A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models.

Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. From a screen of human airway derived cell lines that express varying levels of ACE2/TMPRSS2, we found a subset that express comparably high endogenous levels of ACE2 but surprisingly did not support SARS-CoV-2 replication. Here we report that this resistance is mediated by a basally active cGAS-STING pathway culminating in interferon (IFN)-mediated restriction of SARS-CoV-2 replication at a post-entry step. Pharmacological inhibition of JAK1/2, depletion of the IFN-α receptor and cGAS-STING pathway effectors substantially increased SARS-CoV-2 replication in these cell models. While depletion of cGAS or STING was sufficient to reduce the preexisting levels of IFN-stimulated genes (ISGs), SARS-CoV-2 infection in STING knockout cells independently induced ISG expression. Remarkably, SARS-CoV-2-induced ISG expression in STING knockout cell as well as in primary human airway cultures was limited to uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway, but not viral sensing or IFN production, in productively infected cells. Of note, SARS-CoV-2-infected primary human airway cells also displayed markedly lower levels of STING expression, raising the possibility that SARS-CoV-2 can target STING expression or preferentially infect cells that express low levels of STING. Finally, ectopic ACE2 overexpression overcame the IFN-mediated blocks, suggesting the ability of SARS-CoV-2 to overcome these possibly saturable blocks to infection. Our study highlights that in addition to viral receptors, basal activation of the cGAS-STING pathway and innate immune defenses may contribute to defining SARS-CoV-2 cellular tropism.

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

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.

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

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. We identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2. 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. 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.