LAUSR

Significance SARS-CoV-2 primarily targets the lung and enters the body through ACE2 receptors. However, the expression of ACE2 is extremely low in the airways. In this study, we utilized APEX2 proximity-labeling techniques and identified nonmuscle myosin heavy chain IIA (MYH9) as an ACE2 coreceptor to promote SARS-CoV-2 infection of human lung cells, as well as infection of pan-coronavirus. We demonstrated that MYH9 colocalized with SARS-CoV-2 S primarily at the membrane, which interacts with the S2 subunit and the S1-NTD subunit through its C-terminal domain, and enhances SARS-CoV-2 entry in an ACE2-dependent manner. Our results define MYH9 as a key host factor that facilitates SARS-CoV-2 and pan-coronavirus infection, which may serve as a potential target for future clinical intervention strategies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), binds to host receptor angiotensin-converting enzyme 2 (ACE2) through its spike (S) glycoprotein, which mediates membrane fusion and viral entry. However, the expression of ACE2 is extremely low in a variety of human tissues, especially in the airways. Thus, other coreceptors and/or cofactors on the surface of host cells may contribute to SARS-CoV-2 infection. Here, we identified nonmuscle myosin heavy chain IIA (MYH9) as an important host factor for SARS-CoV-2 infection of human pulmonary cells by using APEX2 proximity-labeling techniques. Genetic ablation of MYH9 significantly reduced SARS-CoV-2 pseudovirus infection in wild type (WT) A549 and Calu-3 cells, and overexpression of MYH9 enhanced the pseudovirus infection in WT A549 and H1299 cells. MYH9 was colocalized with the SARS-CoV-2 S and directly interacted with SARS-CoV-2 S through the S2 subunit and S1-NTD (N-terminal domain) by its C-terminal domain (designated as PRA). Further experiments suggested that endosomal or myosin inhibitors effectively block the viral entry of SARS-CoV-2 into PRA-A549 cells, while transmembrane protease serine 2 (TMPRSS2) and cathepsin B and L (CatB/L) inhibitors do not, indicating that MYH9 promotes SARS-CoV-2 endocytosis and bypasses TMPRSS2 and CatB/L pathway. Finally, we demonstrated that loss of MYH9 reduces authentic SARS-CoV-2 infection in Calu-3, ACE2-A549, and ACE2-H1299 cells. Together, our results suggest that MYH9 is a candidate host factor for SARS-CoV-2, which mediates the virus entering host cells by endocytosis in an ACE2-dependent manner, and may serve as a potential target for future clinical intervention strategies.