TMEM106B Knockout Cell Line (HEK293)

Angiotensin-converting enzyme 2 (ACE2) is the primary entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but ACE2-independent entry has been observed in vitro for strains with the spike-E484D substitution. Here, we conduct a whole-genome CRISPR-Cas9 knockout screen using SARS-CoV-2 mouse adapted 1 (SARS-CoV-2), which carries spike-E484D, to identify the ACE2-independent entry mechanisms. SARS-CoV-2 infection in HEK293T cells relies on heparan sulfate and endocytic pathways, with TMEM106B, a transmembrane lysosomal protein, the most significant contributor. While SARS-CoV-2 productively infects human brain organoids and K18-hACE2 mouse brains, it does not infect C57BL/6J or Ifnar mouse brains. This suggests that ACE2-independent entry via TMEM106B, which is predominantly expressed in the brain, does not overtly increase the risk of SARS-CoV-2 neuroinvasiveness in mice with endogenous Ace2 expression. Importantly, SARS-CoV-2 does not replicate in the Ace2 mouse respiratory tract. Overall, this suggests that robust ACE2-independent infection by SARS-CoV-2 is likely an in vitro phenomenon with no apparent implications for infection in vivo.

TMEM106B is an endolysosomal transmembrane protein not only associated with multiple neurological disorders including frontotemporal dementia, Alzheimer's disease, and hypomyelinating leukodystrophy but also potentially involved in COVID-19. Additionally, recent studies have identified amyloid fibrils of C-terminal TMEM106B in both aged healthy and neurodegenerative brains. However, so far little is known about physiological functions of TMEM106B in the endolysosome and how TMEM106B is involved in a wide range of human conditions at molecular levels. Here, we performed lipidomic analysis of the brain of TMEM106B-deficient mice. We found that TMEM106B deficiency significantly decreases levels of two major classes of myelin lipids, galactosylceramide and its sulfated derivative sulfatide. Subsequent co-immunoprecipitation assay showed that TMEM106B physically interacts with galactosylceramidase. We also found that galactosylceramidase activity was significantly increased in TMEM106B-deficient brains. Thus, our results suggest that TMEM106B interacts with galactosylceramidase to regulate myelin lipid metabolism and have implications for TMEM106B-associated diseases.

Transmembrane protein 106B (TMEM106B) has been identified as a functional receptor facilitating ACE2-independent SARS-CoV-2 entry. However, its role in supporting a broad range of SARS-CoV-2 variants, including the emerging Omicron BA.2.86 and JN.1 subvariants, remains to be determined. To address this question, we generated 293- and A549-derived TMEM106B knockout cell lines and assessed their ability to support viral entry for various SARS-CoV-2 variants (D614G, E484D, Omicron BA.1, BA.2, XBB.1.5, BA.2.86, and JN.1) using pseudoviral infection systems. We also examined the role of transmembrane protease serine 2 (TMPRSS2) and other type II transmembrane serine proteases (TTSP) in viral entry and syncytium formation. Our results showed that TMEM106B knockout significantly reduced viral entry across all tested variants. Additionally, overexpression of TMPRSS2, TMPRSS11F and TMPRSS13 in TMEM106B-expressing cells enhanced viral entry and syncytium formation, including BA.2.86 and JN.1 variants. Importantly, we identified two single-nucleotide polymorphisms (SNPs) that result in G2A and N151S variant, respectively, affects TMEM106B receptor function, indicating that selected genetic polymorphisms of TMEM106B gene may impact cell susceptibility to SARS-CoV-2 infection. These findings highlight TMEM106B as a functional receptor for SARS-CoV-2 across different variants, including the latest Omicron subvariants, and provide new insights for the therapeutic interventions targeting viral entry.