key: cord-1043714-5qd0ws6p
authors: Cheng, Ya-Wen; Chao, Tai-Ling; Li, Chiao-Ling; Wang, Sheng-Han; Kao, Han-Chieh; Tsai, Ya-Min; Wang, Hurng-Yi; Hsieh, Chi-Ling; Chen, Pei-Jer; Chang, Sui-Yuan; Yeh, Shiou-Hwei
title: D614G Substitution of SARS-CoV-2 Spike Protein Increases Syncytium Formation and Viral Transmission via Enhanced Furin-mediated Spike Cleavage
date: 2021-01-28
journal: bioRxiv
DOI: 10.1101/2021.01.27.428541
sha: 5e94c6f9b248adedd3e5bc5719596c777d44ff5a
doc_id: 1043714
cord_uid: 5qd0ws6p

Since the D614G substitution in the spike (S) of SARS-CoV-2 emerged, the variant strain underwent rapid expansion to become the most abundant strain worldwide. Therefore, this substitution may provide an advantage of viral spreading. To explore the mechanism, we analyzed 18 viral isolates containing S proteins with either G614 or D614. Both the virus titer and syncytial phenotype were significantly increased in S-G614 than in S-D614 isolates. We further showed increased cleavage of S at the furin substrate site, a key event that promotes syncytium, in S-G614 isolates. These functions of the D614G substitution were validated in cells expressing S protein. The effect on syncytium was abolished by furin inhibitor treatment and mutation of the furin-cleavage site, suggesting its dependence on cleavage by furin. Our study provides a mechanistic explanation for the increased transmissibility of S-G614 containing SARS-CoV-2 through enhanced furin-mediated S cleavage, which increases membrane fusion and virus infectivity.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes a rapid accumulation of confirmed and fatal cases and poses a threat to public health around the world. A better understanding of viral evolution and characterization of viral genetic variations usually provides valuable insights into the mechanisms linked to pathogenesis, antiviral drug resistance, and immune responses, which also impact the development of new vaccines, antiviral drugs and diagnostic tests (Chellapandi and Saranya, 2020; Sanjuan and Domingo-Calap, 2016; Young et al., 2020) . Therefore, analysis of viral genomes and monitoring of the evolutionary trajectory of SARS-CoV-2 over time have been meticulously conducted to identify any specific genetic variations that contribute to the transmissibility and virulence of SARS-CoV-2 (Chitranshi et al., 2020; Mercatelli and Giorgi, 2020; Pachetti et al., 2020) .

Among the genetic variations that have evolved during the course of the SARS-CoV-2 outbreak, the D614G substitution in the spike (S) protein, which corresponds to a change of the A nucleotide at genome position 23,403 to a G, has been identified as the signature of the A2a clade of SARS-CoV-2, the most prevalent clade (Korber et al., 2020; Yin, 2020) . This substitution emerged at low frequency in early March but had rapidly expanded to become the most abundant clade worldwide by April to May (Korber et al., 2020) , which was thus proposed to provide a selective fitness advantage during the outbreak. Korber B et al. recently reported that the S-G614-containing strain is associated with a higher viral load in the upper respiratory tract in infected individuals, though not increased disease severity (Korber et al., 2020) . Several recent reports further demonstrated that pseudotyped viruses or the engineered viruses containing the S-G614 exhibit significantly higher infectivity than 5 those containing S-D614 (Daniloski et al., 2020; Hu et al., 2020; Plante et al., 2020; Weissman et al., 2020) . Sera from most convalescent COVID-19 patients could neutralize both S-D614 and S-G614 pseudotyped viruses with comparable efficiencies (Korber et al., 2020) . These findings thus raised the possibility that D614G substitution confers increased infectivity and transmissibility, promoting its rapid expansion worldwide, but not through an increased binding affinity for ACE2 or increased escape of immune surveillance. Elucidating the molecular basis for the higher infectivity of D614G virus is urgent to understand its predominance and design an effective treatment strategy for patients.

As documented, release of the fusion peptide from the S protein via cleavage by host proteases, including furin/proprotein convertases (PCs) at the S1/S2 boundary and TMPRSS2 at the S2' site within the S2 domain, is a prerequisite for the membrane fusion of SARS-CoV-2 with the target cells and thus viral infection (Hoffmann et al., 2020; Hou et al., 2020b; Ou et al., 2016) . In our recent study, we found that the cleavage of S by furin/PCs at the S1/S2 boundary is also critical for S-mediated syncytium formation, another pathogenic event that contributes to increased viral transmission (Cheng et al., 2020) . Meanwhile, a recent cryogenic electron microscopy (cryo-EM) analysis suggested that the D614G substitution induces a conformational change in the S protein (Yurkovetskiy et al., 2020) . As noted, the D614G substitution is located at the C-terminal region of the S1 domain of the S protein, close to the furin-cleavage site (between a.a. 685-686). It thus raised a possibility that the D614G substitution might contribute to increase accessibility of the S protein for cleavage by furin through a conformational change and thus increasing the membrane fusion activity, as the basis for the increased infectivity and transmission capability of S-G614-containing SARS-CoV-2. To test this hypothesis, 6 we first compared the virus titer, syncytial phenotype and cleavage of spike protein in 18 clinical SARS-CoV-2 isolates. The effects of the D614G substitution on enhanced syncytium and spike cleavage have been further quantitatively validated in the cells expressing spike protein. This in vitro assay system has been further used to examine the critical role of furin mediated S cleavage for the effect of D614G substitution. We expect the findings will provide a mechanistic explanation for the rapid expansion of S-G614 containing SARS-CoV-2 and help develop therapeutic strategy for intervening their spreading in population.

The virus titer and syncytial phenotype were significantly higher in S-G614 containing viral isolates than in S-D614 containing viral isolates

We first compared the virus titer and syncytial phenotype of 18 clinical SARS-CoV-2 isolates containing either S-D614 or S-G614 protein from infected Vero E6 cells (NTU01 to NUT18, Table S1 ). Interestingly, we found that the S-G614-containing viruses (n=10) had a significantly higher virus titer than the S-D614-containing viruses (n=8), as determined by plaque assay ( Figure 1A ).

Consistently, northern blotting and qRT-PCR analysis confirmed the higher viral RNA levels in Vero E6 cells infected with S-G614-containing viruses compared to S-D614-containing viruses (P=0.0164) ( Figure 1B ). Microscopic observation further showed a higher syncytium level in S-G614-containing viruses than in S-D614-containing viruses ( Figure 1C ). These results obtained with the virus isolates suggest the possible functional effect of the D614G substitution in increasing virus production and syncytium formation.

S-G614-containing viral isolates showed increased cleavage of the S protein than S-D614-containing viral isolates As shown in our previous study, cleavage of the S protein at the S1/S2 boundary by furin/PCs is critical for S-mediated syncytium formation (Cheng et al., 2020) . We thus compared the patterns of S protein cleavage at this site for viruses containing either the S-G614 or S-D614 protein. Viruses in the supernatants of cells infected with either group of viruses were harvested for immunoblot analysis. Interestingly, the results showed significantly increased cleavage of the S protein into the S1 and S2 fragments in S-G614-containing viruses than in S-D614-containing viruses, and the cleaved S/full-length (FL) S ratio was shown to be 4.8 ± 0.7 versus 1.5 ± 0.5, 8 respectively (P=0.0103) ( Figure 1D , upper panel). Consistently, a significant difference in cleavage of S between lysates from cells infected with the two groups of viruses was also demonstrated by immunoblotting, with the cleaved S/FL S ratio found to be 1.6 ± 0.2 versus 0.7 ± 0.3, respectively (P=0.0172) ( Figure 1D , lower panel). Therefore, these results suggested the function of the D614G substitution in enhancing cleavage of the S protein.

The syncytial phenotype and S cleavage were increased in S-G614-expressing cells than in S-D614-expressing cells Genetic heterogeneity beyond the D614G substitution among different SARS-CoV-2 isolates might cause confusion regarding our observation. Therefore, we tested our hypothesis in cultured cells expressing only the S protein. and 24 hr posttransfection, respectively) ( Figure 2B ). These results are consistent with the findings from the virus infection system shown in Figure 1D . 9 The S mediated syncytium was increased in S-G614-expressing cells than in

To confirm the effect of D614G substitution on S and ACE2 binding-mediated cell syncytium formation, we established a luciferase-based reporter assay to quantitatively compare syncytium induction by the S-G614 and S-D614 proteins (schematically illustrated in Figure 2C ). As documented, interaction between S protein and ACE2 at the membrane of infected cells and adjacent cells primes the cleavage of S by host proteases to release the fusion peptide essential for syncytium formation (Bertram et al., 2011) . Therefore, in our experimental design, we selected ACE2-null 293T cells transfected with expression plasmid for either S-D614 or S-G614 as S(+) and ACE2(-) effector cells. Two ACE2(+) target cell lines were selected: 293T cells stably transfected with human ACE2 (293T-hACE2) and Vero E6 cells (with endogenous ACE2 expression). Neither effector cells nor target cells alone developed syncytia due to the lack of the binding partner (ACE2 or the S protein). To quantitatively examine the fusion of effector and target cells, we developed a one hybrid luciferase reporter assay. The pGAL4DBD-hAR-NTD plasmid was co-transfected with an individual S expression plasmid into the effector cells; the pGAL4/UAS-TK-Luc plasmid was transfected into the target cells. The effector and target cells were then co-cultured and harvested for reporter activity analysis. The expression of pGAL4/UAS-TK-Luc in the target cells was activated only upon formation of syncytia consisting of effector and target cells (mediated by interaction of S and ACE2), which is driven by the transcriptional activator encoded by the pGAL4DBD-hAR-NTD plasmid in the effector cells.

As expected, the expression of pGAL4DBD-hAR-NTD or S alone in the effector cells did not activate luciferase expression in the target cells ( Figure 2D , lanes 2-4), 10 which was elevated only when pGAL4DBD-hAR-NTD and the S protein were coexpressed in the effector cells. Expression of S-G614 significantly increased luciferase activity compared to that upon the expression of S-D614 in the effector cells ( Figure Figure 2E . Altogether, these results suggest that the putative function of the D614G mutation in the S protein of SARS-CoV-2 is dependent on enhanced cleavage at the furin substrate motif at S1/S2 boundary, which contributes to an increased membrane fusion activity.

Both virus entrance and syncytium formation during SARS-CoV-2 infection are mediated through membrane fusion between the virus and cell or between cells. As shown in our recent study, cleavage of the S protein by furin/PCs is critical for the membrane fusion of SARS-CoV-2, after the binding of S protein with ACE2 receptor (Cheng et al., 2020) . The current study further revealed that the D614G substitution in S contributes to increasing accessibility to the polybasic RRAR motif for cleavage by furin/PCs, leading to the increased infectivity and syncytium formation of S-G614-containing SARS-CoV-2 viruses. In fact, syncytium formation has also been documented to confer advantages in terms of infectivity and transmission in many coronaviruses (CoVs) (Frana et al., 1985; Nakagaki et al., 2005; Park et al., 2016; Yamada and Liu, 2009) . Direct cell-to-cell spread of CoVs is more efficient than their cell-free spread; the syncytium also allows viruses to evade innate immune surveillance (Sattentau, 2008 (Sattentau, , 2011 . Moreover, an unique structure generated by cleavage of S at furin substrate site was identified to be critical for viral entry and infectivity, via interacting with cell surface Neurophilin-1 (NRP1) (Cantuti-Castelvetri et al., 2020; Daly et al., 2020) . Therefore, the effect of the D614G substitution in increasing cleavage of the furin substrate motif could benefit viral infectivity and transmission and provides a mechanism for the significant expansion 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Shiou-Hwei Yeh (shyeh@ntu.edu.tw)

All materials and reagents will be made available upon instalment of a material transfer agreement (MTA).

The original sequencing datasets for hCoV-19/Taiwan/NTU01/2020 to hCoV-19/Taiwan/NTU18/2020 can be found on the GISAID under Accession ID listed in Table S1 .

Sputum specimens from SARS-CoV-2-infected patients were kept in viral transport medium. Virus isolated from the specimens was propagated in Vero E6 cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 μg/mL tosylsulfonyl phenylalanyl chloromethyl ketone-trypsin. The 18 virus isolates used in the current study were hCoV-19/Taiwan/NTU01/2020 to hCoV-19/Taiwan/NTU18/2020. The sequencing data have been deposited in the GISAID, and the accession codes are listed in Table S1 .

The plaque assay was performed as previously described with minor modifications (Su et al., 2008) . In brief, Vero E6 cells (2 x 10 5 cells/well) were seeded in 24-well tissue culture plates and maintained in DMEM supplemented with 10% FBS and antibiotics. After 24 hr incubation, SARS-CoV-2 virus was treated to the cell monolayer for 1 hr at 37°C. After removed the virus and washed the cell monolayer once with PBS, maintained the cells with medium containing 1% methylcellulose and incubated for 5-7 days. Subsequently, cells fixed with 10% formaldehyde overnight and stained with 0.7% crystal violet in order to count the plaques. The virus titer is from the mean of three independent experiments. 

Western blotting was performed as previously described (Wu et al., 2009) . In brief, Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

The qRT-PCR was conducted following the protocol as described previously (Cheng 21 et al., 2020) . In brief, RNA extracted from SARS-CoV-2 infected VeroE6 cells was reverse transcribed using SuperScript III Reverse Transcriptase System (Thermo Fisher Scientific). Quantitative PCR targeting to E gene was performed using 

The virus titers quantified by plaque assays in triplicate are shown as the mean ± SD.

Results from the reporter assay are shown as data representative of three independent experiments and presented as the mean ± SD. Differences in data from the virus titer, qRT-PCR and reporter assay between each indicated paired sample groups were evaluated by Student's t-test. A P value of 0.05 or lower was used to indicate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001). 

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