key: cord-0693734-jurnkaxe
authors: Liu, Yang; Liu, Jianying; Johnson, Bryan A.; Xia, Hongjie; Ku, Zhiqiang; Schindewolf, Craig; Widen, Steven G.; An, Zhiqiang; Weaver, Scott C.; Menachery, Vineet D.; Xie, Xuping; Shi, Pei-Yong
title: Delta spike P681R mutation enhances SARS-CoV-2 fitness over Alpha variant
date: 2022-04-29
journal: Cell Rep
DOI: 10.1016/j.celrep.2022.110829
sha: 545d85ae11d1a8efb715b3ab7cf69563294a20c1
doc_id: 693734
cord_uid: jurnkaxe

We report that SARS-CoV-2 Delta spike mutation P681R plays a key role in the Alpha-to-Delta variant replacement during the COVID-19 pandemic. Delta SARS-CoV-2 efficiently outcompetes the Alpha variant in human lung epithelial cells and primary human airway tissues. The Delta spike mutation P681R is located at a furin cleavage site that separates the spike 1 (S1) and S2 subunits. Reverting the P681R mutation to wild-type P681 significantly reduces the replication of Delta variant, to a level lower than the Alpha variant. Mechanistically, the Delta P681R mutation enhances the cleavage of the full-length spike to S1 and S2, which could improve cell surface-mediated virus entry. In contrast, the Alpha spike also has a mutation at the same amino acid (P681H), but the cleavage of Alpha spike is reduced compared to the Delta spike. Our results suggest P681R as a key mutation in enhancing Delta variant replication via increased S1/S2 cleavage.

contribute to the enhanced viral fitness of the Delta variant over Alpha. Importantly, the P681R 182 mutation plays a critical role in this fitness advantage and increases the processing of Delta spike 183 to S1 and S2, most likely through an improved furin cleavage when newly assembled virions 184 egress through the trans-Golgi network. Although the original SARS-CoV-2 strain has a functional 185 furin cleavage site with a minimal recognition site of RXXR↓ (Molloy et al., 1992) , adjacent 186 residues influence the cleavage efficiency (Krysan et al., 1999) . Indeed, the Delta P681R 187 substitution increases the intracellular spike cleavage ( Figure 4B) . Corroboratively, purified 188 extracellular Delta virions showed an improved spike cleavage than the wild-type USA/WA1-2020 189 and Alpha virions; reversion of the Delta P681R to wild-type P681 alone reduced the spike 190 cleavage of the purified Delta virion ( Figure 4A ). These results support the hypothesis that, when 191 the Delta variant infects respiratory epithelial cells, it binds to ACE2 receptor via the RBD in S1; 192 already cleaved at the S1/S2 site, the Delta virion facilitates cleavage at S2' by the cell surface 193 protease TMPRSS2, leading to an activation of the S2 fusion peptide (FP) for viral and plasma 194 membrane fusion (Murgolo et al., 2021 

One weakness of the current study is the discrepancy between the cell culture Calu-3/HAE 219 results and the hamster transmission results. A similar discrepancy was observed for Omicron 220 variant when analyzed in cell culture and mouse/hamster models (Halfmann et al., 2022) . These 221 further support our conclusion that spike P681R is a key mutation in enhancing Delta variant 229 replication via increased S1/S2 cleavage. experiments is shown. The densitometry was quantified by ImageLab 6.0.1 (Bio-Rad). The ratios 317 of S1/S2 or S2 subunits over FL spike are indicated at the top of the Western blots. 318

Further information and requests for resources and reagents should be directed to and will be 322 fulfilled by Lead Contact, Dr. Pei-Yong Shi (peshi@utmb.edu) 323

Plasmids and virus generated in this study will be made available on request, but we might 

The full-length cDNA clones of Alpha and Delta variants were constructed through mutagenesis 

The competition results generated by Sanger sequencing were confirmed using NGS methods. 420

Briefly, viral RNA samples from competition groups of (i) Delta versus Alpha and (ii) Delta 421 versus Alpha-spike/Delta-backbone were used for a specific one-step RT-PCR that containing 422 the A23063T mutation site. Viral RNA samples from competition group of Alpha versus Alpha-423 spike/Delta-backbone were quantified by the T14444C mutation. The RT-PCR primers were 424 listed in Table S1 . The PCR products were purified by a QIAquick PCR Purification kit (Qiagen, 425 protocol. The reads were filtered for Q-scores of 37 at the A23063T and T14444C mutation sites 429 and adjacent bases and counted. The input ratios and output ratios of two viruses were 430 obtained and used for the relative replicative fitness analysis ( Table S2 ). The output virus ratios 431 were normalized by the input virus ratios and presented in Figures 1-3 and Figure S2 . 

It is important to identify mutations that account for the emergence of SARS-CoV-2 variants. Liu et al. show that Delta spike mutation P681R enhances the cleavage of full-length spike to S1 and S2, which improves cell surface-mediated virus entry and leads to the Alpha-to-Delta variant replacement.

• SARS-CoV-2 spike P681R mutation contributes to Alpha-to-Delta variant replacement

• Delta P681R mutation enhances the cleavage of full-length spike to S1 and S2

• Mutations affecting S1/S2 cleavage must be closely monitored in variant surveillance The biosensors were then dipped into serially diluted human ACE2 protein or buffers to measure association and dissociation kinetics. The binding affinity-related parameters, including association (Kon), dissociation (Koff), and affinity (KD) are shown. Data derived from a single experiment. The affinity of ACE2 to Alpha RBD (N501Y) is below the detection limit and is presented as KD < 1.0×10 -12 . The result for Alpha RBD and ACE2 binding was adopted from our previous study (Liu et Equal PFU of Alpha and Delta viruses were mixed and inoculated into donor hamsters intranasally at a total titer of 10 5 PUF per hamster. The donor hamsters were co-housed with recipient hamsters 1 day after infection. After 8 h of contact, the donor and recipient animals were separated to individual cages. All hamsters were subjected to nasal washes daily from day 1 to 4. The total RNAs were isolated, amplified by RT-PCR, and quantified for the ratios of Alpha:Delta RNA by NGS. Red dots represent individual hamsters (n=5) from a single experiment; the horizontal lines in each catseye represent the mean; shaded regions represent standard error of the mean; yaxes use a log10 scale. Black numbers above each set of values (catseye) indicate the ratios of two viral RNA species. P values were calculated for group coefficient using linear regression model. ***p<0.001. 

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Vero E6 expressing TMPRSS2 were infected with different SARS-CoV-2 at an MOI of 0.01. At 453 24 h post-infection, the culture medium was collected, purified through a 20% sucrose cushion, 454and analyzed by Western blot as previously described on a 4-20% gradient SDS-PAGE gel 455 (Johnson et al., 2021) . For the intracellular spike cleavage experiments, USA/WA1-2020 spike, 456 P681H mutant, and P681R mutant were inserted to plasmid pVAX1 Vector with a C-terminal HA 457 tag. Two micrograms of each plasmid were transfected and expressed in HEK293T cells for 24 458 h. Cells were harvested and lysed by RIPA buffer for Western blot analysis (anti-HA, Cell 459 Signaling Technology, 2367; anti-GAPDH, Sigma-Aldrich, G9545). Densitometry was performed 460 to quantify the cleavage efficiency of full-length spike to S1/S2 or S2 subunits using ImageLab 461 6.0.1 (Bio-Rad #12012931). The average results of three experiments were presented. 462

The human ACE2 protein was purchased from Sino Biological (Beijing, China; Cat# 10108-464 H08H) and the human IgG1 Fc-tagged RBD proteins were made in-house using a method as 465 previously described (Ku et al., 2021) . The affinity measurement was performed on the ForteBio 466 Octet RED 96 system (Sartorius, Goettingen, Germany). Briefly, the RBD proteins (20 µg/ml) of 467 Alpha or Delta RBDs were captured onto protein A biosensors for 300s. The loaded biosensors 468 were then dipped into the kinetics buffer for 10 s for adjustment of baselines. Subsequently, the 469 biosensors were dipped into serially diluted (from 1.23 to 300 nM) human ACE2 protein for 200 470 s to record association kinetics and then dipped into kinetics buffer for 400 s to record 471 dissociation kinetics. Kinetic buffer without ACE2 was used to correct the background. The 472Octet Data Acquisition 9.0 software was used to collect affinity data. For fitting of KD values, 473Octet Data Analysis software V11.1 was used to fit the curve by a 1:1 binding model using the Group term, which was transformed to the original scale as 10^coefficient. This modeling 496 approach compensates for any correlation due to clustering within experiment similarly to that of 497 corresponding mixed effect models and is effective since the number of experiments was small. 498Statistical analyses were performed using R statistical software (R Core Team, 2019, version 499 3.6.1). In all statistical tests, two-sided alpha=.05. Catseye plots (Cumming, 2014) , which 500