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ACE2-dependent and -independent SARS-CoV-2 entries dictate viral replication and inflammatory response during infection

Nature Cell Biology volume 26, pages 628–644 (2024)Cite this article

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Abstract

Excessive inflammation is the primary cause of mortality in patients with severe COVID-19, yet the underlying mechanisms remain poorly understood. Our study reveals that ACE2-dependent and -independent entries of SARS-CoV-2 in epithelial cells versus myeloid cells dictate viral replication and inflammatory responses. Mechanistically, SARS-CoV-2 NSP14 potently enhances NF-κB signalling by promoting IKK phosphorylation, while SARS-CoV-2 ORF6 exerts an opposing effect. In epithelial cells, ACE2-dependent SARS-CoV-2 entry enables viral replication, with translated ORF6 suppressing NF-κB signalling. In contrast, in myeloid cells, ACE2-independent entry blocks the translation of ORF6 and other viral structural proteins due to inefficient subgenomic RNA transcription, but NSP14 could be directly translated from genomic RNA, resulting in an abortive replication but hyperactivation of the NF-κB signalling pathway for proinflammatory cytokine production. Importantly, we identified TLR1 as a critical factor responsible for viral entry and subsequent inflammatory response through interaction with E and M proteins, which could be blocked by the small-molecule inhibitor Cu-CPT22. Collectively, our findings provide molecular insights into the mechanisms by which strong viral replication but scarce inflammatory response during the early (ACE2-dependent) infection stage, followed by low viral replication and potent inflammatory response in the late (ACE2-independent) infection stage, may contribute to COVID-19 progression.

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Fig. 1: SARS-CoV-2 NSP14 facilitates the activation of NF-κB signalling and inflammatory response.
Fig. 2: SARS-CoV-2 NSP14 upregulates NF-κB signalling by promoting phosphorylation of the IKK complex.
Fig. 3: SARS-CoV-2 ORF6 shuts down NF-κB signalling by blocking the nuclear translocation of p65.
Fig. 4: The expression of ACE2 is distinctly associated with productive viral replication and the inflammatory response in epithelial and myeloid cells.
Fig. 5: ACE2-dependent and -independent SARS-CoV-2 entries dictate the transcription of viral subgenomic RNA in epithelial and myeloid cells.
Fig. 6: Ectopic expression of ACE2 in myeloid cells confers SARS-CoV-2 productive replication but hampers the virus-induced inflammatory response.
Fig. 7: TLR1 is a critical entry factor responsible for SARS-CoV-2 entry and virus-induced inflammatory response in myeloid cells.
Fig. 8: TLR1-mediated SARS-CoV-2 entry in myeloid cells through the recognition and binding of E and M proteins.

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Data availability

RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession code GSE249500. All of the other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank Y. Chen and M. Li from the USC Libraries Bioinformatics Service for help with the bioinformatics analysis of the RNA-seq data. We thank Q. Zhu and J. Xu from Tsinghua University for help with the bioinformatics analysis of the bronchoalveolar lavage fluid macrophage bulk RNA-seq data. We thank W. D. Wallace and M.-L. Chen from the Translational Pathology Core of the USC for providing the autopsy specimens from patients with lung cancer and COVID-19. We thank N. J. Krogan, D. E. Gordon, J. Z. Guo and M. Soucheray from the University of California, San Francisco for providing the plasmids encoding 27 SARS-CoV-2 viral proteins. We thank W. Yuan from the USC for providing the plasmids encoding E proteins of other human coronaviruses. We also thank: P.-Y. Shi from the University of Texas Medical Branch for the generous gift of the SARS-CoV-2-mNeoGreen virus; L. Comai and J. Henley from the USC BSL-3 Core for support and resources; and K. O’Brien from USC Environmental Health and Safety for biosafety support. This work was in part supported by the USC Startup Fund, as well as grants from the NCI/NIH (R01CA101795 and R01CA246547) and Department of Defense Congressionally Directed Medical Research Programs (Breast Cancer Research Program (BC151081) and Lung Cancer Research Program (LC200368)) to R.-F.W.

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  1. These authors contributed equally: Tianhao Duan, Changsheng Xing, Junjun Chu.

Authors and Affiliations

  1. Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    Tianhao Duan, Changsheng Xing, Junjun Chu, Yang Du, Xin Liu, Chen Qian, Bingnan Yin, Helen Y. Wang & Rong-Fu Wang

  2. Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    Xiangxue Deng, Yuzhou Hu & Rong-Fu Wang

  3. Department of Pediatrics, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    Helen Y. Wang & Rong-Fu Wang

  4. Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    Rong-Fu Wang

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Contributions

R.-F.W. supervised the entire study. T.D., C.X., J.C. and R.-F.W. conceived of and designed the study. T.D. performed all of the experiments, with assistance from C.X., J.C., X.D., Y.D., X.L., Y.H., C.Q. and B.Y. in some of the experiments. C.X., J.C. and X.D. performed the SARS-CoV-2 infection studies in the BSL-3 and Animal BSL-3 facilities. T.D., C.X., J.C., H.Y.W. and R.-F.W. collected and analysed the data and wrote the manuscript with input from all of the authors.

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Correspondence to Rong-Fu Wang.

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Extended data

Extended Data Fig. 1 SARS-CoV-2 NSP14 promotes the virus-/TLR-induced inflammatory response.

(a) Quantitative PCR with reverse transcription (RT-PCR) analysis of proinflammatory relative cytokines genes (IL6, IL1B, TNF, IL8) of THP-1 cells with SARS-CoV-2 or VSV-GFP infection at an MOI = 1 for indicated time points. (b) Sixteen nonstructural proteins, four structural proteins, and nine accessory proteins of SARS-CoV-2 were delineated. (c) RT-PCR analysis of SARS-CoV-2 NSP14 in THP-1 or MDM transfected with NSP14-specific siRNAs or control scramble siRNA, followed by SARS-CoV-2 infection at an MOI = 1 for 48 hrs. (d) Expression of proinflammatory genes in THP-1 transfected with NSP14-specific or control scramble siRNA, followed by SARS-CoV-2 infection at an MOI = 1 for 48 hrs. (e) Expression of proinflammatory genes in doxycycline-inducible SARS-CoV-2 NSP14 expression THP-1 cells was measured after being treated with doxycycline (1 μg/ml) or PBS for 24 h, followed by LPS (1 μg/ml) stimulation at indicated periods. Data in (a, c-e) are plotted as mean ± SD (n = 3 independent samples). Statistical analyses were performed using one-way ANOVA followed by Dunnett’s post-test (c, d) or two-way ANOVA followed by Sidak post-test (e).

Source data

Extended Data Fig. 2 SARS-CoV-2 NSP14 promotes the phosphorylation of IKKs in an N7-MTase activity-independent manner.

(a) Co-immunoprecipitation and immunoassay of extracts of HEK293T cells transfected with the plasmids for Flag-tagged MyD88, TRAF6, TAK1 + TAB2, ΝΕΜΟ (inhibitor of NF-κB kinase subunit gamma (IKKγ)) or p65, together with NSP14. (b-c) The recombinant NEMO protein was directly incubated with recombinant NSP14 protein pre-bound on the anti-Flag-beads (b) or Strep-beads (c) overnight at 4 °C, followed by immunoprecipitation. (d, e) HEK293T cells were transfected with an NF-κB-luc, together with the vector for IKKα (d) and IKKβ (e), along with an empty vector or expression vector for NSP14 or its mutants. (f) HEK293T cells were transfected with an NF-κB-luc, together with vector for MyD88 and NSP14, along with DMSO or increasing amounts (wedge) of Nitazoxanide (1 nM, 10 nM, 100 nM) and Sinefungin (100 nM, 1 μM, 10 μM) treatment. (g) Expression of the proinflammatory genes in MDMs treated with DMSO (no wedge) or Nitazoxanide (100 nM) or sinefungin (10 μM), followed by SARS-CoV-2 infection at an MOI = 1 for 48 h. (h) Sequence alignment of NSP14 from seven pathogenic CoVs (NL63, 299E, OC43, HKU1, MERS-CoV, SARS-CoV, and SARS-CoV-2). (i) HEK293T cells were transfected with an NF-κB-luc, together with the vector for IKKα and the adjusted amount of expression vector for SARS-CoV-2 NSP14 or its mutants. (j) Confocal microscopy is shown in HEK293T cells co-transfected with BFP-tagged IKKα and GFP-tagged SARS-CoV-2 NSP14 or its point mutants (Scale bar: 10 μm). (k) The recombinant protein of GFP-IKKβ, along with or without NSP14 WT or its mutant, was directly incubated with recombinant Flag-IKKβ pre-bound on the anti-Flag beads overnight at 4 °C, followed by immunoprecipitation. (l) HEK293T cells were transfected with an NF-κB-luc, together with a vector for IKKα and increasing amounts (wedge) of an expression vector for SARS-CoV-2 ancestral NSP14 or Omicron variant NSP14. Data in (a, b, c, j, k) are the representative of multiple independent experiments, data in (d-g, i, l) are plotted as the mean ± SD (n = 3 independent samples). Statistical analyses were performed using one-way ANOVA followed by Dunnett’s post-test (d, e, l) or two-tailed Student’s t-test (f, g, i). ns, not significant.

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Extended Data Fig. 3 SARS-CoV-2 ORF6 attenuates the TLR-induced inflammatory response through its C-terminal tail.

(a) Western blot to show the expression of GFP-ORF6 in the THP-1 cells. (b) Production of cytokines TNFα and IL-6 in the culture medium of control or SARS-CoV-2 ORF6 THP-1 cells were measured after indicated TLRs ligands treatment for 24 h. (c) Co-immunoprecipitation and immunoassay of extracts of HEK293T cells transfected with the plasmids for Flag-tagged MyD88, TRAF6, TAK1, IKKα, IKKβ, NEMO (IKKγ) or p65, together without or with an expression vector for SARS-CoV-2 ORF6. (d) The fluorescence intensity distribution of GFP signaling and Hochest signaling on the defined red lines in Fig. 3c (GFP-ORF6) were quantified using the NIS-Elements Imaging Software. The red dotted line box marked the fluorescence intensity located on the nuclear membrane. (e) Alignment of representative sequences across different sarbecoviruses species. The C-terminal tail of ORF6 is highly conserved, as illustrated here by WebLogo (http://weblogo.berkeley.edu). (f) HEK293T cells were transfected with an NF-κB-luc, together with the vector for p65, along with an empty vector or adjusted amount of expression vector for SARS-CoV-2 ORF6 or its mutants. (g) The fluorescence intensity distribution of GFP signaling and Hochest signaling on the defined red lines in Fig. 3c (GFP-ORF6 M58A) were quantified using the NIS-Elements Imaging Software. The red dotted line box marked the fluorescence intensity located on the nuclear membrane. (h) HEK293T cells were transfected with plasmids for p65, Myc-KPNA2, HA-KPNB1 and Flag-Nup98 in the indicated combinations, together with or without an expression vector for GFP-tagged SARS-CoV-2 ORF6. After transfection, whole-cell lysates were collected and used for immunoprecipitation with anti-Flag beads. Subsequently, immunoblot analysis was carried out using the specified antibodies. Data in (a, c) are the representative of multiple independent experiments, data in (b) and (f) are plotted as the mean ± SD (n = 3 independent samples). Statistical analyses were performed using two-way ANOVA followed by Sidak post-test (b) or one-way ANOVA followed by Dunnett’s post-test (f).

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Extended Data Fig. 4 SARS-CoV-2 ORF6 downregulates genes associated with the TLR signaling pathway and the NF-κB signaling pathway in myeloid cells.

(a) KEGG pathway enrichment analysis shows that the differentially expressed genes (DEGs) were enriched in multiple innate immune signaling pathways. (b) Gene-set enrichment analysis (GSEA) of genes with differential expression in ORF6-electroporated THP-1 cells compared to EV-electroporated control cells 4 hours after Pam3CSK4 stimulation. (c) The volcano plot displays DEGs when comparing ORF6-electroporated THP-1 cells to EV-electroporated control cells 4 hours after Pam3CSK4 stimulation. The p-value and fold change are calculated using gene-specific analysis (GSA) with the default settings of Partek Flow. (d) The heatmap shows some downregulated DEGs in the TLR and NF-κB signaling pathway.

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Extended Data Fig. 5 Myeloid cells are not efficiently ‘infected’ but are highly activated by SARS-CoV-2.

(a) Quantitative PCR with reverse transcription analysis of SARS-CoV-2 S proteins in Calu-3, Caco-2, NCI-H1355, NHBE, Macrophage or MDM with SARS-CoV-2 infection at an MOI = 0.05 (Caco-2 and NCI-H1355), or an MOI = 0.5 (Calu-3), or an MOI = 1 (NHBE, Macrophage, MDM) for indicated time points. (b) Quantitative PCR with reverse transcription analysis of SARS-CoV-2 S proteins in mouse BMDM (Bone marrow-derived macrophage) with SARS-CoV-2 infection at MOI = 1 for indicated time points. (c, d) Expression of proinflammatory genes in mouse BMDM was measured after SARS-CoV-2 infection at MOI = 1 for indicated time points. (e) Expression of the proinflammatory genes in Calu-3, NCI-H1355, Caco-2, NHBE, Macrophage or MDM with SARS-CoV-2 infection at an MOI = 0.05 (NCI-H1355 and Caco-2), or an MOI = 0.5 (Calu-3), or an MOI = 1 (NHBE, Macrophage and MDM) for indicated time points. Data in (a-e) are plotted as mean ± SD (n = 3 independent samples).

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Extended Data Fig. 6 The subgenomic RNA of SARS-CoV-2 could only be highly transcribed in ACE2-positive epithelial cells but not in ACE2-negative myeloid cells.

(a) Quantitative PCR with reverse transcription (RT-PCR) analysis of RNA of SARS-CoV-2 NSP1, NSP14, ORF6, S protein, and N protein or subgenomic RNA of ORF6, S, and N proteins in epithelial cells (Caco-2 and NCI-H1355) or myeloid cells (Macrophage and MDM) after SARS-CoV-2 infection at an MOI = 0.05 (epithelial cells) or an MOI = 1 (myeloid cells) for 24 h. (b) RT-PCR analysis of subgenomic RNA of SARS-CoV-2 S protein, N protein, and ORF6 in Calu-3, NHBE, Macrophage, and MDM after SARS-CoV-2 infection at an MOI = 0.5 (Calu-3) or an MOI = 1 (NHBE, Macrophage, MDM) for indicated time points. (c, d) Super-resolution microscopic (Airyscan) analysis of NSP12 (c) and S (d) proteins, along with CD68 in the lung autopsy sections from the COVID-19 patients or in normal lung sections from uninfected lung cancer patients (Ctrl). (Scale bars 20 μm). (e) Quantitative PCR with reverse transcription analysis of SARS-CoV-2 ORF6 subgenomic RNA in NCI-H1355 cells transduced with Ctrl-sgRNA or ACE2-sgRNA, followed with SARS-CoV-2 infection at an MOI = 1 for the indicated lengths of time. (f) Quantitative PCR with reverse transcription analysis of SARS-CoV-2 ORF6 subgenomic RNA in Caco-2 cells transduced with Ctrl-sgRNA or ACE2-sgRNA, followed with SARS-CoV-2 infection at an MOI = 1 for the indicated lengths of time. (g) Expression of the proinflammatory genes in NCI-H1355 transduced with Ctrl-sgRNA or ACE2-sgRNA was measured after being treated with SARS-CoV-2 infection at an MOI = 1 for the indicated lengths of time. (h) Expression of the proinflammatory genes in Caco-2 cells transfected with Ctrl-sgRNA or ACE2-sgRNA was measured after being treated with SARS-CoV-2 infection at an MOI = 1 for the indicated lengths of time. Data in (c, d) are the representative of multiple independent experiments, data in (a, b, e-h) are plotted as the mean ± SD (n = 3 independent samples). Statistical analyses were performed using two-way ANOVA followed by Sidak post-test (g, h).

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Extended Data Fig. 7 SARS-CoV-2 enters myeloid cells in a Spike- and ACE2-independent manner.

(a) Quantitative PCR with reverse transcription (RT-PCR) analysis of RNA of SARS-CoV-2 S, RdRp (NSP12), and NSP1 proteins in MDMs, THP-1WT or THP-1ACE2 cells pretreated with Human BD Fc Block™ (Fc Block), along with control IgG or anti-ACE2 blocking antibody (2 μg/ml or 20 μg/ml), followed by SARS-CoV-2 infection at an MOI = 1 for 48 h. (b) RT-PCR analysis of the RNA of SARS-CoV-2 S, RdRp (NSP12), and NSP1 proteins in MDM, THP-1WT or THP-1ACE2 cells pretreated with Fc blocker, along with control IgG or anti-S protein neutralizing monoclonal antibody (2 μg/ml or 20 μg/ml), followed by SARS-CoV-2 infection at an MOI = 1 for 48 h. (c) RT-PCR analysis of RNA of SARS-CoV-2 S protein in THP-1 WT cells, Macrophages, MDMs or THP-1ACE2 cells pretreated with DMSO, MM3122 (2 μM or 10 μM), E64d (2 μM or 10 μM), Chloroquine (2 μM or 10 μM) and EK1C4 (20 nM or 100 nM), followed by SARS-CoV-2 infection at an MOI = 1 for 48 h. (d) RT-PCR analysis of RNA of SARS-CoV-2 RdRp in THP-1 WT cells, Macrophages, MDMs or THP-1ACE2 cells pretreated with DMSO, MM3122 (2 μM or 10 μM), E64d (2 μM or 10 μM), Chloroquine (2 μM or 10 μM) and EK1C4 (20 nM or 100 nM), followed by SARS-CoV-2 infection at an MOI = 1 for 48 h. (e) RT-PCR analysis of RNA of SARS-CoV-2 S protein in THP-1 WT cells, or THP-1ACE2 cells pretreated with Fc blocker, along with normal serum or pooled convalescent serum (0.2 % or 1 %) from monkey (BEI Resources, NR-52401), followed by SARS-CoV-2 infection at an MOI = 1 for 48 h. (f) RT-PCR analysis of RNA of SARS-CoV-2 RdRp in THP-1 WT cells, or THP-1ACE2 cells pretreated with Fc blocker, along with normal serum or pooled convalescent serum (0.2 % or 1 %) from monkey, followed by SARS-CoV-2 infection at an MOI = 1 for 48 h. Data in (a-f) are plotted as the mean ± SD (n = 3 independent samples).

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Extended Data Fig. 8 ACE2-independent SARS-CoV-2 entry in myeloid cells may rely on its interaction with TLR1.

(a) Quantitative PCR with reverse transcription (RT-PCR) analysis of SARS-CoV-2 S and RdRp (NSP12) proteins in MDM cells with live SARS-CoV-2 or heat-inactivated SARS-CoV-2 infection at an MOI = 1 for indicated time points. (b) RT-PCR analysis of SARS-CoV-2 S and RdRp (NSP12) proteins in THP-1 cells with live SARS-CoV-2 or heat-inactivated SARS-CoV-2 infection at an MOI = 1 for indicated time points. (c) RT-PCR analysis of expression of inflammatory genes in THP-1 cells with live SARS-CoV-2 or heat-inactivated SARS-CoV-2 infection at an MOI = 1 for indicated time points. (d) RT-PCR analysis of SARS-CoV-2 S, RdRp (NSP12), and NSP1 proteins in mouse BMDM (Bone marrow-derived macrophage) with SARS-CoV-2 infection at an MOI = 1 for indicated time points. (e) HEK293T ACE2-/- cells were transfected with plasmids containing either a control empty vector (EV) or TLR1, followed by SARS-CoV-2 infection at MOI = 1 for indicated lengths of time and temperature. RT-PCR analysis was then performed to analyze the RNA of SARS-CoV-2 S and RdRp. (f) Lysates of HEK293T cells transfected with the plasmid for TLR1-Flag, together with the empty vector or expression vector of Strep-tagged SARS-CoV-2 structural proteins (E, M, N), were subjected to immunoprecipitation with anti-Strep beads and immunoblot analysis with anti-Flag. (g) Cell proliferation of THP-1 WT or TLR1-/- cells were continuously assessed by counting the cells using flow cytometry for five days. (h) Phase contrast micrographs show the morphology of THP-1 WT or TLR1-/- cells (Scale bars 100 μm). Data in (f) is the representative of multiple independent experiments, data in (a-e, g) are plotted as the mean ± SD (n = 3 independent samples). Statistical analyses were performed using two-way ANOVA followed by Sidak post-test (a-c).

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Extended Data Fig. 9 The entry of SARS-CoV-2 into myeloid cells relies on TLR1 rather than NF-κB signaling.

(a) Quantitative PCR with reverse transcription (RT-PCR) analysis of SARS-CoV-2 S and RdRp (NSP12) in MDMs transfected with Ctrl-siRNA or TLR1-siRNA, followed with SARS-CoV-2 infection at an MOI = 1 for the indicated lengths of time. (b) Ctrl-siRNA or TLR1-siRNA transfected MDMs were infected with SARS-CoV-2 at an MOI = 1 for the indicated lengths of time, and the lysates were harvested for immunoblot analysis with indicated antibodies. (c) Expression of the proinflammatory genes in MDMs transfected with Ctrl-siRNA or TLR1-siRNA was measured after being treated with SARS-CoV-2 infection at an MOI = 1 for the indicated lengths of time. (d) Expression of the proinflammatory genes in MDMs with ethanol or C25-140 (30 μM) treatment, followed by SARS-CoV-2 infection at an MOI = 1 for indicated time points. (e) RT-PCR analysis of SARS-CoV-2 S and RdRp (NSP12) proteins in MDMs pretreated with ethanol or C25-140 (30 μM), followed by SARS-CoV-2 infection at an MOI = 1 for indicated time points. (f) RT-PCR analysis of SARS-CoV-2 S and RdRp (NSP12) proteins in WT or Myd88-/- BMDM infected with SARS-CoV-2 infection at multiple MOIs for 24 h. Data in (a, c-f) are plotted as mean ± SD (n = 3 independent samples). Statistical analyses were performed using two-way ANOVA followed by Sidak post-test (a, c-f). ns, not significant.

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Extended Data Fig. 10 The Omicron variant and SARS-CoV-1 may trigger a hyperinflammatory response in myeloid cells via a mechanism similar to that of SARS-CoV-2.

(a) The recombinant TLR1 protein was directly incubated with Flag-Envelope (E) protein or Flag-Membrane (M) protein pre-bound on the anti-Flag beads overnight at 4 °C, followed by immunoprecipitation. (b) Expression of the proinflammatory genes in alveolar macrophage isolated from mouse bronchoalveolar lavage fluid (BALF) was measured after being stimulated with purified recombinant His-E protein for 4 hours. (c) Expression of the proinflammatory genes in WT or TLR1-/- THP-1 cells was measured after being stimulated with four different sources of purified recombinant E protein of SARS-CoV-2 (1 μg/ml or 2 μg/ml) or E protein of SARS-CoV-1 (equivalent molar quantity as SARS-CoV-2) for 4 hours. (d) Quantitative PCR with reverse transcription (RT-PCR) analysis of proinflammatory relative cytokines genes (IL6, IL1B, TNFA, IL8) of THP-1 cells with HCoV-OC43, HCoV-NL63, and HCoV-229E infection at an MOI = 1 for the indicated lengths of time. (e) The recombinant TLR1 protein was directly incubated with recombinant Flag-M proteins of indicated human coronavirus pre-bound on the anti-Flag beads overnight at 4 °C, followed by immunoprecipitation. (f, g) Expression of the proinflammatory genes in THP-1 (f) or MDM (g) after SARS-CoV-2 omicron variant (BA. 2) infection at multiple MOIs for 24 h. (h, i) RT-PCR analysis of total RNA of SARS-CoV-2 ORF6, S protein, and N protein or subgenomic RNA of ORF6, S, and N proteins in THP-1 cells (h) or MDMs (i) after SARS-CoV-2 omicron variant (BA. 2) infection at multiple MOIs for 24 h. Data in (a, e) are the representative of multiple independent experiments, data in (b-d, f-i) are plotted as mean ± SD (n = 3 independent samples). Statistical analyses were performed using two-way ANOVA followed by Sidak post-test (c).

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Supplementary Table 1. Primer sequences. Supplementary Table 2. siRNA sequences.

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Duan, T., Xing, C., Chu, J. et al. ACE2-dependent and -independent SARS-CoV-2 entries dictate viral replication and inflammatory response during infection. Nat Cell Biol 26, 628–644 (2024). https://doi.org/10.1038/s41556-024-01388-w

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