, 2.5 × 10⁴ MRC5 GFP1-10 cells stably expressing ACE2 or ACE2-GPI were co-cultured with 3.0 × 10⁴ 293T GFP11 cells transfected for D614G or XBB.1.5 Spike or a control plasmid. 18 h post-transfection, fluorescence microscopy images of the cells were acquired using the Opera Phenix High-Content Screening System (PerkinElmer). Hoechst 33342 was added to the culture media at 1:10,000 dilution to perform nuclei counting. GFP area and nuclei number were quantified using the Harmony High-Content Imaging and Analysis Software (PerkinElmer, HH17000012, v.5.0). Surface expression of ACE2, Spike, and TMPRSS2 was analyzed as described in the "Flow cytometry" section.

For SARS-CoV-2 virus cell-to-cell fusion assays, 293T GFP1-10 and GFP11 cells were pooled and transfected with ACE2 or ACE2-GPI or a control plasmid for 24 h. 6.0 × 10⁴ 293T cells were seeded per well in µClear black 96-well plates. MRC5 GFP1-10 and GFP11 cells stably expressing ACE2, ACE2-GPI or control wild-type cells were seeded at a density of 5.0 × 10⁴ cells per well. Cells were then infected with D614G or XBB.1.5 virus at MOI 0.1 or 1.0 for 48 h. 48 h post-infection, cell supernatant was collected for RT-qPCR analysis and cells were fixed with 4% paraformaldehyde (PFA), washed, and stained with Hoechst 33342. Intracellular Spike staining was performed with antibodies diluted in PBS + 1% BSA, 0.1%-sodium azide, 0.05%-saponin (ThermoFisher) using mAb10 anti-S2 antibody (36) as the primary and Alexa fluor 647-conjugated goat anti-human IgG as the secondary. GFP area and nuclei number were quantified on the Opera Phenix High-Content Screen System as described in the previous paragraph.

## SARS-CoV-2 pseudovirus entry

Pseudoviruses were produced by co-transfection of 293T cells with plasmids encoding SARS-CoV-2 Spike (D614G), lentiviral proteins, and a luciferase reporter plasmid.
Pseudoviruses were harvested 48 h post-transfection and titers were quantified by measuring infectivity. The assessment of pseudovirus entry was carried out in 293T cells transfected for 24 h with ACE2 or ACE2-GPI or control plasmid and in MRC5 cells stably expressing ACE2 or ACE2-GPI or wild-type control cells. 4 × 10⁴ 293T cells or 3 × 10⁴ MRC5 cells were seeded per well in white Cellstar 96-well cell culture plates (Greiner Bio-One). Pseudoviruses were added to the cell media at a 1:50 and 1:250 dilution. 48 h after pseudovirus infection, 50 µL Bright-Glo substrate (Promega) was added to each well and incubated in the dark for 5 minutes prior to plate reading. Luciferase activity was measured using an EnSpire plate reader (PerkinElmer).

## MβCD drug treatment assays

The impact of MβCD on cell-to-cell fusion and SARS-CoV-2 pseudovirus entry was carried out in 293T GFP-split cells and wild-type cells, respectively. For cell-to-cell fusion assays, 293T GFP1-10 cells were transfected with ACE2 or control plasmid, and GFP11 cells were transfected with D614G Spike or control plasmid for 24 h. Cells were then treated with 2 mM MβCD or the equivalent volume of DMSO for 3 h on a shaking incubator at 1,000 rpm 37°C. Cells were then pelleted, washed, and seeded at 6.0 × 10⁴ cells per well (50:50 ratio of both GFP1-10 and GFP11 cells). GFP quantification was carried out 3 h after seeding on the Opera Phenix High-Content Screen System. For pseudovirus entry, 293T cells were transfected with ACE2 or control plasmid for 24 h. Cells and D614G Spike pseudovirus were treated with 2 mM MβCD or the equivalent volume of DMSO for 3 h, cells on a shaking incubator at 1,000 rpm 37°C. Cells were pelleted, washed, and seeded. Cells were infected with 1:50 pseudovirus and luciferase activity was measured as described in the earlier "SARS-CoV-2 pseudovirus entry" section.

## Membrane flotation assay and western blotting

For flotation assays, 1.5 × 10⁷ cells were cultured and lysed per condition. Cells were lifted and resuspended in 300 µL NTE buffer (50 mM Tris-HCL [pH 7.4], 50 mM NaCl, 5 mM EDTA) to wash. Cells were then centrifuged at 500 g and resuspended in NTE buffer +1% Triton X-100 +Roche complete protease inhibitor on ice for 1 h to lyse cells. Cell lysates were then transferred to a Dounce homogenizer and passed 10 times with the pestle avoiding bubble formation. Lysates were transferred to tubes and centrifuged at 2,500 g, 4°C for 10 minutes to remove cell debris and nuclei. Lysates were gently mixed with 2 mL of 60% OptiPrep iodixanol density gradient medium (Sigma-Aldrich) within 15 mL ultracentrifuge tubes. A density gradient was then created by slowly adding 6 mL of 30% OptiPrep and then 2.5 mL of 5% OptiPrep diluted in NTE buffer. Samples were loaded into an ultracentrifuge and spun at 100,000 g for 18 h. Samples were then aliquoted gently into fractions and stored at -20°C prior to western blot analysis.

For western blot analysis, 10 µL of fraction was loaded per well. Samples were reduced using Laemmli sample buffer 4x (Bio-Rad) and NuPAGE Sample reducing agent 10x (ThermoFisher) for 5 minutes at 95°C. NuPAGE 4-12% Bis-Tris Gels (Invitrogen) were used to run samples. PageRuler prestained protein ladder (ThermoFisher) was used as a kDa reference. Following the transfer, membranes were blocked in PBS + 5% BSA overnight at 4°C. Antibodies were incubated for 1 h at room temperature and diluted in PBS + 1% BSA, 0.05%-Tween, and 0.1%-sodium azide. Primary antibodies used were for ACE2—goat anti-hACE2 (R&D systems, 1 µg/mL) and for flotillin-1—rabbit anti-flotillin-1 (ThermoFisher, 1 µg/mL). Membranes were washed three times in PBS + 0.05%-Tween between each antibody incubation. Secondary antibodies used were anti-goat IgG DyLight 680 (Life Technologies, 1:5,000) and anti-rabbit IgG DyLight 800 (Invitrogen, 1:5,000). Membranes were revealed using the Licor imager and analyzed using Image Studio Lite v5.2.5 software.
## Flow cytometry

Surface staining was performed on live cells and all primary and secondary antibodies were diluted in MACS buffer (PBS + 0.5% BSA, EDTA 2 mM) and incubated at 4°C for 30 min. Primary antibodies used were as follows: for ACE2—1 µg/mL anti-ACE2 VHH-B07-Fc (Brelot et al., manuscript in preparation), for Spike—1 µg/mL LY-CoV404 (37), for TMPRSS2—anti-TMPRSS2 VHH-A01-Fc (38). Cells were washed in 100 µL PBS between primary and secondary antibody incubations. Alexa fluor 647-conjugated goat anti-human IgG was used as the secondary antibody (Invitrogen, A-21445, 1:500). For phospholipase C (PLC) treatment, cells were incubated with 100 or 200 U/µL PLC for 2 h at 37°C on a shaking incubator at 1,000 rpm. Cells were washed and resuspended before analysis of ACE2 surface levels as described above. For soluble SARS-CoV-2 spike RBD-binding analysis, cells were stained with 4.5 µg/mL soluble Wuhan RBD-biotinylated (36) at 4°C for 30 min. Cells were washed in 100 µL PBS and then Alexa fluor 488-conjugated streptavidin (ThermoFisher, S11223, 1:500) was incubated at indicated timepoints at 4°C for 30 min. All cells were fixed in 4% PFA and resuspended in 200 µL PBS before acquisition on the Attune NtX flow cytometer (ThermoFisher). Data were analyzed using FlowJo software (BDBioSciences).

## RT-qPCR

For quantification of viral RNA release, cell supernatants were collected and inactivated by diluting 1:4 in H₂O and incubating at 80°C for 20 min. For amplification SARS-CoV-2 E-gene forward (5'- ACAGGTACCTTAATAGTTAATAGCGT-3') and reverse (5'-ATATTGCAGCA GTACGCACACA-3') primers were used at 10 µM (39). Luna One-step RT-qPCR kit (New England Biolabs) was used with 1 µL supernatant. A standard curve was generated by 1:10 serial dilution of EURM-019 ssRNA SARS-CoV-2 fragments (European Commission). Analysis was performed with the QuantStudio 6 Flex Real-Time PCR machine (ThermoFisher).

## Statistical analysis

Calculations were performed using Excel 365 (Microsoft). Figures and statistical analyses were conducted using GraphPad Prism 9. Statistical significance between different groups was calculated using the tests indicated in each figure legend. No statistical methods were used to predetermine the sample size.

## RESULTS

### Cholesterol depletion reduces SARS-CoV-2 pseudovirus entry and Spike induced syncytia formation

We first investigated the requirement of cholesterol in the entry of SARS-CoV-2 (virus-cell fusion) and in the formation of Spike-induced syncytia (cell-to-cell fusion). To investigate this in SARS-CoV-2 entry, we incubated ACE2-expressing 293T cells and SARS-CoV-2 pseudovirus with 2 mM MβCD prior to infection. Following cholesterol depletion in the ACE2 expressing target cells, the mean RLU decreased significantly by 50% while treatment of the pseudovirus had no significant effect on entry (Fig. 1A). Next, we investigated the role of cholesterol in Spike-mediated cell-to-cell fusion. For this, we used the GFP-split system (6, 40, 41) as a reporter of fusion. We treated GFP11 cells expressing Spike and GFP1-10 cells expressing ACE2 with 2 mM MβCD prior to co-culturing the cells. GFP quantification showed a significant 45% and 47% reduction in GFP area when either Spike or ACE2 expressing cells are depleted for cholesterol, respectively, and a cumulative 76% decrease when both cells are treated (Fig. 1B). Taken together, cholesterol within the cell plasma membrane, but not the virus envelope, is required to allow entry of SARS-CoV-2 pseudovirus and Spike-mediated cell-to-cell fusion.
<figure><img src="image_3.png" /><figcaption>FIG 1 Plasma membrane cholesterol depletion reduces SARS-CoV-2 fusion and entry. (A) 293T cells were transfected with ACE2 24 h before SARS-CoV-2 D614G pseudovirus infection. Treatment of pseudovirus with 2 mM MβCD or DMSO control was carried out for 3 h prior to the addition of the pseudovirus. Cells were infected for 48 h prior to the addition of luciferase substrate and luminescence quantification. Data were normalized to the ACE2 DMSO condition. Data are means ± s.d. of four independent experiments. (B) 293T GFP-split cells were transfected with SARS-CoV-2 D614G Spike (GFP 1–10 expressing cells) or ACE2 (GFP 11 expressing cells) 24 h before MβCD or DMSO treatment. Treatment of cells with 2 mM MβCD or DMSO control was carried out prior to the co-culturing of cells as shown. GFP area was quantified 3 h after co-culture for a readout of cell fusion. Data were normalized to the Spike + ACE2 DMSO condition. Data are means ± s.d. of three independent experiments. Below: Immunofluorescence images of 293T GFP-split cells taken from one of three independent experiments. Scale bar: 200 µm. For A and B, ordinary One-Way ANOVA tests were performed with Tukey's multiple comparison test to compare to DMSO treatment, \*P < 0.05, \*\*P < 0.001, \*\*\*P < 0.0001, ns = not significant.</figcaption></figure>

## GPI-anchored ACE2 localizes to lipid rafts while wild-type ACE2 is non-raft associated

Cholesterol-depleting agents impact a variety of cellular processes and do not specifically act on lipid rafts (33, 35). Therefore, to investigate the role of lipid rafts during SARS-CoV-2 entry and fusion without cholesterol depletion, we designed an ACE2 construct in which the transmembrane domain (TD) and CT were exchanged for a Thy-1 GPI-anchored protein (herein referred to as ACE2-GPI; Fig. 2A). We asked whether the construct was similarly expressed on the cell surface compared to wild-type ACE2. 293T
<figure><img src="image_4.png" /><figcaption>**FIG 2** ACE2-GPI construct cell surface expression and lipid raft localization. (A) Schematic of the ACE2 and ACE2-GPI constructs used in this study. For ACE2-GPI, the transmembrane domain and cytoplasmic tail of ACE2 were replaced with Thy1, a GPI-anchored protein, to localize ACE2 to lipid rafts. (B) Surface expression of ACE2 and ACE2-GPI by flow cytometry analysis by ACE2 surface staining with anti-ACE2 VHH-B07-Fc. A serial dilution of the ACE2-expression vector was used to transfect 293T cells before surface staining. Shown is the total percentage of ACE2-positive cells (left) and representative contour plots taken from one independent repeat (right). Data are means ± s.d. of three independent experiments. (C) 293T cells expressing ACE2 or ACE2-GPI were incubated with recombinant biotinylated-SARS-CoV-2 RBD (Wuhan variant). Cell surface staining with streptavidin AF-488 was carried out at indicated timepoints. Data were normalized to 0 h post-RBD staining. Data are means ± s.d. of three independent experiments. (D) Treatment of 293T cells with phospholipase C at indicated concentrations for 2 h at 37°C. ACE2 surface levels were assessed by flow cytometry. Data were normalized to NT conditions. Data are means ± s.d. of three independent experiments. (E) Histogram plots from flow cytometry analysis of ACE2 cell surface staining of 293T cells (left) and MRC5 cells (right) stably expressing ACE2 or ACE2-GPI. Results are representative of at least three independent experiments. (F) Cell lysates of 293T cells (above) and MRC5 cells (below) stably expressing ACE2 or ACE2-GPI were ultracentrifuged in an iodixanol (OptiPrep) gradient to isolate cellular membranes on their lipid content. \* =Lipid raft-associated protein, flotillin-1, was used as a control for lipid raft localization. For B, C, and D, Mann-Whitney tests were performed to compare ACE2 and ACE2-GPI conditions, \*\*\*P < 0.0001, ns = not significant.</figcaption></figure>
cells were transfected with a serial dilution of either ACE2 or ACE2-GPI plasmid and expression was examined by surface staining with an anti-ACE2 antibody. Analysis by flow cytometry revealed that both the percentage of ACE2-positive cells and the mean fluorescence intensity (MFI) of the cells showed no significant differences between ACE2 and ACE2-GPI surface levels (Fig. 2B). We next investigated the rate of endocytosis of ACE2-GPI compared to ACE2. 293T cells, expressing either protein, were incubated with SARS-CoV-2 biotinylated-RBD, washed, and incubated with streptavidin-AF-488 at the indicated timepoints. Over the course of 24 h, both ACE2 and ACE2-GPI surface levels decreased at the same rate, indicating both constructs display similar surface turnover (Fig. 2C). To further validate the ACE2-GPI construct, cells were treated with phospholipase C (PLC), an enzyme that cleaves GPI-anchored proteins. PLC (100 and 200 U/µL) significantly reduced ACE2-GPI surface staining and had no impact on ACE2 (Fig. 2D).

To investigate ACE2 membrane domain localization, we generated 293T and MRC5, stably expressing ACE2 or ACE2-GPI cell lines. Both cell lines showed comparable surface levels of ACE2 and ACE2-GPI by flow cytometry (Fig. 2E). To assess localization, we used a membrane flotation assay. Flotation assays use an iodixanol gradient to separate membranes based on their lipid content (42). Cells are lysed mechanically to preserve the membranes and lysate is loaded at the bottom of the gradient. During ultracentrifugation, membranes with higher lipid content, such as lipid rafts, will move up the gradient. Separation of the cell membranes revealed in both the 293T and MRC5 stable cell systems, ACE2 localized to non-raft fractions, whereas ACE2-GPI primarily localized to raft fractions as confirmed by flotillin-1 staining (Fig. 2F). A similar raft profile was observed in 293T cells transiently transfected with ACE2 or ACE2-GPI (Fig. S1A). Flotillin-1 staining showed non-specific bands to non-raft fractions. Therefore, a western blot was performed on fraction number 3 from each cell line. Here, clear flotillin-1 and ACE2-GPI bands were seen, while ACE2 was absent (Fig. S1B). Thus, ACE2 was non-raft associated in the cell lines tested while ACE2-GPI localized to the raft compartment of the cell membrane.

## SARS-CoV-2 displays similar entry, fusogenicity, and replication in ACE2 and ACE2-GPI-expressing cells

Localization of ACE2 to lipid rafts is suggested to enhance SARS-CoV-2 entry. To investigate this, we infected transfected 293T and stable MRC5 cells with SARS-CoV-2 D614G variant pseudovirus expressing a luciferase reporter (Fig. 3A). 293T cells were co-transfected with TMPRSS2 and ACE2 or ACE2-GPI and infected with two doses of D614G pseudovirus. The luciferase signal (RLU) correlated with the viral inoculum. Both ACE2 and ACE2-GPI 293T cells showed no significant differences in the mean RLU in the absence of TMPRSS2 (Fig. 3B). However, in the presence of TMPRSS2, viral entry was increased in the ACE2-GPI condition by 2.5-fold compared to ACE2, independent of the viral inoculum (Fig. 3B). Conversely, Spike-mediated pseudovirus entry in ACE2 or ACE2-GPI stable MRC5 cells showed no significant difference when overexpressing ACE2 or ACE2-GPI (Fig. 3C). To investigate this finding further, we tested pseudovirus entry at different expression levels of the receptor in 293T cells. ACE2 expression was assessed by flow cytometry (Fig. 3D; Fig. S2A). Here, we saw no significant difference between ACE2 and ACE2-GPI in facilitating D614G pseudovirus entry, for both doses, at lower and higher surface levels. Thus, receptor raft localization had little impact on SARS-CoV-2 entry.

We next explored the impact of ACE2 localization on the fusogenicity (i.e., capacity to induce syncytia formation) and replication of the D614G and XBB.1.5 variants. Stable MRC5 GFP-split cells were infected with D614G or XBB.1.5 variants at MOI 0.1. Syncytia formation was assessed by GFP quantification and replication from the supernatant of the same cells was assessed by RT-qPCR (Fig. 4A). Cells were stained intracellularly with an anti-Spike antibody which showed colocalization of Spike with the GFP-positive syncytia (Fig. 4B). GFP area quantification showed no significant differences in fusion-induced D614G or XBB.1.5 in MRC5 cells expressing ACE2 or ACE2-GPI (Fig. 4C). In
<figure><img src="image_5.png" /><figcaption>**FIG 3** SARS-CoV-2 D614G pseudovirus exhibits similar entry in ACE2- and ACE2-GPI-expressing cells. (A) Schematic of experimental workflow. (B) 293T cells were transfected with ACE2 or ACE2-GPI 24 h prior to D614G pseudovirus infection. Infection was performed at a virus dilution of 1:50 (left) or 1:250 (right) for 48 h before the addition of the luciferase substrate and measurement of luciferase activity. Data are means ± s.d. of five independent experiments. (C) As shown in B using MRC5 cells stably expressing ACE2 or ACE2-GPI. Data are means ± s.d. of six independent experiments. (D) 293T cells were transfected with the indicated amounts of ACE2 or ACE2-GPI plasmid prior to D614G pseudovirus infection for 48 h before the addition of luciferase substrate. Surface expression was assessed using flow cytometry by ACE2 surface staining with anti-ACE2 VHH-B07-Fc. For B, C, and D, Mann-Whitney tests were performed to compare ACE2 and ACE2-GPI conditions, \*P < 0.05, \*\*P < 0.001, ns = not significant.</figcaption></figure>

accordance, viral RNA copies in the cell supernatant showed no significant differences (Fig. 4D). We next tested SARS-CoV-2 virus fusogenicity and replication in 293T cells. Again, GFP-positive syncytia colocalized with Spike expression (Fig. 4E). SARS-CoV-2 replicated inefficiently at MOI 0.1 in these cells in the absence of TMPRSS2 (Fig. 4G). Therefore, the cells were infected at MOI 0.1 and 1.0. Both D614G and XBB.1.5 displayed similar fusogenicity in ACE2 and ACE2-GPI-expressing target cells (Fig. 4F). Fusion was collectively increased by the presence of TMPRSS2, yet levels remained similar between the two ACE2 proteins. Concurrently, virus replication showed no significant differences in cells expressing ACE2 or ACE2-GPI at either MOI for either variant (Fig. 4G). Therefore, in accordance with the pseudovirus entry, the membrane localization of ACE2 had little effect on fusogenicity or viral replication of SARS-CoV-2.
<figure><img src="image_6.png" /><figcaption>FIG 4 SARS-CoV-2 virus displays similar fusogenicity and replication in ACE2- and ACE2-GPI-expressing cells. (A) Schematic of experimental workflow. (B) Immunofluorescence images of MRC5 GFP-split cells expressing ACE2 infected with SARS-CoV-2 D614G for 48 h and stained intracellularly for Spike. Scale bar: 100 µm. (C) MRC5 GFP-split cells stably expressing ACE2 or ACE2-GPI were infected with SARS-CoV-2 D614G or XBB.1.5 variants at MOI 0.1. 48 hours (Continued on next page)</figcaption></figure>
Fig 4 (Continued)

post-infection (hpi) cells were fixed and the GFP area was quantified. Data were normalized to D614G fusion with ACE2. Data are means ± s.d. of four or six independent experiments. (D) Viral RNA quantification by RT-qPCR from the cell supernatant of infected MRC5 cells taken from the same cells as panel C, 48 hpi. Primers target the SARS-CoV-2 envelope protein. Data are means ± s.d. of three independent experiments. (E) Immunofluorescence images of 293T GFP-split cells expressing ACE2 infected with SARS-CoV-2 D614G for 48 h and stained intracellularly for Spike. Scale bar: 100 µm. (F) 293T GFP-split cells transiently expressing ACE2 or ACE2-GPI were infected with SARS-CoV-2 D614G or XBB.1.5 variants at MOI 0.1 or MOI 1.0. 48 hpi, cells were fixed and the GFP area was quantified. Data were normalized to D614G fusion with ACE2. Data are means ± s.d. of three independent experiments. (G) Viral RNA quantification by RT-qPCR from the cell supernatant of infected 293T cells taken from the same cells as panel F, 48 hpi. Primers target the SARS-CoV-2 envelope protein. Data are means ± s.d. of three independent experiments. For C, D, F, and G, Mann-Whitney tests were performed to compare ACE2 and ACE2-GPI conditions, ns = not significant.

ACE2 raft localization does not impact Spike-mediated syncytia formation

To further verify the impact of ACE2 localization on SARS-CoV-2 cell-to-cell fusion, we examined syncytia formation through the expression of Spike alone. 293T GFP-Split were transfected with D614G or XBB.1.5 Spike, ACE2 or ACE2-GPI in the presence or absence of TMPRSS2. Fusion was assessed by GFP area quantification (Fig. 5A). In 293T cells, ACE2-GPI significantly increased fusion by 1.5-fold and 3.0-fold compared to ACE2 with the D614G and XBB.1.5 Spikes, respectively (Fig. 5B). However, in the presence of TMPRSS2, this increase with ACE2-GPI was less marked. The surface expression of ACE2 and ACE2-GPI was similar, and the levels of fusion were non-saturating (Fig. S3A). As MRC5 cells are difficult to transfect, we transfected 293T GFP11 cells with Spike and co-cultured them with stable ACE2 or ACE2-GPI MRC5 GFP1-10 cells. Here, no significant difference was seen in the level of Spike-induced cell-to-cell fusion with ACE2 or ACE2-GPI for either D614G or XBB.1.5 Spike (Fig. 5C; Fig. S3D), supporting the previous results on virus infection.

To further clarify the impact of ACE2 localization on Spike-induced syncytia formation, we carried out the same fusion assay in two additional cell lines, U2OS and HeLa cells. Here, for both cell lines, we saw no difference in the levels of fusion between ACE2 and ACE2-GPI with either D614G or XBB.1.5 Spikes (Fig. 5D and E). Levels of fusion were non-saturating and ACE2 and ACE2-GPI surface expression were not significantly different (Fig. S3B and C). While TMPRSS2 increased the overall levels of fusion, there were no significant differences between ACE2 and ACE2-GPI fusion. Finally, we explored the impact of receptor level on cell-to-cell fusion by diluting the plasmids. Surface expression was assessed by flow cytometry (Fig. S2C). Here, there was a small but mostly non-significant increase in cell fusion for ACE2-GPI compared to ACE2 for both D614G and XBB.1.5 in the absence of TMPRSS2 (Fig. 5F). However, in the presence of TMPRSS2, there was no significant difference at higher and lower ACE2 and ACE2-GPI surface levels with D614G and XBB.1.5 Spikes. Altogether, the localization of ACE2 into lipid rafts had little impact on Spike-induced fusion. In 293T cells, there was a trend to increase fusion with raft localization, but this was less marked in the presence of TMPRSS2.

DISCUSSION

We describe the role of cholesterol and ACE2 lipid raft localization during SARS-CoV-2 fusion. We show that cholesterol depletion in the target cell membrane significantly reduces pseudovirus entry. This is consistent with previous reports (27, 43). Interestingly, treatment of pseudovirus with MβCD did not impact virus entry. Some viruses such as canine distemper virus are dependent on virus envelope cholesterol for infection, while others including ecotropic murine leukemia virus are not (44, 45). Further investigations into the role of cholesterol in the SARS-CoV-2 virion envelope would be of interest.

Cholesterol is required for membrane fusion of various enveloped viruses including lentiviruses, flaviviruses, and coronaviruses, consistent with our results when depleting either the Spike or ACE2-expressing cells of cholesterol (46–48). Increasing membrane cholesterol by media supplementation increases SARS-CoV-2 pseudovirus entry while cholesterol depletion using agents such as MβCD, statins, or cholesterol 25-hydroxylase has the reverse effect (26, 27, 49, 50). In addition, increasing cholesterol intake into the
A

<figure><img src="image_7.png" /></figure>

B

293T transfected

<figure><img src="image_8.png" /></figure>

C

293T-Spike transfected x MRC5-ACE2 stable

<figure><img src="image_9.png" /></figure>

D

U2OS transfected

<figure><img src="image_10.png" /></figure>

E

HeLa transfected

<figure><img src="image_11.png" /></figure>

F

<figure><img src="image_12.png" /></figure>

FIG 5 SARS-CoV-2 Spike exhibits similar fusogenicity with ACE2-GPI compared to ACE2. (A) Schematic of experimental workflow. (B) 293T GFP-split cells were transfected with SARS-CoV-2 D614G or XBB.1.5 Spikes and ACE2 or ACE2-GPI in the presence or absence of TMPRSS2 for 18 h. Fusion was quantified by GFP area and data were normalized to D614G fusion with ACE2. Data are means ± s.d. of three, four, or five independent experiments. (C) 293T GFP1-10 cells were transfected with Spike and co-cultured with MRC5 GFP11 cells stably expressing ACE2 or ACE2-GPI for 18 h. Fusion was quantified by the area of GFP, and data (Continued on next page)
Fig 5 (Continued)

were normalized to D614G fusion with ACE2. Data are means ± s.d. of three independent experiments. (D) As shown in B but using U2OS GFP-split expressing cells. Data were normalized to D614G fusion with ACE2. Data are means ± s.d. of three independent experiments. (E) As shown in B but using HeLa GFP-split expressing cells. Data were normalized to D614G fusion with ACE2. Data are means ± s.d. of three independent experiments. (F) 293T GFP-split cells were transfected with indicated amounts of ACE2 or ACE2-GPI plasmid for 18 h, with D614G or XBB.1.5 Spike in the presence or absence of TMPRSS2. Fusion was quantified by GFP area and data were normalized to ACE2 10 ng. Data are means ± s.d. of three independent experiments. For B, C, D, E, and F, Mann-Whitney tests were performed to compare ACE2 and ACE2-GPI conditions, \*P < 0.05, \*\*P < 0.001, ns = not significant.

target cell *via* ApoE increases SARS-CoV-2 pseudovirus entry (51). This is in line with clinical reports describing the reduction of COVID-19 disease severity by statins (52, 53). As statins modulate a variety of cellular functions, including an anti-inflammatory effect, further investigations are required to decipher these mechanisms (54). Similar clinical studies have been proposed for β-cyclodextrins (55). Cholesterol is required in membrane fusion due to its intrinsic ability to stabilize curved fusion intermediates and facilitate the penetration of fusion peptides into the target membrane (34). While cholesterol is a key component of lipid rafts, its depletion is not specific to rafts despite MβCD often being referenced as a lipid raft inhibitor (33, 56). Thus, cholesterol depletion may disturb various cellular functions and membrane properties, notably those involved in viral fusion.

To assess the role of ACE2 localization without cholesterol depletion and lipid raft disruption, we design an ACE2-GPI construct. This led to the re-localization of ACE2 from non-lipid raft regions to lipid raft regions. The GPI anchor is a post-translational modification comprised of a phosphoethanolamine linker attached to a glycan core and phospholipid tail that preferentially inserts into the outer leaflet of lipid rafts (57). Attaching a GPI anchor to angiotensin-converting enzyme (ACE) sequesters the protein to lipid rafts while wild-type ACE does not and treatment of the cells with PLC, releases the protein from the surface (17). Furthermore, the cytoplasmic tail of ACE2 is not required for either SARS-CoV or SARS-CoV-2 entry and should thus not impact the function of ACE2-GPI as a receptor (58).

Through membrane flotation assays, we show that ACE2 localized to non-raft membrane regions while ACE2-GPI localized preferentially within lipid rafts. ACE2 has been previously described as a lipid-raft-associated protein in studies exploring SARS-CoV and SARS-CoV-2 entry in VeroE6 and Caco2 cell lines (26, 27). Conversely, other studies in VeroE6 and CHO cells conclude that ACE and ACE2 are found predominantly in the detergent-soluble membrane domains, separate from the lipid rafts (28, 29). From our results, we postulate that ACE2 is a non-raft-associated protein in the human cell lines that we tested. This warrants further investigation including the localization of ACE2 in primary human cells and tissue which remains unexplored.

We describe little effect of ACE2 raft localization on SARS-CoV-2 entry, or virus and Spike-induced syncytia formation in several cell lines. 293T cells expressing ACE2-GPI exhibited a slight but general increase in these processes compared to ACE2-expressing cells, an observation that requires further research. Regarding viral entry, internalization of receptors in rafts occurs *via* caveolae-mediated endocytosis as opposed to the classical clathrin-dependent pathway (46). While several studies suggest ACE2-raft localization is important for SARS-CoV-2 entry and fusion, SARS-CoV-2 utilizes both endosomal pathways to mediate entry into the host cell, together with cell surface fusion, suggesting ACE2 raft localization is not essential for this process (2, 59).

Palmitoylation is a post-translational modification of cysteine residues within the CT of envelope proteins including SARS-CoV-2 Spike (60), influenza hemagglutinin (61), MLV env (62), and HIV-1 gp160 (63). The addition of fatty acids allows the anchoring of the envelope proteins to the plasma membrane and mutation of these residues strongly decreases viral infectivity. In addition, aromatic SARS-CoV Spike residues at the membrane interface facilitate interaction with membrane cholesterol (64, 65). As with ACE2, Spike requires cholesterol for fusion and this mechanism is independent of lipid rafts (43). Thus, we propose a model by which, while cholesterol in the plasma
membrane is required for SARS-CoV-2 fusion, the localization of ACE2 into cholesterol-
rich lipid rafts does not enhance this process.

This study contains limitations. First, due to the artificial nature of the ACE2-GPI
construct, we rely on overexpression systems and cell-line-based models to assess
viral entry and syncytia formation. Further research may look to more physiologically
relevant models, for example, using super-resolution microscopy to further investigate
the membrane localization of ACE2 in primary airway cells, and examining the impact of
cholesterol depletion on viral replication. In addition, we do not factor the localization
of other proteins required for SARS-CoV-2 fusion such as proteases including TMPRSS2
and cathepsins. Finally, we demonstrate ACE2 localization through membrane flotation
assays; however, this would be supported by techniques such as fluorescence micro-
scopy through co-localization analysis.

Overall, the data presented here show the requirement of cholesterol to facilitate
entry of SARS-CoV-2 as well as Spike-mediated cell-to-cell fusion. ACE2 localizes outside
of lipid rafts in the cell line models we tested. Re-localization of ACE2 to lipid rafts
had little or no impact on viral entry and fusion. The independence of ACE2 lipid raft
localization provides insight into the mechanisms of SARS-CoV-2 entry and fusion.

ACKNOWLEDGMENTS

We thank the members of the Virus and Immunity Unit (Institut Pasteur) for their
discussion and help in this project. We also thank the staff at the UtechS Photonic
Biolmaging core facility (Institut Pasteur), the Theravectys Unit (Institut Pasteur) for kindly
supplying SARS-CoV-2 pseudovirus, and the Humoral Immunology Unit (Institut Pasteur)
for kindly supplying the soluble SARS-CoV-2 RBD.

W.B. was supported by a stipend from the Pasteur-Paris University (PPU) International
PhD program.

Work in the UVI unit is funded by Institut Pasteur, Urgence COVID-19 Fundraising Campaign of Institut Pasteur, Fondation pour la Recherche Médicale, ANRS-MIE, the Vaccine Research Institute (ANR-10-LABX-77), Labex IBEID (ANR-10-LABX-62-IBEID), ANE/FRM Flash COVID PROTEO-SARS-CoV-2, ANR Coronamito, the HERA Project DURABLE (grant 101102733), and the HERA project LEAPS. The Opera Phenix system was co-funded by Institut Pasteur and the Région Ile de France (DIM1Health). The funders of this study had no role in the study design, data collection, analysis, interpretation, or writing of the article.

Conceptualization: O.S., J.B., W.B., and N.C. Methodology: W.B., J.B., O.S., I.M., N.C., F.G.-B., F.P., and A.B. Investigation: W.B., J.B., O.S., I.M., and N.C. Data collection and analysis: W.B., I.M., C.P., and J.B. Manuscript writing and editing: W.B., J.B., and O.S.

AUTHOR AFFILIATIONS

AUTHOR ORCIDs

William Bolland http://orcid.org/0000-0002-8886-0448

Olivier Schwartz http://orcid.org/0000-0002-0729-1475

Julian Buchrieser http://orcid.org/0000-0003-4790-7577

AUTHOR CONTRIBUTIONS

William Bolland, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and

<footnote n="1">¹Virus and Immunity Unit, Institut Pasteur, Université Paris Cité, CNRS UMR3569, Paris, France</footnote>

<footnote n="2">²Université Paris Cité, Paris, France</footnote>

<footnote n="3">³Dynamic of Host-Pathogen Interactions Unit, Institut Pasteur, Université Paris Cité, CNRS UMR3691, Paris, France</footnote>

<footnote n="4">⁴Vaccine Research Institute, Créteil, France</footnote>
editing | Inès Marechal, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization | Chloé Petiot, Data curation, Formal analysis, Investigation, Methodology, Validation | Françoise Porrot, Investigation, Methodology | Florence Guivel-Benhassine, Investigation, Methodology | Anne Brelot, Methodology, Resources | Nicoletta Casartelli, Conceptualization, Formal analysis, Investigation, Methodology, Supervision | Olivier Schwartz, Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – review and editing | Julian Buchrieser, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – review and editing

## DATA AVAILABILITY

This study includes no data deposited in external repositories.

## ADDITIONAL FILES

The following material is available online.

## Supplemental Material

Figures S1 to S3 (JVI01823-24-s0001.pdf). Western blot, flow cytometry analysis, and raw microscopy analysis.

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