The SARS-CoV-2 Delta variant spike protein facilitates cell–cell fusion

doi:10.21203/rs.3.rs-1736792/v1

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The SARS-CoV-2 Delta variant spike protein facilitates cell–cell fusion

https://doi.org/10.21203/rs.3.rs-1736792/v1

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Objective

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates virus entry by binding to the host cell receptor, human angiotensin converting enzyme 2 (hACE2), and catalyzing virus–host membrane fusion. The S protein also mediates cell–cell fusion, potentially allowing the virus to spread virion-independently. Here, we compared the fusogenicity of SARS-CoV-2 variant S proteins using a cell–cell fusion assay.

Results

In cells overexpressing hACE2, cell–cell fusion ability of all tested SARS-CoV-2 variants was similar to that of the Wuhan-Hu-1 strain. However, in cells with endogenous hACE2, SARS-CoV-2 variants, especially the Delta variant, stimulated significantly greater cell–cell fusion than the original strain. Our results showed that the Delta variant S protein is the most fusogenic and can spread rapidly by utilizing small amounts of hACE2.

SARS-CoV-2

variant

cell–cell fusion

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing respiratory coronavirus disease 2019 pandemic [5]. SARS-CoV-2 is classified as a member of the subgenus Sarbecovirus within the Betacoronavirus genus [34]. It is thought to have originated from bats and infected humans via intermediate hosts such as pangolins [24, 32, 33]. Common signs of infection in humans include respiratory symptoms such as fever, cough, and shortness of breath [10, 15]. In severe cases, infections can be associated with pneumonia, severe acute respiratory syndrome, and renal failure leading to death [10, 15].

SARS-CoV-2 is an enveloped virus containing positive-sense single-stranded RNA [20]. Virus particles are composed of four structural proteins: spike (S), envelope, membrane, and nucleocapsid proteins [20]. Among them, the S protein plays a major role in virus entry and virion-independent cell-to-cell spread [13, 31]. The S protein ectodomain consists of two domains [11]. S1 contains two receptor-binding domains (RBDs) and interacts with a variety of sugars and proteins [17]. The S2 domain contains a hydrophobic fusion peptide and two heptad repeats, which are typical features of class I viral fusion proteins [4]. Between S1 and S2, there are two cleavage sites, called the S1/S2 and S2` cleavage sites [1, 13]. The SARS-CoV-2 S protein contains a furin cleavage motif at the S1/S2 cleavage site; therefore, it is cleaved during biosynthesis [21, 25]. S2` cleavage is required for membrane fusion to occur and is mediated by various host proteases, such as TMPRSS2 and endosomal cathepsins [3, 22].

Once SARS-CoV-2 was first identified in Wuhan, China, in December 2019, over time, multiple SARS-CoV-2 variants have been reported [9, 26]. The WHO highlights variants of SARS-CoV-2 that may be more dangerous than previous strains as variants of concern [9]. These variants have featured multiple mutations in S proteins and showed increases of 25 to 100% in viral transmissibility compared to the original isolates [6–8, 26]. Furthermore, they have also shown modest resistance against existing antibodies and vaccines [23, 28, 30].

The emergence of new SARS-CoV-2 variants is a serious concern, especially since many of the mutations in these variants occur within the S protein and appear to increase infectivity and transmissibility. Given the role of the S protein in viral infection, it is likely that the S mutation in the SARS-CoV-2 variant promotes viral propagation and infection by altering the entry characteristics of the virus. In this study, to understand the molecular mechanisms of the enhanced transmissibility of SARS-CoV-2 variants, we investigated the fusogenic ability of four SARS-CoV-2 variants (Alpha, Beta, Gamma, and Delta) in comparison with the SARS-CoV-2 original strain (Wuhan-hu-1) and SARS-CoV-1.

To investigate whether SARS-CoV-2 variants affect membrane fusion kinetics, we performed a dual-split protein (DSP)-based cell–cell fusion assay. DSP is a pair of chimeric reporter proteins composed of split GFP and split Renilla luciferase (RL) [12]. Both GFP and RL are activated only after association of the pair.

Genes encoding codon-optimized SARS-CoV-2 S (GenBank accession nos. NC045512, MZ344997, MW598419, MZ169911, MZ359841) with a C-terminal C9 tag were purchased from GenScript (Hong Kong) and cloned into the pcDNA3.1 vector with EcoRI/NotI restriction enzyme sites. Plasmids encoding DSP_1 − 7, DSP_8 − 11, SARS-CoV-1 S-C9, and hACE2 were obtained from Thomas Gallagher of Loyola University, Chicago, IL.

Effector HEK293T cells were transfected with plasmids encoding the DSP_1− 7 and S proteins. Target HEK293T cells were transfected with plasmids encoding DSP_8− 11 and hACE2. At 24 h post-transfection, effector and target cells were trypsinized and mixed at a 1:1 ratio. At the indicated time points after cell–cell fusion, GFP expression was monitored with a digital imaging system (CELENA® S, Logosbiosystems). The average green object size (syncytia) was measured using ImageJ and is presented as the mean ± SD. Statistical significance was calculated using the Holm–Sidak multiple Student’s t test procedure. A P value of < 0.05 was considered statistically significant. All experiments were independently repeated three times.

Figure 1 shows the cell–cell fusion assay in hACE2-expressing cells. Very little GFP expression was observed in cells lacking S proteins. When SARS-CoV-1 and SARS-CoV-2 S proteins were expressed in effector cells, GFP expression was significantly increased. Compared to SARS-CoV-1 S, all SARS-CoV-2 S-expressing cells showed increased GFP expression, indicating that SARS-CoV-2 S induces cell–cell fusion more efficiently. Cells expressing SARS-CoV-2 variant S proteins showed a non-significant increase in GFP expression compared to those expressing SARS-CoV-2 reference strain S proteins. Among variants, Alpha and Delta showed higher fusion activity than Beta and Gamma.

Next, we investigated cell–cell fusion in cells expressing endogenous levels of hACE2 (Fig. 2). In cells expressing endogenous levels of hACE2s, the Delta variant showed the highest fusion activity. Other variants showed levels of fusion kinetics similar to those of the reference viruses. SARS-CoV-1 showed lower fusion activity than all SARS-CoV-2 strains. The results indicated that the level of hACE2 expression can affect the fusogenicity of S proteins and that the Delta variant can utilize very low amounts of hACE2 for S-mediated membrane fusion.

S-mediated membrane fusion is a crucial step for virus entry and cell–cell spread. Receptor interaction is needed to induce conformational changes in S proteins to expose the S2` cleavage site and trigger fusion [2, 18]. We present two possible reasons that SARS-CoV-2, especially the Delta S protein, exhibits high cell–cell fusion. First, we speculate that the high binding affinity between the S protein and hACE2 may promote cell–cell fusion. The binding affinity of S proteins and hACE2 has been well documented in previous reports. The N501Y (Alpha, Beta, Gamma), E484K (Beta, Gamma), L452R (Delta), and T478K (Delta) mutations increased binding affinity to hACE2, whereas K417N resulted in weaker binding [14, 16, 27]. RBDs with triple mutation of N501Y, E484K, and K417N showed only slightly enhanced binding affinity [16]. These results are consistent with our observations that the Alpha and Delta variants generally induced greater cell–cell fusion than the Beta and Gamma variants.

Additionally, S mutations outside the RBD may influence the dynamics of the conformational changes that occur after receptor binding. Previously, it was shown that communication between the amino acid at position 330 (in S1) and the amino acid at position 1114 (in S2) is important for the efficient fusion activity of murine hepatitis virus in BHK-R2 cells [19]. Although both the Alpha and Delta RBDs have mutations that increase hACE2 binding affinity, only Delta S proteins showed significantly enhanced cell–cell fusion. Unlike Alpha variant S proteins, Delta variant S proteins contain additional mutations in the NTD domains of S1 and S2. Thus, it is possible that these mutations affect the kinetics of conformational changes in S proteins. To demonstrate the molecular mechanisms of increased cell–cell fusion of Delta S proteins, further studies with single S mutations are needed.

Recently, it was demonstrated that SARS-CoV-2 can spread through cell–cell fusion transmission [31]. Cell-to-cell transmission is critical for the robust replication of SARS-CoV-2 in human lung and other tissues, as well as the rapid spread of SARS-CoV-2, including some variants of concern. In a previous study, the Alpha and Beta variants, as well as the D614G mutant, exhibited similar efficiencies of cell-to-cell transmission compared with the wild type [29, 31]. Here, we found that the cell–cell fusion promoted by the Alpha, Beta, and Gamma variants was similar to that of reference strains. However, Delta variants exhibited stronger cell–cell fusion than all the tested strains. More importantly, the Delta variant seems to induce cell–cell fusion even in the presence of very low amounts of hACE2, as evidenced in our results.

In conclusion, we evaluated the cell–cell fusion ability of SARS-CoV-2 variants in comparison with SARS-CoV-1 and SARS-CoV-2 reference strains. We found that S has evolved to favor receptor-induced conformational changes and that the facilitated conformational changes induce accelerated cell–cell fusion. Our results improve the molecular understanding of earlier S variants underlying virus transmissibility and infection and further provide a molecular basis of our knowledge and prediction of SARS-CoV-2 evolution.

The study was conducted using cells overexpressing the S proteins. In addition, cell-cell fusion was performed in HEK293T cells. Therefore, to fully understand cell-cell fusion of SARS-CoV-2 variants, it would be necessary to investigate cell-cell fusion under virus infection in more relevant cells and tissues.

Ethics approval and consent to participate

Not applicable.

Consent to publish

Not applicable.

Availability of data and materials

Data sets used and/or analyzed in this study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no conflict of interest.

Funding

This work was supported with a National Research Foundation of Korea (NRF) grant founded by the Korean government (MSIT) (2020R1C1C1012854), the Korean Government (MOE), and BK21 FOUR Program by Chungnam National University Research Grant, 2021.

Authors' Contributions

JK and SK conceived the experiments and collected data. JK and JEP analyzed results and wrote the manuscript.

Acknowledgements

Not applicable.

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The SARS-CoV-2 Delta variant spike protein facilitates cell–cell fusion

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