Host and viral determinants for efficient SARS-CoV-2 infection of the human lung

Abstract

Understanding the factors that contribute to efficient SARS-CoV-2 infection of human cells may provide insights on SARS-CoV-2 transmissibility and pathogenesis, and reveal targets of intervention. Here, we analyze host and viral determinants essential for efficient SARS-CoV-2 infection in both human lung epithelial cells and ex vivo human lung tissues. We identify heparan sulfate as an important attachment factor for SARS-CoV-2 infection. Next, we show that sialic acids present on ACE2 prevent efficient spike/ACE2-interaction. While SARS-CoV infection is substantially limited by the sialic acid-mediated restriction in both human lung epithelial cells and ex vivo human lung tissues, infection by SARS-CoV-2 is limited to a lesser extent. We further demonstrate that the furin-like cleavage site in SARS-CoV-2 spike is required for efficient virus replication in human lung but not intestinal tissues. These findings provide insights on the efficient SARS-CoV-2 infection of human lungs.

Fig. 1. Heparan sulfate (HS) serves as an attachment factor for SARS-CoV-2 infection.

a–b Calu3 and Caco2 cells were inoculated with HS-pretreated SARS-CoV-2 or SARS-CoV at 0.2 MOI for 2 h at 37 °C. Cell lysates and supernatants were harvested at 24hpi and virus replication was determined with qRT-PCR (n = 3). c Schematic of the attachment assay. d Calu3 and Caco2 cells were inoculated with HS-pretreated SARS-CoV-2 or SARS-CoV at 0.2 MOI for 2 h at 4 °C. Cell lysates were harvested after the 2 h incubation and virus attachment was determined with qRT-PCR (n = 3). e–f Calu3 and Caco2 were pretreated with Heparinase I (4U/ml), Heparinase III (0.5U/ml) or both for 1 h at 37 °C, followed by virus infection at 0.2 MOI. Cell lysates and supernatants were harvested at 24hpi for qRT-PCR analysis (n = 3). g–h Human lung tissues were challenged with HS-pretreated SARS-CoV-2 or SARS-CoV at an inoculum of 1 × 10⁷ PFU/ml for 2 h. Tissues were harvested and homogenized at 24hpi for viral gene copy detection (n = 3). Data represented mean and standard deviations from the indicated number of biological repeats. Statistical significance between groups was determined with one way-ANOVA (a and b supernatant panels, e and f) or two-sided unpaired Student’s t test (a and b cell lysate panels, d and h). * represented p < 0.05, ** represented p < 0.01, *** represented p < 0.001, **** represented p < 0.0001. ns not significant. Source data are provided as a Source Data file.

Fig. 2. Sialic acids play differential roles on attachment and replication of SARS-CoV-2, SARS-CoV, and MERS-CoV.

a–c Calu3, Caco2, and VeroE6 cells were treated with neuraminidase (NA) for 1 h at 37 °C, followed by SARS-CoV-2, SARS-CoV, or MERS-CoV inoculation at 0.2 MOI for 2 h. Cell lysates and supernatants were harvested at 24hpi for qRT-PCR analysis and plaque assay titration (n = 6 for SARS-CoV-2 and SARS-CoV cell lysate or supernatant samples in a and b, n = 3 for other panels). d Schematic of attachment assay. e NA-pretreated Calu3 cells were incubated with the viruses at 4 °C for 2 h. Cell lysates were harvested at 2hpi for qRT-PCR analysis (n = 3). f NA- or mock-treated Calu3 or Caco2 cells were challenged with SARS-CoV-2 or SARS-CoV at 0.2 MOI. Infected cells were fixed at 16hpi and immunolabeled for virus nucleocapsid (N) protein (green) and DAPI (blue). Images were taken at ×20 magnification. Bars represented 50 µm. Data represented mean and standard deviations from the indicated number of biological repeats. The experiment in f was repeated three times independently with similar results. Statistical significance between groups was determined with two-sided unpaired Student’s t-test. * represented p < 0.05, ** represented p < 0.01, *** represented p < 0.001, **** represented p < 0.0001. ns not significant. Source data are provided as a Source Data file.

Fig. 3. SARS-CoV-2 partly overcomes sialic acid-mediated restriction in ex vivo human lung tissues.

a SLC35A1^WT and SLC35A1^KO 293 cells were fixed and stained with Sambucus Nigra Lectin (green) and DAPI (blue) for cell surface sialic acid detection. Bars represented 50 µm. b Schematic of attachment assay. c SLC35A1^WT and SLC35A1^KO 293 cells with or without hACE2 overexpression were inoculated with SARS-CoV-2 or SARS-CoV at 0.2 MOI for 2 h at 4 °C. Cell lysates were harvested at 2hpi for qRT-PCR analysis (n = 3). d–e Mock- or NA-treated ex vivo human lung tissues were infected with SARS-CoV-2 or SARS-CoV at an inoculum of 1 × 10⁷PFU/ml. Supernatants were collected at 2, 24, and 48hpi and tissue samples were collected at 48hpi for qRT-PCR analysis (n = 3). f Supernatants at 48hpi were titrated by plaque assays (n = 3). g Representative images of human lung tissues challenged with SARS-CoV-2 or SARS-CoV with or without NA treatment. Viral N proteins were detected with anti-SARS-CoV-2-N or rabbit anti-SARS-CoV-N immune serum (arrows). Bars represented 100 µm. Data represented mean and standard deviations from the indicated number of biological repeats. The experiments in a and g were repeated three times independently with similar results. Statistical significance between groups was determined with two way-ANOVA. * represented p < 0.05, ** represented p < 0.01, *** represented p < 0.001, **** represented p < 0.0001. ns not significant. Source data are provided as a Source Data file.

Fig. 4. Sialic acids present on ACE2 precluded perfect spike/ACE2-interaction.

a Schematic of the N-linked and O-linked ACE2 glycosylation mutants. b Western blot of ACE2 glycosylation mutants overexpressed in BHK21 cells. c BHK21 cells were transfected with the ACE2 mutants for 24 h before inoculating the cells with SARS-CoV-2-S-pseudovirus. Pseudovirus entry was determined at 24 h post inoculation (n = 3). d Entry of VSV-G-pseudovirus in BHK21 cells transfected with the ACE2 mutants (n = 3). e BHK21 cells expressing the ACE2 mutants were infected with SARS-CoV-2. Virus replication in the cell lysate samples at 24hpi was evaluated with qRT-PCR (n = 8 for EV, n = 12 for del N + O, n = 14 for other groups). f Virus replication in the supernatant samples at 24hpi was evaluated with qRT-PCR (n = 8 for EV, n = 12 for N690Q, n = 14 for other groups). g The binding affinity between SARS-CoV-2-S RBD and human ACE2 was determined with SPR. h The binding affinity between SARS-CoV-2-S RBD and NA-pretreated human ACE2 was determined with SPR. Data represented mean and standard deviations from the indicated number of biological repeats. The experiments in b, g, and h were repeated three times independently with similar results. Statistical significance between groups was determined with one way-ANOVA. * represented p < 0.05 and ** represented p < 0.01. ns not significant, EV empty vector. Source data are provided as a Source Data file.

Fig. 5. The inserted furin-like cleavage site or furin overexpression does not promote SARS-CoV-2 replication in BHK21 (non-permissive) and Huh7 (permissive) cells.

a Human ACE2 and furin overexpression in BHK21 cells was confirmed with western blot. b–c BHK21 cells with or without human ACE2 or furin overexpression were infected with SARS-CoV-2 or SARS-CoV. Cell lysates and supernatants were collected at 2 and 24hpi for qRT-PCR analysis (n = 3). d Amino-acid sequence alignment of residues around the S₁/S₂ cleavage site of SARS-CoV-2 reference strain (GenBank accession number: MN908947), SARS-CoV-2 HKU001a (MT230904), SARS-CoV-2 HKU001a S₁/S₂mut (MT621560), SARS-CoV GZ50 (AY304495), SARSr-CoV WIV1 (KF367457), and bat-CoV RaTG13 (MN996532). e Furin overexpression in Huh7 cells was confirmed with western blot. f–g Huh7 cells with or without furin overexpression were infected with SARS-CoV-2, SARS-CoV-2 S₁/S₂mut, or SARS-CoV. Cell lysates and supernatants were collected at 2 and 24hpi for qRT-PCR analysis (n = 3). Data represented mean and standard deviations from the indicated number of biological repeats. The experiment in a and e was repeated three times independently with similar results. Statistical significance between groups was determined with two way-ANOVA. ns not significant. Source data are provided as a Source Data file.

Fig. 6. The furin-like cleavage site in SARS-CoV-2 spike is required for efficient SARS-CoV-2 replication in human lungs.

a Seven cell types of human and non-human origin, including Caco2 (human intestine), Calu3 (human lung), Huh7 (human liver), CRFK (cat), RK13 (rabbit), PK15 (pig), and VeroE6 (monkey) were infected with SARS-CoV-2 or SARS-CoV-2 S₁/S₂mut at 0.2 MOI. Cell lysates were collected at 24hpi for viral genome copy analysis by qRT-PCR (n = 3). b The expression of TMPRSS2, cathepsin L, cathepsin B, and furin from Caco2, Calu3, Huh7, and VeroE6 were analyzed with qRT-PCR (n = 3). c Schematic of ex vivo human lung and intestinal tissue infection. d–g Human lung and intestine tissues were infected with SARS-CoV-2 or SARS-CoV-2 S₁/S₂mut. Cell lysate and supernatant samples were harvested at the indicated time points for qRT-PCR analysis (n = 3). h The expression of ACE2, TMPRSS2, cathepsin L, cathepsin B, and furin from human lung and intestinal tissues were determined with qRT-PCR (n = 8 for TMPRSS2 in intestine and n = 9 for other groups). Data represented mean and standard deviations from the indicated number of biological repeats. Statistical significance between groups was determined with two way-ANOVA (d and f), one way-ANOVA (b), or two-sided unpaired Student’s t test (a, e, g, and h). * represented p < 0.05, ** represented p < 0.01, *** represented p < 0.001, **** represented p < 0.0001. ns not significant. Source data are provided as a Source Data file.

Fig. 7. Schematic of the host and viral determinants for SARS-CoV-2 infection of the human lung as revealed in this study.

a SARS-CoV-2 can utilize cell surface heparan sulfate proteoglycan as an attachment receptor. b Sialic acids present on ACE2 precluded perfect spike/ACE2-interaction but SARS-CoV-2 is affected to a lesser extent by the sialic acid-mediated restriction in lung cells in comparison with SARS-CoV. c Furin cleavage of SARS-CoV-2 spike is essential for efficient SARS-CoV-2 replication in the human lungs, which is potentially due to the relatively low level of TMPRSS2 expression in the lungs.