TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to cause significant respiratory disease, particularly in vulnerable populations. Although ACE2 is the primary receptor for viral entry, previous studies have identified a naturally occurring, ACE2-independent entry pathway that depends on cell surface heparan sulfates and the E484D substitution within the receptor-binding domain of Spike (S). In this study, we expand the panel of airway-derived cell lines that support ACE2-independent, S-E484D-dependent entry and identify the host lysosomal transmembrane protein TMEM106B as the alternative receptor for infection. Specifically, we show that knockout of TMEM106B completely abolishes infection by SARS-CoV-2 SE484D in NCI-H522 and NCI-H661 cells. Ectopic expression of the luminal C-terminal domain (CTD) of TMEM106B-either alone or redirected to the plasma membrane - is sufficient to enable viral entry largely independent of cell surface heparan sulfates in non-permissive cells. We show that S-E484D is required for TMEM106B utilization, whereas other Variants of Concern substitutions at S-E484 are unable to mediate entry. Fc-TMEM106B-CTD fusion protein binds to a chimeric VSV-SARS-CoV-2 SE484D with a higher affinity than WT virus and blocks infection by SARS-CoV-2 SE484D, supporting a direct interaction between S-E484D and TMEM106B-CTD. Serial passaging experiments with VSV-SARS-CoV-2 SE484D identify a V951A mutation within the heptad repeat 1 region that enhances viral spread and fusogenicity in the ACE2-independent cell models. Together, these findings demonstrate that TMEM106B is a key mediator of a naturally occurring ACE2-independent pathway in multiple airway-derived cell lines and suggest that variation in the S protein can expand receptor usage by SARS-CoV-2.