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

ABSTRACT

Background: Established in vitro models for SARS-CoV-2 infection are limited and include cell lines of non-human origin and those engineered to overexpress ACE2, the cognate host cell receptor. Although Calu-3, a human lung cell line which endogenously expresses ACE2, supports SARS-CoV-2 replication, they are significantly less permissive to infection than other models. Furthermore, ACE2 expression in the respiratory tract is low and emerging evidence suggests the utilization of alternative host cell receptors and attachment factors may compensate for low ACE2 expression levels in the lung. We identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2. Methods: A panel of 10 cell lines, with variable expression levels of ACE2 and TMPRSS2 were infected with SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020. Viral replication was monitored through assessment of cell-associated and cell-free viral RNA (vRNA) by QRT-PCR as well as N staining by FACS and in situ hybridization. Effect of blocking S protein by neutralizing antibodies and an ACE2-Fc decoy peptide, ACE2 blocking by a specific antibody, and ACE2 knockout by CRISPR on SARS-CoV-2 replication was determined by Q-RT-PCR for vRNAs. Various viral entry inhibitors were used to pathway of SARS-CoV-2 entry in H522 cells. RNA sequencing and proteomics was used to study the cell and innate immune responses in infected H522 cells. siRNA-mediated knockdown was utilized to further characterize the pathway of immune sensing. Results: Infection of H522 cells required the SARS-CoV-2 spike protein, though in contrast to ACE2-dependent models, spike alone was not sufficient for H522 infection. Temporally resolved transcriptomic and proteomic profiling revealed alterations in cell cycle and the antiviral host cell response, including MDA5- dependent activation of type-I interferon signaling. Focused chemical screens point to important roles for clathrin-mediated endocytosis and endosomal cathepsins in SARS-CoV-2 infection of H522 cells. Conclusion: These findings imply the utilization of an alternative SARS-CoV-2 host cell receptor which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.