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.

严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 是 2019 年冠状病毒病 (COVID-19) 的病原体，通过其介导膜的尖峰 (S) 糖蛋白与宿主受体血管紧张素转换酶 2 (ACE2) 结合融合和病毒进入。然而，ACE2 在多种人体组织中的表达极低，尤其是在气道中。因此，宿主细胞表面的其他辅助受体和/或辅助因子可能导致 SARS-CoV-2 感染。在这里，我们使用 APEX2 邻近标记技术将非肌肉肌球蛋白重链 IIA (MYH9) 确定为人类肺细胞 SARS-CoV-2 感染的重要宿主因子。MYH9 的基因消融显着减少野生型 (WT) A549 和 Calu-3 细胞中的 SARS-CoV-2 假病毒感染，和 MYH9 的过表达增强了 WT A549 和 H1299 细胞中的假病毒感染。MYH9 与 SARS-CoV-2 S 共定位，并通过 S2 亚基与 SARS-CoV-2 S 直接相互作用，并通过其 C 端结构域（称为 PRA）与 S1-NTD（N 端结构域）直接相互作用。进一步的实验表明，内体或肌球蛋白抑制剂可有效阻断 SARS-CoV-2 病毒进入 PRA-A549 细胞，而跨膜蛋白酶丝氨酸 2 (TMPRSS2) 和组织蛋白酶 B 和 L (CatB/L) 抑制剂则不能，表明 MYH9促进 SARS-CoV-2 内吞作用并绕过 TMPRSS2 和 CatB/L 途径。最后，我们证明 MYH9 的缺失减少了 Calu-3、ACE2-A549 和 ACE2-H1299 细胞中真正的 SARS-CoV-2 感染。总之，我们的结果表明 MYH9 是 SARS-CoV-2 的候选宿主因子，