The cell entry of SARS-CoV-2 has emerged as an attractive drug repurposing target for COVID-19. Here we combine genetics and chemical perturbation to demonstrate that ACE2-mediated entry of SARS-Cov and CoV-2 requires the cell surface heparan sulfate (HS) as an assisting cofactor: ablation of genes involved in HS biosynthesis or incubating cells with a HS mimetic both inhibit Spike-mediated viral entry. We show that heparin/HS binds to Spike directly, and facilitates the attachment of Spike-bearing viral particles to the cell surface to promote viral entry. We screened approved drugs and identified two classes of inhibitors that act via distinct mechanisms to target this entry pathway. Among the drugs characterized, Mitoxantrone is a potent HS inhibitor, while Sunitinib and BNTX disrupt the actin network to indirectly abrogate HS-assisted viral entry. We further show that drugs of the two classes can be combined to generate a synergized activity against SARS-CoV-2-induced cytopathic effect. Altogether, our study establishes HS as an attachment factor that assists SARS coronavirus cell entry and reveals drugs capable of targeting this important step in the viral life cycle.

SARS-CoV-2 的细胞进入已成为 COVID-19 有吸引力的药物再利用靶点。在这里，我们结合遗传学和化学扰动来证明 ACE2 介导的 SARS-Cov 和 CoV-2 的进入需要细胞表面硫酸乙酰肝素 (HS) 作为辅助辅因子：消融参与 HS 生物合成的基因或用 HS 模拟物孵育细胞两者都抑制刺突介导的病毒进入。我们发现，肝素/HS 直接与 Spike 结合，并促进带有 Spike 的病毒颗粒附着到细胞表面，从而促进病毒进入。我们筛选了已批准的药物并确定了两类抑制剂，它们通过不同的机制作用于该进入途径。在所表征的药物中，米托蒽醌是一种有效的 HS 抑制剂，而舒尼替尼和 BNTX 会破坏肌动蛋白网络，从而间接消除 HS 辅助的病毒进入。我们进一步表明，这两类药物可以结合起来产生针对 SARS-CoV-2 诱导的细胞病变效应的协同活性。总而言之，我们的研究将 HS 确定为辅助 SARS 冠状病毒进入细胞的附着因子，并揭示了能够针对病毒生命周期中这一重要步骤的药物。