key: cord-0787178-zu2jrptn authors: Daniloski, Zharko; Jordan, Tristan X.; Ilmain, Juliana K.; Guo, Xinyi; Bhabha, Gira; tenOever, Benjamin R.; Sanjana, Neville E. title: The Spike D614G mutation increases SARS-CoV-2 infection of multiple human cell types date: 2020-07-07 journal: bioRxiv DOI: 10.1101/2020.06.14.151357 sha: fa3fd8f9ba734f939ad05dfa6537d49173eb36e9 doc_id: 787178 cord_uid: zu2jrptn A novel isolate of the SARS-CoV-2 virus carrying a point mutation in the Spike protein (D614G) has recently emerged and rapidly surpassed others in prevalence. This mutation is in linkage disequilibrium with an ORF1b protein variant (P314L), making it difficult to discern the functional significance of the Spike D614G mutation from population genetics alone. Here, we perform site-directed mutagenesis to introduce the D614G variant and show that in multiple cell lines, including human lung epithelial cells, that the D614G mutation is up to 8-fold more effective at transducing cells than wild-type. We demonstrate increased infection using both Spike-pseudotyped lentivirus and intact SARS-CoV-2 virus. Although there is minimal difference in ACE2 receptor binding between the Spike variants, we show that the G614 variant is more resistant to proteolytic cleavage in vitro and in human cells, suggesting a possible mechanism for the increased transduction. This result has important implications for the efficacy of Spike-based vaccines currently under development in protecting against this recent and highly-prevalent SARS-CoV-2 isolate. seen whether the D614G variant alters neutralization sensitivity to other classes of anti-Spike antibodies 23 (Yurkovetskiy et al., 2020) . 24 The first sequenced SARS-CoV-2 isolate (GenBank accession MN908947.3) and the majority of 25 viral sequences acquired in January and February 2020 contained an aspartic acid at position 614 of the 26 2 Spike protein (Figure 1a) . Beginning in February 2020, an increasing number of SARS-CoV-2 isolates 27 with glycine at position 614 of the Spike protein were identified. We found that ~72% of 22,103 SARS- 28 CoV-2 genomes that we surveyed from the GISAID public repository in early June 2020 contained the 29 G614 variant (Shu and McCauley, 2017) . Previously, Cardozo and colleagues reported a correlation 30 between the prevalence of the G614 variant and the case-fatality rate in individual localities using viral 31 genomes available through early April 2020 (Becerra-Flores and Cardozo, 2020) . Using a ~10-fold larger 32 dataset, we found a smaller yet significant positive correlation between the prevalence of G614 in a country 33 with its case-fatality rate (r = 0.29, p = 0.04) (Figure 1b) . There has been little consensus on the potential 34 function of this mutation and whether its spread may or may not be due to a founder effect (Bhattacharyya 35 et al., Dorp et al., 2020) . Recently, two separate groups at the University of Sheffield and at the 36 University of Washington have found that in COVID-19 patients there is a ~3-fold increase in viral RNA 37 during quantitative PCR-based testing for those patients with the G614 variant (Korber et al., 2020; Wagner 38 et al., 2020) (Figure 1c, d) . Although there is a consistent difference in qPCR amplification between the 39 sites (~5 Ct) potentially due to different sampling procedures, RNA extraction methods, qRT-PCR reagents 40 or threshold cycle settings (Figure 1c) , the difference in amplification (DDCt) between G614 and D614 41 variants is remarkably consistent (1.6 Ct for Sheffield, 1.8 Ct for Washington), suggesting that this may be 42 due to a biological difference between COVID-19 patients with specific Spike variants (Figure 1d ). 43 Given these findings, we wondered whether the G614 variant may confer some functional 44 difference that impacts viral transmission or disease severity. To address this question, we used a 45 pseudotyped lentiviral system similar to those developed previously for SARS-CoV-1 (Moore et al., 2004) . 46 Using site-directed mutagenesis and a human-codon optimized SARS-CoV-2 spike coding sequence 47 (Shang et al., 2020) , we constructed EGFP-expressing lentiviruses either lacking an attachment protein or 48 pseudotyped with D614 Spike or G614 Spike (Figure 2a) . After production and purification of these viral 49 particles, we transduced human cell lines derived from lung, liver and colon. Others have observed 50 increased S-virus transduction in cells that overexpress the angiotensin-converting enzyme 2 (ACE2) 51 receptor (Li et al., 2003; Moore et al., 2004) ; we also found that S-virus is much more efficient at 52 3 transducing human cell lines when the human ACE2 receptor is overexpressed (Supplementary Fig. 1) . 53 Given this, for two of the human cell lines (A549 lung and Huh7.5 liver), we overexpressed the ACE2 54 receptor to boost viral transduction. After transduction with 4 different viral volumes, we waited 3 days and then performed flow 56 cytometry to measure GFP expression (Figure 2b) . We found in all 3 human cell lines at all viral doses 57 that G614 S-Virus resulted in a greater number of transduced cells than D614 S-virus (Figure 2c) . 58 Lentivirus lacking an attachment protein resulted in negligible transduction (Figure 2c) . With the G614 59 Spike variant, the maximum increase in viral transduction over the D614 variant was 2.4-fold for Caco-2 60 colon, 4.6-fold for A549-ACE2 lung, and 7.7-fold for Huh7.5-ACE2 liver (Figure 2d ). To control for any 61 potential differences in viral titer, we also measured viral RNA content by qPCR. We observed only a small 62 difference between D614 and G614 pseudotyped viruses using 2 independent primer sets (average of 7% 63 higher viral titer for D614), which may result in a slight underestimation of the increase in transduction 64 efficacy of the G614 pseudotyped virus (Supplementary Fig. 2) . 65 We next sought to understand the mechanism through which the G614 variant increases viral 66 transduction of human cells. Like SARS-CoV-1, the SARS-CoV-2 Spike protein has both a receptor-67 binding domain and also a hydrophobic fusion polypeptide that is used after binding the receptor (e.g. 68 ACE2) to fuse the viral and host cell membranes (Heald-Sargent and Gallagher, 2012) (Figure 3a) . Initially 69 we hypothesized that the increased viral transduction of the G614 variant may be due to enhanced binding 70 of the ACE2 receptor. To determine if greater transduction efficiency results from increased affinity of 71 Spike G614 to its receptor, we used bio-layer interferometry to measure the binding kinetics of the Spike 72 protein with and without the variant. We observed similar binding profiles of soluble D614 and G614 Spike 73 to immobilized hACE2 (Figure 3b, c) binding affinities of the Spike D614 -ACE2 interaction (Walls et al., 2020; Yi et al., 2020) , and is similar 77 4 for the Spike G614 variant. This suggests our observed transduction phenotype is independent of Spike 78 protein affinity for the ACE2 receptor. In order for SARS-CoV-2 to enter cells, the Spike protein must be cleaved at two sites by host 80 proteases. It is thought that Spike must first be cleaved into S1 and S2 fragments, which exposes another 81 cleavage site (Bestle et al., 2020; Hoffmann et al., 2020) . The second cleavage event (creating the S2' 82 fragment) is thought to enable membrane fusion with the host cell. We transfected both D614 and G614 83 Spike variants into human HEK293FT cells to see if Spike cleavage might differ between these variants. Both constructs were tagged at their C-termini with a C9 tag to visualize full-length, S2, and S2' fragments 85 via western blot (Figure 3d) . To measure cleavage, we quantified the ratio of cleaved Spike (S2 + S2') to 86 full-length Spike (Figure 3e ). We found that the G614 variant is ~2.5-fold more resistant to cleavage in the 87 host cell than the D614 variant (Figure 3f ). This suggests that the 2.4-to 7.7-fold increased transduction 88 observed with G614 S-virus (Figure 2d ) may be due to superior stability and resistance to cleavage of the 89 G614 variant during Spike protein production and viral capsid assembly in host/producer cells. Previous work showed that cleavage by the host protease furin at the Spike S1/S2 site in SARS- CoV-2 is essential for cell-cell fusion and viral entry (Hoffmann et al., 2020) . To test for differences in 92 furin-mediated cleavage, we performed in vitro digestion of both Spike variants after pull-down. We 93 immunoprecipitated D614 and G614 Spike protein from HEK293FT cell lysates and then performed on-94 bead digestion using different concentrations of purified furin protease. Over a range of furin 95 concentrations, we found that the G614 variant was more resistant to cleavage than the D614 variant 96 (Supplementary Fig. 3) . Importantly, the cleaved S2 and S2' fragments might still be incorporated into 97 new virions since they contain the required C-terminal transmembrane domain; however, they cannot 98 functionally bind receptor due to lack of a N-terminal receptor binding domain (Figure 3a) . Thus, the 99 greater fraction of uncleaved G614 Spike may allow each newly-assembled virion to include more receptor 100 binding-capable Spike protein. Given the global efforts underway to develop a COVID-19 vaccine, we also sought to understand To understand if the observed change in transduction efficiency that we found with our 115 pseudotyped lentivirus also impacts full SARS-CoV-2 virus, we sought to develop an isogenic system for 116 testing the Spike variant. Naturally-occurring isolates from patient samples that carry the Spike variant also 117 carry a linked mutation in ORF1b, which makes it challenging to perform this experiment in an isogenic 118 fashion. In lieu of a reverse genetics system to generate a SARS-CoV-2 variant and building on the 119 observation by several groups that most cell lines require ACE2 overexpression for efficient SARS-CoV-2 120 infection (Hoffmann et al., 2020; Ou et al., 2020; Shang et al., 2020; Ziegler et al., 2020) , we developed a 121 novel trans-complementation assay in which we co-transfect either D614 or G614 Spike along with human 122 ACE2 into HEK293T cells (Figure 4a ). Twenty-four hours later, these cells were infected with SARS- CoV-2 at a low multiplicity of infection (MOI): In this manner, only transfected cells, which express ACE2 124 (and one of the Spike variants), can be readily infected by SARS-CoV-2. 125 We performed this experiment at two different MOIs (0.01 and 0.1) and measured differences in In summary, we have demonstrated that the recent and now dominant mutation in the SARS-CoV-135 2 spike glycoprotein D614G increases the efficiency of cellular entry for the virus across a broad range of 136 human cell types, including cells from lung, liver and colon. We demonstrated increased entry efficiency 137 using both a pseudotyped lentiviral model system and also replication-competent SARS-CoV-2 virus. Given the concordance between the pseudotyped lentiviral system and SARS-CoV-2 virus, this suggests 139 that changes in Spike protein are well represented using the pseudovirus, which should enable a much 140 broader group of laboratories to use and study Spike variants. 141 We also found that G614 Spike is more resistant to proteolytic cleavage during production of the 142 protein in host cells, suggesting that replicated virus produced in human cells may be more infectious due 143 to a greater proportion of functional (uncleaved) Spike protein per virion. Using bio-layer interferometry 144 with purified Spike and ACE2 proteins, we showed that there is no difference in binding kinetics with the 145 ACE2 receptor resulting from the D614G mutation. Since our initial preprint, several other groups have now confirmed that the D614G results in 147 greater infection efficiency Ozono et al., 2020; Yurkovetskiy et al., 2020; Zhang et al., 148 2020) . Despite the emerging consensus the G614 results in faster viral spread (Korber et al., 2020) , it is still 149 uncertain whether this will have a clinical impact on COVID-19 disease progression. Two studies that have 150 examined potential differences in clinical severity or hospitalization rates did not see a correlation with 151 Spike mutation status (Korber et al., 2020; Wagner et al., 2020) , although one study found a small but not 152 significant enrichment of G614 mutations among intensive care unit (ICU) patients (Korber et al., 2020) . Given its rapid rise in human isolates and enhanced transduction across a broad spectrum of human cell 154 types, the G614 variant merits careful consideration by biomedical researchers working on candidate 155 7 therapies, such as those to modulate cellular proteases, and on vaccines that deliver Spike D614 nucleic 156 acids or peptides. assays. X.G. analyzed SARS-CoV-2 genomes from patient isolates. All authors contributed to drafting and reviewing the manuscript, provided feedback and approved the manuscript in its final form. For temporal tracking of D614G mutations in SARS-CoV-2 genomes, we used the Nextstrain analysis tool (https://nextstrain.org/ncov) with data obtained from GISAID (https://www.gisaid.org/) (Hadfield et al., 2018; Shu and McCauley, 2017) . With the Nextstrain webtool, we visualized 3,866 genomes using the "clock" layout with sample coloring based on Spike 614 mutation status. All complete SARS-CoV-2 genomes submitted before June 2nd 2020 were obtained from GISAID. To express the D614 Spike, we used an existing CMV-driven SARS-CoV-2 plasmid (pcDNA3.1-SARS2-Spike, Addgene 145032) (Shang et al., 2020) . To express the G614 Spike, we cloned pcDNA3.1-SARS2-SpikeD614G using the Q5 site-directed mutagenesis kit (NEB E0554S) and the following primers: 5'-CTGTACCAGGgCGTGAATTGCAC-3' and 5'-CACGGCCACCTGGTTGCT-3'. To make spike-pseudotyped lentivirus, we co-transfected a d2EGFP-containing transfer plasmid Viral RNA was isolated from 100 mL of 100x-concentrated Spike D614 or G614 pseudotyped lentiviruses using 500 mL Trizol (Thermo 15596026) and following the Zymo Direct-zol RNA MicroPrep kit protocol. RNA was eluted with 15 mL RNase-free water. The RNA was then diluted 1:50 and 2 mL were used to perform a one-step qPCR protocol using Luna Universal One-step qPCR kit (NEB). Two primer sets were used: 5'-CGCTATGTGGATACGCTGC-3' and 5'-GCGAAAGTCCCGGAAAGGAG-3' that amplify WPRE, and 5'-CGTGCAGCTCGCCGACCAC-3' and 5'-CTTGTACAGCTCGTCCATGCC-3' that amplify EGFP. qPCR was performed following the Luna Universal One-step qPCR kit protocol on a ViiA 384-well qPCR machine. We plated 50,000 cells per well of a 48-well plate. The cells were transduced the following morning using the indicated pseudotyped lentiviral amounts plus media supplemented with polybrene 8 µg/mL to a final volume of 150 µL per well. The media was changed 8 hours post-transduction. The cells were analyzed by flow cytometry 72 hours post-transduction. To generate pLenti-ACE2-Hygro, we amplified human ACE2 (hACE2) from pcDNA3.1-ACE2 (Addgene 1786) and cloned it into a lentiviral transfer pLEX vector carrying the hygromycin resistance gene using Gibson Assembly Master Mix (NEB E2611L). A 2A epitope tag was added to hACE2 at the Cterminus. Huh7.5-ACE2 and A549-ACE2 cell lines were generated by lentiviral transduction of ACE2. The protocol for lentiviral production was the same as above except we used the common lentiviral pseudotype (VSV-g) using plasmid pMD2.G (Addgene 12259). Transduced cells were selected with hygromycin at 50 ug/mL for Huh7.5-ACE2 and 500 ug/mL for A549-ACE2 for 10 days before use. Cells were harvested and washed with Dulbecco's phosphate-buffered saline (Caisson Labs) twice. Cell acquisition and sorting was performed using a Sony SH800S cell sorter with a 100 µm sorting chip. We used the following gating strategy: 1) We excluded the cell debris based on the forward and reverse scatter; 2) Doublets were excluded. For all samples, we recorded at least 5000 cells that pass the gating criteria described above. Gates to determine GFP+ cells were set based on control GFP-cells, where the percent of GFP+ cells was set as <0.5% (background level). Flow cytometry analyses were performed using FloJo v10. The kinetics of the D614 and G614 Spike protein variants with hACE2 were analyzed using biolayer interferometry on an Octet system (ForteBio, Octet RED96). Recombinant His-tagged and biotinylated human ACE2 protein (Sino Biological, Cat # 10108-H08H-B) was immobilized on a Streptavidin (SA) coated sensor. Loaded sensors were dipped into recombinant SARS-Cov-2 His-tagged Spike protein (D614 or D614G, Sino Biological, Cat # 40591-V08H and 40591-V08H3). All proteins were diluted in kinetics buffer (0.1% w/v BSA, 0.02% Tween-20 in 1x PBS). Sensors were equilibrated in kinetics buffer for ten minutes at room temperature preceding data acquisition, and experiments were performed at 30 ℃. Prior to ligand load, a baseline level was established for 60 s. hACE2 was loaded onto the sensor at 2.5μg/mL for 180 s, followed by a sensor wash (180 s) and a second baseline establishment (60 s) in kinetics buffer. Analyte in concentrations ranging from 200nM to 6.25nM were associated for 240 s and dissociated for 240 s. To determine KD values for each variant, a reference sensor with loaded ligand but no analyte was subtracted from the data before fitting. Data was fit using a 2:1 heterogeneous ligand model from association and dissociation rates. The analysis was carried out with Octet ForteBio Analysis 9.0 software. Using a 2:1 model, two KDs were obtained. The higher affinity KDs are consistent with previously published values, and the second KD values may represent a small amount of non-specific binding. HEK293FT cells were transiently transfected with equal amounts of spike or ACE2 vectors using PEI. Cells were collected 18-24 hours post-transfection with TrypLE (Thermo), washed twice with PBS (Caisson Labs) and lysed with TNE buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Nonidet P-40) supplemented with protease inhibitor cocktail (Bimake B14001) for 1 hour on a rotator at 4°C. Cells lysates were spun for 10 min at 10,000 g at 4°C, and protein concentration was determined using the BCA assay (Thermo 23227). Whole cell lysates (10 μg protein per sample) were denatured in Tris-Glycine SDS sample buffer (Thermo LC2676) and loaded on a Novex 4-12% Tris-Glycine gel (Thermo XP04122BOX). PageRuler pre-stained protein ladder (Thermo 26616) was used to determine the protein size. The gel was run in 1x Tris-Glycine-SDS buffer (IBI Scientific IBI01160) for about 120 min at 120V. Protein transfer was performed using nitrocellulose membrane (BioRad 1620112) using prechilled 1x Tris-Glycine transfer buffer (Fisher LC3675) with 20% methanol for 100 min at 100V. Membranes were blocked with 5% skim milk dissolved in PBST (1x PBS + 1% Tween 20) at room temperature for 1 hour. Primary antibody incubations were performed overnight at 4°C using the following antibodies: rabbit anti-GAPDH 14C10 (0.1 μg/mL, Cell Signaling 2118S), mouse anti-rhodopsin antibody clone 1D4 (1 μg/mL, Novus NBP1-47602) which recognizes the C9-tag added to the Spike proteins. Following the primary antibody, the blots were incubated with IRDye 680RD donkey anti-rabbit (0.2 μg/mL, LI-COR 926-68073) or with IRDye 800CW donkey anti-mouse (0.2 μg/mL, LI-COR 926-32212) for 1 hour at room temperature. The blots were imaged using Odyssey CLx (LI-COR). Band intensity quantification was performed by first converting Odyssey multichannel TIFFs into 16-bit grayscale image (Fiji) and the then selecting lanes and bands in ImageLab 6.1 (BioRad). In ImageLab, background subtraction was applied uniformly across all lanes on the same gel. We transiently transfected 10-cm plates with 80% confluent HEK293FT with 10 µg of either spike D614 or G614 using PEI. About 24 hours later, cells were collected and lysed with 800 mL TNE buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Nonidet P-40) supplemented with protease inhibitor cocktail (Bimake B14001) for 1 hour on a rotator at 4°C. Cells lysates were spun for 10 min at 10,000 g at 4°C. Spike was immunoprecipitated using 2 µg C9 antibodies (Novus NBP1-47602) per sample and incubated on a rotator at 4°C for at least 4 hours. Recombinant Protein G Sepharose 4B beads (Thermo 101241) were washed twice with 1 mL TNE buffer and then were added to the immunoprecipitated cell lysate and incubated on a rotor at 4°C for 2 hours. Beads were then spun using a prechilled centrifuge at 4°C for 1 min at 2,000 rpm and washed 3x with 1 mL TNE. After the final spin, the beads were washed twice with 1 mL of furin reaction buffer (100 mM HEPES pH 7.5, 1 mM CaCl2, 1 mM b-Mercaptoethanol). Finally, the beads were resuspended in 150 µL and split equally in microcentrifuge tubes. The indicated amount of furin protease (NEB P8077) was added per reaction tube in a final volume of 20 µL. The reaction was incubated at 37°C for 1 hour and was occasionally mixed by gently tapping the tubes. Then the beads were denatured in Tris-Glycine SDS sample buffer (Thermo LC2676) and incubated at 95°C for 5 min. Samples were then loaded on a Novex 4-12% Tris-Glycine gel (Thermo XP04122BOX). Western blotting was performed as described above using mouse anti-rhodopsin antibody clone 1D4 (1 μg/mL, Novus NBP1-47602) which recognizes the C9-tag added to the Spike proteins. Following the primary antibody, the blots were incubated with IRDye 800CW donkey anti-mouse (0.2 μg/mL, LI-COR 926-32212) for 1 hour at room temperature. The blots were imaged using Odyssey CLx (LI-COR). Band intensity quantification was performed by first converting Odyssey multichannel TIFFs into 16-bit grayscale image (Fiji) and the then selecting lanes and bands in ImageLab 6.1 (BioRad). In ImageLab, background subtraction was applied uniformly across all lanes on the same gel. Since 9mer epitopes are most commonly presented by MHC receptors (Sarkizova et al., 2020) , we constructed all possible 9mers surrounding the D/G 614 site in the Spike protein. We predicted binding affinities for 5 common HLA-A alleles and 7 common HLA-B alleles using the NetMHC 4.0 prediction webserver (Andreatta and Nielsen, 2016) (http://www.cbs.dtu.dk/services/NetMHC/). For each peptide, we computed the difference in predicted affinity between the D614 and G614 variant using R/RStudio and visualized them using the pheatmap R package. Data analysis was performed using R/Rstudio 3.6.1 and GraphPad Prism 8 (GraphPad Software Inc.). Specific statistical analysis methods are described in the figure legends where results are presented. Values were considered statistically significant for p values below 0.05. All plasmids cloned for this study will be available on Addgene . Tables and table legends Table 1 . (nM) (1/M * s) (1/s) (nM) (1/M * s) (1/s) Gapped sequence alignment using artificial neural networks: application to the MHC class I system SARS-CoV-2 viral spike G614 mutation exhibits higher case fatality rate TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets Global Spread of SARS-CoV-2 Subtype with Spike Protein Mutation D614G is Shaped by Human Genomic Variations that Regulate Expression of TMPRSS2 and MX1 Genes No evidence for increased transmissibility from recurrent mutations in SARS-CoV-2 Nextstrain: real-time tracking of pathogen evolution Ready, set, fuse! The coronavirus spike protein and acquisition of fusion competence A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells The D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity and decreases neutralization sensitivity to individual convalescent sera Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2 Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus Developing Covid-19 Vaccines at Pandemic Speed Retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme 2 Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV Naturally mutated spike proteins of SARS-CoV-2 variants show differential levels of cell entry A large peptidome dataset improves HLA class I epitope prediction across most of the human population Structural basis of receptor recognition by SARS-CoV-2 GISAID: Global initiative on sharing all influenza data -from vision to reality Comparing viral load and clinical outcomes in Washington State across D614G mutation Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies DNA vaccine protection against SARS-CoV-2 in rhesus macaques SARS-CoV-2 Spike protein variant D614G increases infectivity and retains sensitivity to antibodies that target the receptor binding domain The D614G mutation in the SARS-CoV-2 spike protein reduces S1 shedding and increases infectivity SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues We thank the entire Sanjana laboratory for support and advice. We are grateful to T.