RESUMO
Background: We previously screened 10 human lung and upper airway cell lines expressing variable levels of endogenous ACE2/TMPRSS2. We found that H522 human lung adenocarcinoma cells supported SARS-CoV-2 replication independent of ACE2, whereas the ACE2 positive cell lines were not permissive to infection. Type I/III interferons (IFNs) potently restrict SARS-CoV-2 replication through the actions of hundreds of interferon-stimulated genes (ISGs) that are upregulated upon IFN signaling. Here we report that a number of ACE2 positive airway cell lines are unable to support SARS-CoV-2 replication due to basal activation of the cGAS-STING DNA sensing pathway and subsequent upregulation of IFNs and ISGs which restrict SARS-CoV-2 replication. Method(s): SARS-CoV-2 WT strain 2019-nCoV/USA-WA1/2020 viral replication was detected through analysis of cell associated RNA. RNA sequencing was used to study the basal level of genes in the type-I IFN pathway in the 10 cell lines, which was further validated by western blotting and qRT-PCR. A panel of 5 cell lines, with varying expression levels of ACE2 and TMPRSS2, were pre-treated with Ruxolitinib, a JAK1/2 inhibitor. A siRNA-mediated screen was used to determine the molecular basis of basally high expression of ISGs in cell lines. CRISPR knockout of IFN-alpha receptor and cGAS-STING pathway components was conducted in parallel Results: Here we show that higher basal levels of IFN pathway activity underlie the inability of ACE2+ cell lines to support virus replication. Importantly, this IFN-induced block can be overcome by chemical inhibition and genetic disruption of the IFN signaling pathway or by ACE2 overexpression, suggesting that one or more saturable ISGs underlie the lack of permissivity of these cells. Ruxolitinib treatment increased SARS-CoV-2 RNA levels by nearly 3 logs in OE21 and SCC25. Furthermore, the baseline activation of the STING-cGAS pathway accounts for the high ISG levels and genetic disruption of the cGAS-STING pathway enhances levels by nearly 2 and 3 logs of virus replication in the two separate ACE2+ cell line models respectively. Conclusion(s): Our findings demonstrate that cGAS-STING-dependent activation of IFN-mediated innate immunity underlies the inability of ACE2+ airway cell lines to support SARS-CoV-2 replication. Our study highlights that in addition to ACE2, basal activation of cGAS-STING pathway, IFNs and ISGs may play a key role in defining SARS-CoV-2 cellular tropism and may explain the complex SARS-CoV- 2 pathogenesis in vivo.
RESUMO
Background: Lung cell lines to model SARS-CoV-2 replication in vitro are greatly limited hampering the rigorous study of SARS-CoV-2-host interactions. We analyzed a panel of 10 airway cell lines with various levels of ACE2 expression to identify models of SARS-CoV-2 infection. We found that none of the ACE2 expressing cell lines supported replication, whereas the H522 human lung adenocarcinoma cells were naturally permissive to SARS-CoV-2 infection despite detectable expression of ACE2. We confirmed that SARS-CoV-2 replication is indeed completely independent of ACE2 in H522s but dependent on heparan sulfates and the E484D substitution within the Spike. Further, we show that many of the ACE2 positive non-permissive cell lines express high basal levels of interferon-stimulated genes, which can be overcome by inhibition of the JAK/STAT pathway or by ACE2 overexpression. Together, our findings highlight ACE2-independent pathways can control the cellular tropism of SARS-CoV-2. Methods: Conventional molecular virology assays have been conducted to study the permissiveness of a panel of 10 cell lines expressing various levels of ACE2. ACE2 independence of SARS-CoV-2 replication was validated by antibody blocking, Fc-ACE2 decoy peptide and CRISPR-based approaches in H522 cells. RNA sequencing was used to study the basal level of genes in the type-I IFN pathway in the panel of 10 cell lines, which was further validated by western blotting and qRT-PCR. A panel of 5 cell lines, with varying expression levels of ACE2 and TMPRSS2, were pre-treated with Ruxolitinib, a JAK inhibitor, and infected with SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 and spike variants. Viral replication was detected through analysis of cell associated RNA Results: H522 human lung adenocarcinoma supports SARS-CoV-2 replication in a completely ACE2-independent manner. Transcriptomic analysis revealed basal high level of expression of interferon response pathway genes in some ACE2-positive cells recalcitrant to SARS-CoV-2 infection. Infection of OE21 and SCC25 cells required blocking of the IFN response pathway or ACE2 overexpression to allow SARS-CoV-2 infection. Conclusion: These findings suggest that SARS-CoV-2 replication can proceed in complete absence of ACE2 and that the innate immunity is a key determinant of SARS-CoV-2 cellular tropism. These findings may explain the complex SARS-CoV-2 pathogenesis in vivo as it shows that factors independent of ACE2 can define cellular tropism.
RESUMO
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.