Panoply™ Human AXL Over-expressing Stable Cell Line

Although the role of ACE2 as a receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is well-established, studies have shown that ACE2 is expressed at extremely low levels in various human tissues, particularly the respiratory tract. Therefore, other host receptors and/or co-receptors may exist to facilitate SARS-CoV-2 entry into respiratory cells. Here, researchers discovered that the tyrosine protein kinase receptor UFO (AXL) specifically interacts with the N-terminal domain of the SARS-CoV-2 S protein. Using both a SARS-CoV-2 virus pseudotype and authentic SARS-CoV-2, they found that overexpression of AXL in HEK293T cells promoted SARS-CoV-2 invasion as effectively as overexpression of ACE2, while knockout of AXL significantly reduced SARS-CoV-2 infection in H1299 lung cells and human primary lung epithelial cells. Soluble recombinant human AXL was able to block SARS-CoV-2 infection in cells with high AXL expression. In bronchoalveolar lavage fluid cells from COVID-19 patients, AXL expression levels were significantly positively correlated with SARS-CoV-2 S protein levels. Therefore, these findings suggest that AXL is a novel candidate receptor for SARS-CoV-2 and may play an important role in promoting viral infection of the human respiratory system.

HEK293T cells typically exhibit extremely low expression levels of ACE2 and AXL, making them unsuitable for infection by SARS-CoV-2 pseudoviruses. However, the results showed that ACE2 overexpression significantly enhanced viral infection in HEK293T cells (Figure 1a, b), consistent with previous findings and confirming ACE2 as a receptor for SARS-CoV-2. Viral infection was significantly enhanced in AXL-overexpressing HEK293T cells, while overexpression of EGFR or LDLR did not have this effect (Figure 1a, b). Furthermore, MER and FGFR failed to promote viral entry, indicating that AXL's function in promoting viral entry is highly specific (Figure 1c-e). Two hours after infection with AXL-overexpressing HEK293T cells, SARS-CoV-2 viral pseudotype particles were observed on their surface, suggesting that AXL promotes the binding of SARS-CoV-2 viral pseudotypes to the cell surface (Figure 1f). In fact, 4 hours post-infection, the SARS-CoV-2 S protein partially co-localized with AXL and host cell endocytosis and vesicle transport markers (including Caveolin1 (CAV1), early endosome antigen 1 (EEA1), DCC interacting protein 13-α (APPL1), clathrin heavy chain 1 (CLTC), and Syntaxin-6 (STX6)) (Figure 1g). Knockout of the AXL gene in H1299 cells blocked the co-localization of the S protein with host cell endocytosis and vesicle transport markers, indicating that SARS-CoV-2 is internalized and penetrates early endosomes in a clathrin-dependent manner after binding to AXL. AXL not only promotes the entry of SARS-CoV-2 viral pseudotypes into cells but also promotes viral replication. Overexpression of AXL in HEK293T cells significantly increased the number of SARS-CoV-2 viral pseudovirus particles 24 hours post-infection (Figure 1h). To determine the region where AXL binds to the SARS-CoV-2 S protein, researchers constructed multiple truncated AXL mutants and found that the extracellular NTD (rather than the kinase domain) of AXL is responsible for the interaction with the SARS-CoV-2 S protein (Figure 1i) and the process of viral entry into host cells (Figure 1j, k). These results indicate that AXL, like ACE2, can effectively promote SARS-CoV-2 invasion.

Figure 1. AXL binds to SARS-CoV-2 S and facilitates SARS-CoV-2 entry into host cells. (Wang S, et al., 2021)