key: cord-0846801-9m3lc4y8
authors: Ozono, Seiya; Zhang, Yanzhao; Ode, Hirotaka; Tan, Toong Seng; Imai, Kazuo; Miyoshi, Kazuyasu; Kishigami, Satoshi; Ueno, Takamasa; Iwatani, Yasumasa; Suzuki, Tadaki; Tokunaga, Kenzo
title: Naturally mutated spike proteins of SARS-CoV-2 variants show differential levels of cell entry
date: 2020-06-26
journal: bioRxiv
DOI: 10.1101/2020.06.15.151779
sha: 8e8ddd7d4f47059c6bafb8a4b192bffbdf578f91
doc_id: 846801
cord_uid: 9m3lc4y8

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is steadily mutating during continuous transmission among humans. Such mutations can occur in the spike (S) protein that binds to the angiotensin-converting enzyme-2 (ACE2) receptor and is cleaved by transmembrane protease serine 2 (TMPRSS2). However, whether S mutations affect SARS-CoV-2 infectivity remains unknown. Here, we show that naturally occurring S mutations can reduce or enhance cell entry via ACE2 and TMPRSS2. A SARS-CoV-2 S-pseudotyped lentivirus exhibits substantially lower entry than SARS-CoV S. Among S variants, the D614G mutant shows the highest cell entry, as supported by structural observations. Nevertheless, the D614G mutant remains susceptible to neutralization by antisera against prototypic viruses. Taken together, these data indicate that the D614G mutation enhances viral infectivity while maintaining neutralization susceptibility.

suggests that the expression of ACE2 alone is insufficient to support cell entry of SARS-CoV-2. 1 Because accumulated evidence has shown that the expression of TMPRSS2 enhances both 2 SARS-CoV and SARS-CoV-2 infection (5, 6), we next tested whether the coexpression of ACE2 3 and TMPRSS2 could mediate efficient infection of 293T cells with SARS2-S-pseudotyped 4 lentiviruses. Notably, the dual expression of ACE2 and TMPRSS2 markedly facilitated 5 SARS2-S-mediated cell entry while moderately enhancing entry by the SARS-S pseudovirus 6 ( Fig. 1A) . These results indicate that SARS2-S-mediated entry into cells is more highly 7 dependent on TMPRSS2 coexpression than that of SARS-CoV, suggesting that they may have 8 different cellular tropisms to some extent. 9 Nevertheless, ~25-fold differences in cell entry between SARS-S and SARS2-S 10 pseudoviruses were observed (Fig. 1A) , leading to the hypothesis that SARS2-S might be less 11 incorporated into lentiviral particles than SARS-S, probably due to reduced compatibility with 12 lentiviral particles. Compared with SARS-S, SARS2-S has one (alanine-to-cysteine at position 13 1247) and two (valine-to-isoleucine at position 1216 and leucine-to-methionine at position 1233) 14 amino acid differences in the cytoplasmic tail (CT) and transmembrane (TM) domain, 15 respectively ( Fig. S2A ). Thus, we created a SARS2-S C1247A mutant and a chimeric SARS2-S 16 harboring the TM/CT domains of SARS-S. All SARS2-S proteins showed comparable levels of 17 cell entry (Fig. S2B ) and actual virion incorporation (Fig. S2C) . These results indicate that the 18 significantly lower rate of cell entry of the SARS2-S pseudovirus was not due to differences in 19 the efficiency of S protein incorporation into virions but rather to the intrinsic nature of the 20 SARS2-S protein. 21 To further assess differences between the SARS-S and SARS2-S proteins, we next addressed 22 5 whether these S proteins might differ in their ability to utilize a given level of cell surface ACE2 1 or TMPRSS2. Based on lentiviral infection of 293T cells expressing a high and constant level of 2 ACE2 together with a range of expression levels of TMPRSS2 and vice versa, 3 SARS2-S-mediated infection required higher levels of cell-surface ACE2 and TMPRSS2 4 expression than SARS-S to attain maximum levels of infectivity ( Fig. 1B and 1C ). Therefore, it 5 is likely that SARS2-S is less adapted to ACE2 due to the inefficient usage of this host protein 6 and essentially requires sufficient levels of TMPRSS2 expression. 7 Next, we investigated using our assay system whether naturally occurring mutations in the S 8 protein affect the cell entry of SARS-CoV-2 We created plasmids expressing five different S 9 variants that were initially identified in China (H49Y (12)), Europe (V367F (13) and D614G 10 (14-16)), and the United States (G476S and V483A (13)) ( Fig. 1D ) and examined the effects of 11 these mutations on entry into cells expressing ACE2 and TMPRSS2, by comparison with that of 12 wild-type (WT) S protein. These naturally occurring S mutations resulted in reduced (G476S), 13 equal (V483A), or enhanced (D614G, V367F, and H49Y) cell entry. Remarkably, the D614G 14 mutant displayed the highest level of entry activity compared with that of WT S protein (Fig. 1E) . 15 This result is particularly important because the D614G mutation defines the clade A2a (also 16 called G) that is rapidly spreading worldwide, accounting for the great majority of isolates 17 (17) (18) (19) (20) . 18 To analyze differences among WT/mutant SARS2-S and SARS-S, we performed structural 19 analyses on complex models between ACE2 and these S proteins ( Fig. 2A and 2B ). Interestingly, 20 the SARS-S trimer showed an open conformation, providing a larger contact area in the 21 receptor-binding domain (RBD) that specifically interacts with ACE2; this might lead to higher 6 accessibility of the SARS-S RBD to ACE2 than that of SARS2-S. This finding is indeed 1 consistent with the recent report that the SARS-CoV-2 S protein has lower ACE2 binding 2 affinity than SARS-CoV S (21). It is also likely that to a large extent, the structures of SARS2-S 3 mutants reflect differential cell entry ( Fig. 2C ; see details in the legend). Notably, the aspartic 4 acid residue at position 614 located in the S1 subunit of the WT protein (D614) is able to form a 5 hydrogen bond with a threonine residue at position 859 (T859), as recently reported (19), and/or 6 a salt bridge with a lysine residue at position 854 (K854) located in the S2 subunit of the other 7 protomer (Fig. 2D ). This finding suggests in turn that the mutation of this residue to a glycine 8 (G614) can provide flexible space between two protomers due to the short side chain, allowing 9 the S1 subunit to be dissociated more smoothly from the S2 subunit, and/or likely providing 10 conformational flexibility to the overall structure of the S trimer, which might lead to improved 11 accessibility of ACE2 into the RBD. These results are fully consistent with the high entry 12 activity of the virus harboring this mutation (Fig. 1E ). 13 Given that the D614G S protein is biologically and structurally different from the WT protein, 14 we hypothesized that this mutation might affect the antigenicity of the S protein. To examine this 15 possibility, we performed neutralization assays to compare the neutralizing sensitivity of the WT 16 and D614G S proteins to anti-SARS-CoV-2 sera. For the neutralization assays, we utilized serum 17 samples derived from five patients confirmed to be infected with prototype viruses and a control 18 serum from a healthy donor. Regardless of serum concentration, the anti-SARS-CoV-2 patient 19 sera but not the control serum efficiently neutralized both viruses pseudotyped with the 20 SARS2-S WT and D614G mutant proteins (Fig. 3) . These results indicate that the D614G 21 mutation in the SARS2-S protein maintains neutralization sensitivity to the anti-SARS2-S 7 antibodies, i.e., its antigenicity per se. 1 In this study, we employed a novel entry assay system that we recently developed to quantify 2 cell entry of lentiviral pseudoviruses (10), in which we can precisely normalize viral input unlike 3 for retrovirus-or vesicular stomatitis virus-based pseudoviruses, leading to experimental 4 accuracy in determining levels of viral entry. By using this system, we first showed that 5 SARS2-S-mediated cell entry strongly depends on TMPRSS2 coexpression, without which that 6 of SARS-S still proceeds to some extent. Indeed, SARS-CoV-2 efficiently infects respiratory and 7 intestinal cells (9, 22) that coexpress ACE2 and TMPRSS2 (8), whereas SARS-CoV can target 8 multiple cell types in several organs (23) expressing ACE2, probably even without TMPRSS2. 9 We also found that SARS2-S-mediated cell entry is considerably lower than that of SARS-S, human to human than the prototype viruses, even though further investigations are necessary to 1 determine the correlation between viral infectivity and transmissibility among humans. In fact, 9 pandemic while SARS ended up with epidemics in the past could be, at least in part, that the S 1 protein, a key viral protein for the first step in transmission, of the causative agent SARS-CoV-2, 2 displays suboptimal levels of cell entry activity as shown in this study. Accordingly, this 3 phenomenon would occasionally result in inefficient replication among humans, presumably 4 leading to a higher rate of asymptomatic infection, as described above. In such cases, 5 unknowingly infected subclinical individuals could be virus spreaders who still have the 6 potential to cause lethal respiratory disease to others, as recently reported (25). Consequently, 7 during its continuous transmission from human to human, the Wuhan prototype might have 8 acquired more widely prevalent phenotypes represented by the A2a clade that harbors the 9 D614G mutation, possibly with enhanced speed of global transmission. If this scenario is correct, 10 fatal pathogens for which we should be concerned are those with such characteristics rather than 11 the obviously deadly viruses that can be readily detected. Although in the present case, it is likely 12 that the mutation did not influence the antigenicity of the S protein, we may need further 13 worldwide surveillance for virological changes of SARS-CoV-2 in the human population. pWPI-Luc2, using FuGENE 6 (Promega). Sixteen hours later, the cells were washed with 13 phosphate-buffered saline, and 1 ml of fresh complete medium was added. After 24 h, the 14 supernatants were harvested and treated with 37.5 U/ml DNase I (Roche) at 37°C for 30 minutes. 15 The lentivirus levels in viral supernatants were measured by the HiBiT assay, as previously 16 described (10). Briefly, a lentivirus stock with known levels of p24 antigen was serially diluted. (obtained in February 2020) and heat-inactivated at 56°C for 30 minutes. Two-fold serially 10 diluted sera were mixed with an equal volume of 1 ng of 24 antigen of the WT or D614G mutant 11 SARS2-S-pseudotyped virus and incubated at 37°C for 1 h. The mixture was added to 293T cells 12 transiently coexpressing ACE2 and TMPRSS2 (seeded into a 96-well plate). After 48 h, cells 13 were lysed and subjected to luciferase assays, as described above, to determine the levels of and SARS-CoV-2 (B) that bind to the host receptor ACE2 (cyan). One protomer of the S protein is shown as a ribbon in navy (S1 subunit), orange (receptor-binding domain (RBD) in S1 subunit), and green (S2 subunit), while two other protomers are shown as gray transparent surfaces and ribbons. The structures are viewed from two different angles. (C) Comparison between wild-type (WT) and mutant S proteins from globally spread SARS-CoV-2 variants. (Inset) Enlargement of the region in which each amino acid is mutated, with comparison with the WT S protein. H49Y; as the histidine at position 49 is located distant from the RBD and putative cleavage sites, the effect of this mutation on S's function is likely limited. V367F; the substitution from a valine to a phenylalanine at position 367 in the RBD introduces a larger side chain at a protomer-protomer interface, which might provide a more rigid RBD structure. G476S; the substitution at position 476 in the RBD results in a protruded surface, which appears to interfere with the ACE2-RDB interaction. V483A; both valine and alanine residues have short side chains, likely sharing similar phenotypes. D614G; details are depicted in Fig. 2D . Note that these structural bases are largely consistent with the results of cell entry activity shown in Fig. 1D . (D) Structural difference between WT and D614G SARS-CoV-2 S proteins. WT (left); an aspartic acid (D614) in the S1 subunit (navy) of a protomer binds to a threonine (T859) and/or a lysine (K854) in the S2 subunit (green) of the other protomer though electrostatic interaction between the pairs of these residues. D614G (right); the short nonpolar side chain of glycine (G614), which does not bind to T859 and K854, provides flexible space between the two protomers. The figures were drawn with PyMOL ver. 2.4 (https://pymol.org).

Lentiviruses pseudotyped with either the WT or D614G mutant SARS2-S were preincubated with two-fold serially diluted human sera (80-fold to 10, 240-fold) obtained from a healthy donor (Ctrl) or collected at 15-30 days postsymptom onset from confirmed case patients (#1 -#5) infected with the prototypic viruses. The mixture was used for infection of 293T cells coexpressing ACE2 and TMPRSS2, and cell entry levels of pseudoviruses in the presence of diluted patient sera were determined by luciferase assays. Representative data from two independent experiments are shown as percent neutralization (mean ± s.d., n = 3 technical replicates). Figure S1 . ACE2 expression alone is insufficient to support SARS2-S-mediated cell entry. To prepare Spseudotyped lentiviruses, 293T cells were transfected with the HiBiT-tagged lentiviral packaging plasmid, the firefly luciferase-reporter lentiviral transfer plasmid, and either a SARS-CoV S (SARS-S) or SARS-CoV-2 S (SARS2-S) expression plasmid. The viruses produced were assessed by HiBiT assays, and S-pseudotyped viruses normalized based on HiBiT activity were used for infection of 293T cells expressing the host receptor ACE2 only. Cell entry was determined by firefly luciferase activity in cell lysates. Data from three experiments are shown (mean ± s.d., n = 3 technical replicates). The p value was calculated using paired two-tailed Student's t-test, ***p < 0.001. Figure S2 . The lower level of SARS2-S-mediated entry is not due to differences in the efficiency of S incorporation into virions. (A) Amino acid sequence alignments of the C-terminal sequence of S proteins from SARS-CoV (Urbani strain) and SARS-CoV-2 (Wuhan-Hu-1 strain). Amino acid differences are boxed in several colors. EC, extracellular domain; TM, transmembrane domain; CT, cytoplasmic tail. SARS2-S-C1247A and SARS2-S-TM/CT1 were created by mutating a cysteine to an alanine at the CT (indicated by a red arrow) and by additionally mutating an isoleucine and a methionine to a valine and a leucine at the TM (indicated by blue arrows), respectively. (B) 293T cells were transfected with the HiBiT-tagged lentiviral packaging plasmid, the firefly luciferase-reporter lentiviral transfer plasmid, and the plasmid expressing either the SARS2-S wild-type (WT) protein or one of the two mutants (SARS2-S-C1247A or SARS2-S-TM/CT1). Viruses produced were assessed by HiBiT assays, and S-pseudotyped viruses normalized based on HiBiT activity were used for infection of 293T cells expressing ACE2 and TMPRSS2. Cell entry was determined by firefly luciferase activity in cell lysates. Data from three experiments are shown as a percentage of cell entry of the SARS-S-pseudotyped viruses (mean ± s.d., n = 3 technical replicates).

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