The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

Abstract

The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards the human respiratory tract. First, the S proteins exhibit an intrinsic temperature preference, corresponding with the temperature of the upper or lower airways. Pseudoviruses bearing the SARS-CoV-2 spike (SARS-2-S) were more infectious when produced at 33°C instead of 37°C, a property shared with the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV and MERS-CoV favored 37°C, in accordance with virus preference for the lower airways. Next, SARS-2-S-driven entry was efficiently activated by not only TMPRSS2, but also the TMPRSS13 protease, thus broadening the cell tropism of SARS-CoV-2. Both proteases proved relevant in the context of authentic virus replication. TMPRSS13 appeared an effective spike activator for the virulent coronaviruses but not the low pathogenic HCoV-229E virus. Activation of SARS-2-S by these surface proteases requires processing of the S1/S2 cleavage loop, in which both the furin recognition motif and extended loop length proved critical. Conversely, entry of loop deletion mutants is significantly increased in cathepsin-rich cells. Finally, we demonstrate that the D614G mutation increases SARS-CoV-2 stability, particularly at 37°C, and, enhances its use of the cathepsin L pathway. This indicates a link between S protein stability and usage of this alternative route for virus entry. Since these spike properties may promote virus spread, they potentially explain why the spike-G614 variant has replaced the early D614 variant to become globally predominant. Collectively, our findings reveal adaptive mechanisms whereby the coronavirus spike protein is adjusted to match the temperature and protease conditions of the airways, to enhance virus transmission and pathology.

Fig 1. Study panel of wild-type and S1/S2 site mutant spikes, and the D614G mutant of SARS-2-S.

(A) The CoV S protein contains two main cleavage sites: the S1/S2 site separates the S1 and S2 subunits, whereas S2′ cleavage liberates the fusion peptide (FP). (B) Structure of the SARS-CoV-2 spike trimer, based on PDB 6ZGE [90], in which we modelled the cleavage loop using SWISS-MODEL [91]. The amino acids shown in magenta were substituted or deleted, to create three S1/S2 loop mutants. The inset on the right shows residue D614, which forms a hydrogen bond with residue T859 in the S2 subunit of another protomer [22,23]. (C) Amino acid sequences around the S1/S2 and S2′ cleavage sites of the CoV spikes and mutant forms created in this study. Basic Arg (R) and Lys (K) residues are shown in bold.

Fig 2. S protein level and infectivity of pseudovirions are dependent on the production temperature.

(A) Experiment set-up. S-bearing pseudoviruses were produced in HEK293T cells at either 33°C or 37°C, and the released particles were pelleted to determine S content by western blot. In parallel, they were used to transduce HEK293T target cells expressing the appropriate receptor and TMPRSS2. (B) The graphs show S content relative to that of MLV-gag (mean ± SEM of four independently produced stocks). Representative blots show bands of uncleaved and cleaved S protein. (C) Particle infectivity was measured by luminescence read-out at day 3 post transduction (mean ± SEM of three independently produced stocks). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001 (two-tailed unpaired t-test; 37°C versus 33°C).

Fig 3. Temperature impacts particle infectivity of replicating SARS-CoV-2 virus.

Calu-3 cells were infected with SARS-CoV-2 virus bearing variation S^D614 or S^G614, and incubated at 33°C or 37°C. (A) Number of viral genome copies in the supernatant, determined by RT-qPCR. (B) Infectivity of produced virus particles, expressed as the ratio of infectious titer (determined by virus titration in Vero E6 cells) over viral genome copy number. ns, P > 0.05; *, P ≤ 0.05; ****, P ≤ 0.0001 (two-tailed unpaired t-test; 37°C versus 33°C). Results are the mean ± SEM; N = 4, performed in duplicate.

Fig 4. S1/S2 cleavage efficiency of WT and mutant spikes, expressed in HEK293T cells.

The SARS-2-S, SARS-S and MERS-S proteins were expressed in HEK293T cells and at 48 h post transfection, extracts were made for western blot analysis of the V5-tagged proteins in uncleaved (S0) or S1/S2 cleaved form. β-Actin served as loading control.

Fig 5. Entry of WT and S1/S2 mutant pseudoviruses in cells with different spike-activating proteases.

(A) Western blot detection of TMPRSS2, cathepsin B and cathepsin L proteases in human lung tissue, human nasal tissue (each from three donors), Calu-3 cells and Vero E6 cells (loading control: clathrin). (B) The broad serine protease inhibitor camostat prevents fusion activation by TTSPs like TMPRSS2, whereas E64d inhibits cathepsin B/L-mediated fusion after virus uptake by endocytosis. (C) The graphs show entry efficiency (top panels) or % inhibition relative to the DMSO solvent control (bottom panels), by 50 μM camostat in Calu-3 cells (left panel) or 50 μM E64d in Vero E6 cells (right panel). ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001, ****, P ≤ 0.0001 (unpaired two-tailed t-test; mutant forms versus WT). Results are the mean ± SEM from three experiments. nd, not determined.

Fig 6. Virus bearing variation S^G614 is more effective at using the cathepsin L route.

Control (Calu-3-EMPTY) and cathepsin L-expressing (Calu-3-CTSL) Calu-3 cells were (A) checked for expression of cathepsin L and (B) infected with SARS-CoV-2 virus bearing spike variation D614 or G614, in the presence of 20 μM E64d; 100 μM camostat; 100 μM camostat plus 20 μM E64d; or 10 μM GS-441524. At 3 days p.i., the inhibitory effect of the compounds on virus replication was quantified by immunofluorescence for dsRNA. **, P ≤ 0.01 (unpaired two-tailed t-test; SARS-CoV-2^S-G614 versus SARS-CoV-2^S-D614). Results are the mean ± SEM from three experiments.

Fig 7. Temperature and pH stability of pseudovirions with WT or mutant S proteins.

(A) The S-pseudotyped particles were incubated at the indicated temperatures for 1 h, followed by 2 h entry into HEK293T target cells and luminescence reading after 72 h. The Y-axis shows particle infectivity, relative to the condition incubated at 4°C (mean ± SEM, N = 3, performed in duplicate). Left: analysis of the four CoV pseudotypes; right: comparison of the S^D614 and S^G614 variants of SARS-2-S, and the three S1/S2 loop mutants.*, P ≤ 0.05; **, P ≤ 0.01 (Fisher’s LSD test; different strains or mutants versus SARS-2-S^D614 at each temperature). (B) The pseudovirus stocks were adapted to pH 6.3, 7.5 or 8.0 and incubated for 1 h at 4°C. Next, their infectivity was determined in HEK293T target cells, as above. The Y-axis shows particle infectivity (mean ± SEM, N = 3, performed in triplicate), relative to the optimal pH condition (i.e. giving the highest luminescence signal). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (unpaired two-tailed t-test; versus optimal pH condition).

Fig 8. Activation of pseudovirus entry by different human TTSPs.

(A) Experiment set-up. One day before transduction, HEK293T target cells were transfected with the appropriate receptor and one of the TTSPs. To block the cathepsin route, E64d was added at 2 h before and during transduction. (B) SARS-2-S activating capacity of the 18 human TTSPs. At the top of the graph, the four TTSP subfamilies are indicated. (C, D) The four TTSPs that proved active in panel B were evaluated for activation of wild-type and mutant forms of SARS-2-S (panel C), or SARS-S, MERS-S and 229E-S (panel D). An ordinary one-way ANOVA with Dunnett’s correction was used to analyze differences for empty versus TTSP plasmid conditions (panel B); and for SARS-2-S mutants versus D614 WT (panel C). Panel D: unpaired two-tailed t-test (WT versus mutant forms). *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. Results are the mean ± SEM from three experiments.

Fig 9. Knockdown of TMPRSS2 and TMPRSS13 reduces SARS-CoV-2 replication in Calu-3 cells.

(A) Western blot analysis confirmed expression of TMPRSS13 in Calu-3 cells, and in human lung tissue and nasal tissue (each from three donors). HEK293T cells transfected with TMPRSS13 served as positive control. (B) Calu-3 cells were transfected with siRNA for TMPRSS2, TMPRSS13 or a scrambled control, and 48 h later infected with SARS-CoV-2 virus carrying S^D614 or S^G614. At 72 h p.i., virus replication was quantified by immunofluorescence for dsRNA; the pictures show representative images. The bar graphs show the number of infected cells, quantified by high-content imaging and expressed relative to the scrambled siRNA control. Results are the mean ± SEM (N = 3 with five or six replicates). *, P ≤ 0.05; ***, P ≤ 0.001 (one-way ANOVA, followed by Dunnett’s test; specific siRNA versus scrambled control).