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. 2022 Dec 2;186(1):112–130.e20. doi: 10.1016/j.cell.2022.11.030

Figure 2.

Figure 2

SARS-CoV-2 attaches to the cilia during the initial stage of infection

(A) StcE mucinase reduces MUC1 mucin levels and increases virus infection efficiency. (Upper panel) Representative IF staining of MUC1, phalloidin in control-, and StcE (10 mg/mL)-treated (6 h) HNEs (Donor 6). Quantification of MUC1 from control- or drug-treated HNEs from Donors 6–8 (lower panel). Error bars represent mean ± SD (Lower panel) SARS-CoV-2-treated HNEs were treated with StcE (10 mg/mL, 6 h) before SARS-CoV-2 infection. Representative IF staining of SARS-CoV-2 SP, ACTUB, and phalloidin staining in control versus StcE-treated infected HNEs (Donor 6). Quantified percentages of SP-positive ciliated HNEs. Error bars represent mean ± SD (3,000–4,000 cells quantified from control- or drug-treated SARS-CoV-2-infected HNEs, Donor 6–8). ∗p < 0.05, paired, two-tailed Student’s t test. Each dot represents one donor.

(B and C) SARS-CoV-2 binds to the motile cilia during early viral infection. Mock-treated or SARS-CoV-2-infected HNEs (Donor3) (MOI 0.3) were fixed after 6 hpi. Representative images of SARS-CoV-2 virions attaching to motile cilia as observed by scanning electron microscope (SEM; B) or transmission electron microscopy (TEM; C). Arrowhead: virus particles. Similar results from Donors 4 and 5.

(D and E) SARS-CoV-2 neutralizing antibody inhibits SARS-CoV-2 attachment to cilia and decreases infected cells. (D) Representative IF images of SARS-CoV-2 SP with ACTUB after pre-treating HNEs (2 h) with control antibody (left) versus SP neutralizing antibodies (right) before SARS-CoV-2 inoculation (MOI 0.3). HNEs were fixed 6 hpi and stained for SARS-CoV-2 SP and ACTUB (left panel). Quantification is percentage of ciliated HNEs with cilia-attached SARS-CoV-2 (right panel). Error bars represent mean ± SD (2,000–3,000 cells quantified, HNEs from Donors 5–8). (E) Quantified percentages of SP-positive ciliated HNEs (48 hpi). Error bars represent mean ± SD (3,000–4,000 cells quantified, Donors 6–8). ∗∗p < 0.01, paired, two-tailed Student’s t test. Each dot represents one donor.

(F) Depleting cilia decreases HNE infection. Infected HNEs (MOI 0.3) were stained after 24 (left) and 48 (right) hpi. Representative IF staining for SARS-CoV-2 SP and ACTUB with phalloidin in shControl versus shCEP83 HNEs. Quantified percentages of SP-positive HNEs (right panel). Error bars represent mean ± SD (3,000–4,000 cells quantified from infected shControl and shCEP83 HNEs Donors 1–4).

(G) SARS-CoV-2 viral entry requires TMPRSS2 and ciliary dynein. SARS-CoV-2-infected HNEs were treated: DMSO, Ciliobrevin D (60 μM), camostat mesylate (25 μM), or HCQ (25 μM) 2 h before SARS-CoV-2 infection. Representative IF staining of SARS-CoV-2 SP, ACTUB, and phalloidin staining in DMSO- versus drug-treated infected HNEs at 24 hpi (left) and 48 hpi (right), Donor 6. Quantified percentages of SP-positive ciliated HNEs. Error bars represent mean ± SD (3,000–4,000 control- or drug-treated SARS-CoV-2-infected HNEs, Donor 6–8).

(H) Model for motile cilia during SARS-CoV-2 entry. (A and F) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, NS represents not significant, paired, two-tailed Student’s t test.

(G) ∗p < 0.05, ∗∗p < 0.01, NS represents not significant, paired, one-way ANOVA with Tukey’s post-test. Scale bars: 200 nm (C), 1 μm (B), 5 μm (A [top figure] and D), 20 μm (F), and 50 μm (A [down figure] and G). Each dot represents one donor. See Figure S2 and Table S1.