Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, United States of America.
Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.
PLoS Pathog. 2021 Nov 19;17(11):e1009743. doi: 10.1371/journal.ppat.1009743. eCollection 2021 Nov.
Phosphatidylserine (PS) receptors enhance infection of many enveloped viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2 in cells that express low surface levels of ACE2. Expression of members of the TIM (TIM-1 and TIM-4) and TAM (AXL) families of PS receptors enhance SARS-CoV-2 binding to cells, facilitate internalization of fluorescently-labeled virions and increase ACE2-dependent infection of SARS-CoV-2; however, PS receptors alone did not mediate infection. We were unable to detect direct interactions of the PS receptor AXL with purified SARS-CoV-2 spike, contrary to a previous report. Instead, our studies indicate that the PS receptors interact with PS on the surface of SARS-CoV-2 virions. In support of this, we demonstrate that: 1) significant quantities of PS are located on the outer leaflet of SARS-CoV-2 virions, 2) PS liposomes, but not phosphatidylcholine liposomes, reduced entry of VSV/Spike pseudovirions and 3) an established mutant of TIM-1 which does not bind to PS is unable to facilitate entry of SARS-CoV-2. As AXL is an abundant PS receptor on a number of airway lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of Vero E6 cells as well as multiple human lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block endosomal acidification and cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. We extended our observations to the related betacoronavirus mouse hepatitis virus (MHV), showing that inhibition or ablation of AXL reduces MHV infection of murine cells. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and suggest that inhibition of the PS receptor AXL has therapeutic potential against SARS-CoV-2.
磷脂酰丝氨酸（PS）受体通过与病毒相关的 PS 结合增强许多包膜病毒的感染，这种结合被称为凋亡模拟。在这里，我们表明这种广泛共享的摄取机制被 SARS-CoV-2 利用，用于表达低表面 ACE2 水平的细胞。PS 受体 TIM（TIM-1 和 TIM-4）和 TAM（AXL）家族成员的表达增强了 SARS-CoV-2 与细胞的结合，促进了荧光标记病毒的内化，并增加了 ACE2 依赖性的 SARS-CoV-2 感染；然而，PS 受体本身并不能介导感染。我们无法检测到 PS 受体 AXL 与纯化的 SARS-CoV-2 刺突蛋白的直接相互作用，与之前的一份报告相反。相反，我们的研究表明 PS 受体与 SARS-CoV-2 病毒粒子表面的 PS 相互作用。支持这一点，我们证明：1）大量 PS 位于 SARS-CoV-2 病毒粒子的外叶；2）PS 脂质体，但不是磷脂酰胆碱脂质体，减少了 VSV/Spike 假病毒的进入；3）一种不能结合 PS 的 TIM-1 建立突变体不能促进 SARS-CoV-2 的进入。由于 AXL 是许多气道细胞上丰富的 PS 受体，我们评估了 AXL 信号通路的小分子抑制剂，如 bemcentinib，以评估其抑制 SARS-CoV-2 感染的能力。bemcentinib 强烈抑制了 Vero E6 细胞以及表达 AXL 的多种人类肺细胞系的病毒感染。这种抑制与阻断内体酸化和组织蛋白酶活性的抑制剂非常吻合，这与 AXL 介导的 SARS-CoV-2 进入内体区室的摄取一致。我们将我们的观察结果扩展到相关的β冠状病毒鼠肝炎病毒（MHV），表明 AXL 的抑制或消融减少了 MHV 对鼠细胞的感染。总的来说，我们的研究结果提供了证据表明 PS 受体促进了大流行冠状病毒 SARS-CoV-2 的感染，并表明抑制 PS 受体 AXL 具有针对 SARS-CoV-2 的治疗潜力。
