TMEM106B-mediated SARS-CoV-2 infection allows for robust ACE2-independent infectionin vitrobut notin vivo

Abstract

ABSTRACTAngiotensin converting enzyme 2 (ACE2) serves as the primary entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, ACE2-independent entry has been observedin vitrofor SARS-CoV-2 strains containing the E484D amino acid substitution in the spike protein. In this study, we conducted a whole genome CRISPR-Cas9 knockout screen using a SARS-CoV-2 strain containing the spike-E484D substitution (SARS-CoV-2MA1) to identify the ACE2-independent entry mechanisms. Our findings revealed that SARS-CoV-2MA1infection in HEK293T cells relied on heparan sulfate and endocytic pathways, with TMEM106B emerging as the most significant contributor. While SARS-CoV-2MA1productively infected human brain organoids and K18-hACE2 mouse brains, it did not infect C57BL/6J orIfnar-/-mouse brains. This suggests that ACE2-independent entry via TMEM106B, which is a protein that is predominantly expressed in the brain, did not overtly increase the risk of SARS-CoV-2 neuroinvasiveness in wild-type mice. Importantly, SARS-CoV-2MA1did not replicate inAce2-/-mouse respiratory tracts. Overall, this suggests that robust ACE2-independent infection by SARS-CoV-2E484Dis likely a phenomenon specific toin vitroconditions, with no apparent clinical implications.