Identification of ACE2 modifiers by CRISPR screening

Abstract

SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. We identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2 in HuH7 cells. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual cell lines with disruption of SMAD4, EP300, PIAS1, or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry and suggest potential targets for therapeutic development.

SARS-CoV-2 remodels the Golgi apparatus to facilitate viral assembly and secretion

“…How SARS-CoV-2, an enveloped beta-coronavirus, modulates the endomembrane system for its assembly and exit is an important but unanswered question. To address this question, we performed morphological analyses of multiple subcellular organelles in the secretory pathway, including the ER, ERGIC, Golgi, autophagosome, endosome, and lysosome, in SARS-CoV-2-infected Huh7-ACE2 cells, a permanent cell line established from male hepatoma tissue that is often used in studies of SARS-CoV-2 infection (Sherman et al, 2021). After 24 h infection by SARS-CoV-2 (USA-WA1 strain, same as below unless specified), the ER structure marked by calreticulin was not dramatically altered (Table 1, Figure S1A-C).…”

“…ACE2-expressing Huh7 cells were sorted, enriched, and selected by flow cytometry (FACS) to guarantee a high infection rate of SARS-CoV-2 (Sherman et al, 2021). Huh7-ACE2 and Vero E6 (ATCC CRL 1586) cells were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen) at 37°C with 5% CO 2 .…”