Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

Keywords: ACE2-independent; COVID-19; RIG-I-like receptors; SARS-CoV-2; clathrin-mediated endocytosis; heparan sulfate; proteomics; spike variants; type I interferon; virus-host interactions.

Graphical abstract

Figure 1

The H522 cell line is null for ACE2 expression and is permissive to SARS-CoV-2 infection (A) Normalized RNA-seq-derived read counts for ACE2, TMPRSS2, FURIN, CTSB, CTSL, and NRP1 are given for the indicated cell lines. See also Figure S1 and Table S1. (B qRT-PCR expression normalized to 1 μg of input RNA for each cell line. Each bar represents mean; error bars indicate SEM (n = 3). (C) ACE2 immunoblot across 10 lung and head/neck cancer cell lines and Vero E6 cells (representative of n = 3). Relative ACE2 expression levels are indicated below the immunoblots. (D) qRT-PCR for cell-associated SARS-CoV-2 RNA at 4 and 72 hpi at an MOI of 0.015 or 0.15 (based on Vero titers). Error bars represent SEM (n = 3). ^∗p < 0.05 using a two-way ANOVA test and the Šidák correction for multiple comparisons. (E) qRT-PCR for cell-associated SARS-CoV-2 RNA (viral RNA [vRNA]) in H522 cells across various time points and MOIs. Error bars represent SEM (n = 2). (F) qRT-PCR for SARS-CoV-2 RNA in the supernatant of H522 cells across various time points and MOIs. Error bars represent SEM (n = 2). (G) Percentage of SARS-CoV-2-infected H522 and Vero E6 cells determined by fluorescence-activated cell sorting (FACS) for nucleocapsid (N)-positive cells across various time points and MOIs. Error bars represent SEM (n = 2). (H) Plaque assays on H522 and Vero cells using two viral dilutions (10⁻² and 10⁻¹). Data are representative of n = 3 experiments. (I) Representative images of H522 cells infected with SARS-CoV-2 at an MOI of 1. H522 cells were fixed and stained for SARS-CoV-2 RNA (green) and N protein (red) at 4 and 96 hpi and imaged by confocal microscopy (representative of n = 2). See also Figure S2. (J) Representative images using transmission electron microscopy (TEM) on Vero E6 and H522 cells infected with SARS-CoV-2 (MOI of 0.1 PFU/cell and 24 hpi for Vero, MOI of 1 PFU/cell and 96 hpi for H522).

Figure 2

The SARS-CoV-2 S protein is necessary for viral entry in H522 cells, and the E484D mutation enhances infection (A) Representative immunoblot showing ACE2 expression and vinculin as the loading control in Vero E6, H522, H522-ACE2, basal HBEC, and basal HBEC-ACE2 cells. (B) SARS-CoV-2 (MOI of 0.1 equivalent) was pre-treated with increasing concentrations of S mAb (2B04) for 1 h and used to infect cells in the presence of the mAb. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 24 hpi and was normalized to mock-treated cells (n = 3). (C) SARS-CoV-2 was pre-treated with increasing amounts of ACE2-Fc for 1 h as in (A) and used to infect cells in the presence of ACE2-Fc. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 24 hpi and was normalized to mock-treated cells (n = 3). (D) Representative images of cells infected with VSV-SARS-CoV-2-S_Δ21 at 0 and 8 hpi (n = 3). Percentages of GFP-positive cells seeded in triplicate were quantified over time, with the shaded gray region indicating SD. (E) Quantification of vRNA in H522 cells infected with WT SARS-CoV-2 and SARS-CoV-2 containing the E484D S substitution. (F) Percentage of GFP-positive H522 cells quantified by FACS upon infection (24 hpi) with increasing MOIs of VSV-SARS-CoV-2-S_Δ21 containing the indicated S variants. (G) Relative titers of VSV-SARS-CoV-2-S_Δ21 bearing the indicated S variants on HEK293T-ACE2 cells (6 hpi). Titers were normalized relative to WT after quantification by FACS. (H) Representative images of H522 cells infected with the indicated variants of VSV-SARS-CoV-2-S_Δ21 at 0 and 24 hpi. GFP intensity was assessed as in (D). (I) Percentage of GFP-positive cells upon infection (48 hpi) of H522 cells with 150 μL (MOI of 15–30 IU/cell) of lentiviral pseudoparticles containing the indicated S variants. (J) Percentage of GFP-positive cells upon infection (48 hpi) of HEK293T-ACE2 cells, with the indicated volumes of lentiviral pseudoparticles bearing the indicated S variants. (K and L) SARS-CoV-2 (MOI of 0.1) was pre-treated with the indicated concentrations of anti-S mAbs for 1 h before inoculation of Vero E6 (K) or H522 cells (L) as in (B) (see also Table S2). Amount of cell-associated vRNA was quantified as above. RBD, receptor-binding domain; NTD, N-terminal domain. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 where significance was assessed by a one-way (I, K, and L) or two-way ANOVA (B, C, F, and J) and the Dunnett correction for multiple comparisons.

Figure 3

H522 cells are permissive to SARS-CoV-2 infection independent of ACE2 expression but require surface heparan sulfates (A) Cells were pre-treated with 20 μg/mL of the indicated antibodies for 1 h and infected with SARS-CoV-2 at an MOI of 0.1 in the presence of antibodies. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 72 hpi (n = 3). (B) Polyclonal populations of H522 and Calu-3 (ACE2^+/+ and ACE2^−/−) cells were infected with SARS-CoV-2 virus, and cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 4 and 72 hpi (n = 8). Error bars indicate the SEM. See also Figure S3. (C) Polyclonal populations of H522 and Calu-3 (ACE2^+/+ and ACE2^−/−) cells were pre-treated with 20 μg/mL of the indicated antibodies for 1 h and then infected with SARS-CoV-2 at an MOI of 0.1 in their presence. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 72 hpi (n = 3). Error bars indicate the SEM. See also Figure S3. (D) Monoclonal populations from H522 ACE2^+/+ (six clones), ACE2^−/− (two clones), and ACE2^+/− (one clone) were infected with SARS-CoV-2 at an MOI of 0.1, and cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 4 and 72 hpi (n ≥ 3). Error bars indicate the SEM. See also Figure S3. (E) H522 cells were transfected with non-targeting (NT) siRNAs or siRNAs specific against NRP1 and AXL 48 h prior to infection. Cells were infected with SARS-CoV-2 at an MOI of 1 IU/cell, and the cell-associated vRNA level assessed by qRT-PCR. See also Figure S3. (F) H522 cells were passaged in the absence or presence of 50 mM sodium chlorate to deplete heparan sulfates before infection with SARS-CoV-2 at an MOI of 1 IU/cell. Cell-associated vRNA levels were quantitated at 4 and 72 hpi (n = 5–9). (G) H522 cells were treated with a combination of heparinases I, II, and III from two vendors (Sigma and R&D Systems) for 90 min and then infected with SARS-CoV-2 at an MOI of 0.1 IU/cell. Cell-associated vRNA levels were quantitated at 72 hpi (n = 4). The gray dot indicates an outlier (Grubbs’ method, α = 0.0001). ^∗∗∗p < 0.001 using one-way ANOVA (G), two-way ANOVA and a Dunnett’s test (A), or Tukey’s correction for multiple comparisons (B, C, and F).

Figure 4

H522 infection by SARS-CoV-2 is dependent on clathrin-mediated endocytosis and endosomal cathepsins (A) H522 cells were pre-treated with bafilomycin A, SGC-AAK1-1, E64D, apilimod, or camostat mesylate for 1 h and then infected with SARS-CoV-2 at an MOI of 1 in the presence of the inhibitors. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR 24 hpi and normalized to DMSO-treated cells (n ≥ 3). See also Figure S4. (B) Immunoblot showing pAP2M1 (T156), AP2M1, and AAK1 levels in H522 cells infected with SARS-CoV-2 (representative of n = 2). pAP2M1 (T156) levels were normalized to total AP2M1 and set relative to the 4-h mock control. Quantification is shown. (C) Basal HBECs from five different donors were pre-treated with increasing concentrations of SGC-AAK1-1 for 2 h and then infected with SARS-CoV-2 in its presence. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 72 hpi and normalized to DMSO-treated cells. (D and E) SARS-CoV-2 (MOI of 0.1 for Vero E6 and 1 IU/cell for H522) was pre-incubated with a 1:40 dilution of convalescent sera and used to infect H522 and Vero E6 cells. Cell-associated vRNA levels were analyzed at 72 hpi for H522 and 18 hpi for Vero E6 cells. ^∗p < 0.05, ^∗∗∗p < 0.001 compared to mock using one-way ANOVA and Dunnett’s correction for multiple comparisons. (F and G) SARS-CoV-2 (MOI of 0.1 for Vero E6 and 1 IU/cell for H522) was pre-incubated with a 1:40 to 1:320 dilution of sera from vaccine recipients and used to infect H522 and Vero E6 cells. Cell-associated vRNA levels were analyzed at 72 hpi for H522 and 18 hpi for Vero E6 cells. ^∗∗∗p < 0.001 compared to the respective day 0 vaccinated sera at 1:40 dilution using two-way ANOVA and Tukey’s correction for multiple comparisons.

Figure 5

H522 transcriptomic response to SARS-CoV-2 infection (A) Experimental design of transcriptomic experiments, performed in duplicate. (B) Relative expression of SARS-CoV-2 RNA versus H. sapiens RNA from H522 (n = 2). (C) Principal-component analysis (PCA) of highly expressed genes from MOIs of 0.25 and 1 across all time points. (D) Volcano plot of gene expression changes comparing mock infection to 96 hpi of MOIs of 0.25 and 1. Selected changes in IFN response genes (purple) and SARS-CoV-2 genes (salmon) are highlighted. See also Table S3. (E) Hierarchical clustering of differentially expressed genes (DEGs) after infection. Genes were filtered for an absolute log₂ fold change >2 and adjusted p value <0.005 at any time point. (F) Log₂ fold changes of DEGs as grouped by clustering. The colored lines represent quantification of an individual gene whereas a solid black line represents the cluster mean. (G) Hypergeometric enrichment analysis of biological gene sets in the identified gene clusters (D and E). See also Table S4. (H) Rank-based gene set enrichment analysis. Gene sets were queried when identified by hypergeometric analysis RNA-seq (F) or proteomics data (see Figure 6E). Display indicates p-adjusted < 0.05. N.E.S., normalized enrichment score.

Figure 6

H522 infection with SARS-CoV-2 results in proteome changes within the type I IFN, cell cycle, and DNA replication pathways (A) Experimental design of proteomic experiments. Peptides labeled with TMT10 reagents were analyzed by liquid chromatography-mass spectrometry. (B) PCA of whole-cell proteomics of H522 cells infected with SARS-CoV-2 across a 4-day time course (n = 3). (C) Quantification of total ion intensities for SARS-CoV-2 proteins over time and normalized to the 4-h mock control. The shaded gray regions represent SEM. (D) Volcano plot of protein abundance at 96 hpi compared to the 96-h mock control. See also Table S5. (E) Differentially expressed proteins from (D) were clustered based on the Z score. (F) Quantification of total ion intensities normalized to the 4-h mock control for each protein across the seven identified clusters in (D). The colored lines represent quantification of an individual protein, whereas the solid black and dashed black lines represent the means of infected and mock samples, respectively. (G) Hypergeometric enrichment analysis from three different databases for each individual cluster in (D) (Hallmark, Reactome, Gene Ontology [GO]). Color represents significance (q-value); size indicates the percentage of the cluster represented in the pathway. See also Table S6. (H) Distribution of Pearson’s correlation coefficient between a gene’s transcript and protein log₂ fold change over 4-h mock control for all proteins and differentially expressed proteins. Correlations used the matching time points of 4, 24, 48, 72, and 96 hpi. (I) Rank-based gene set enrichment analysis. Differentially expressed proteins were ranked by their correlation to transcript levels. (J) Protein complexes of differentially expressed H522 and SARS-CoV-2 proteins associated with DNA replication and cell cycle checkpoint. Complexes and functions were extracted from the CORUM database. The colors correspond to the whole-cell proteomic clusters identified in (D). See also Figure S5. (K) Protein interaction network of differentially expressed H522 and SARS-CoV-2 proteins associated with the IFN response. Interactions were determined from the BioGRID multi-validated datasets. Interferon-related functions were extracted from GO terms in MSigDB. The colors correspond to the whole-cell proteomic clusters identified in (D). See also Figure S5.

Figure 7

MDA5 mediates the IFN response to SARS-CoV-2 infection (A) Immunoblot depicting the IFN response in H522 cells infected with SARS-CoV-2 over time (representative of n = 2). (B) H522 cells were either mock transfected or transfected with a NT siRNA 24 h prior to infection. qRT-PCR analysis of ISG expression in H522 cells infected with SARS-CoV-2 (MOI of 1) at 96 hpi is shown. (C) H522 cells were transfected with a NT siRNA or a panel of siRNAs targeting genes involved in RNA sensing 24 h prior to infection. ISG mRNA levels were detected by qRT-PCR in H522 cells infected with SARS-CoV-2 at 96 hpi. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 compared to NT using two-way ANOVA and Dunnett’s correction for multiple comparisons. See also Figure S6. (D) qRT-PCR for cell-associated SARS-CoV-2 RNA in H522 cells (96 hpi) following siRNA transfection as in (C).