这里,我们提供的证据表明,使用LDLR的Caveolae介导的内吞作用是SARS-CoV-2病毒在眼部细胞系ARPE-19中内化的途径。首先,我们发现,而血管紧张素转换酶2(ACE2)在这些细胞中表达,通过抗体治疗阻断ACE2并不能预防SARS-CoV-2尖峰假病毒粒子的感染,也没有抗体阻断细胞外波形蛋白和其他富含胆固醇的脂筏蛋白。接下来,我们通过显示将细胞与不同的环糊精和氧固醇25-羟基胆固醇(25-HC)一起孵育可以抑制ARPE-19的假病毒颗粒感染,从而暗示了胆固醇稳态在感染中的作用.然而,25-HC的作用可能不是通过胆固醇生物合成,因为与洛伐他汀的孵育没有明显影响感染。此外,它不太可能是25-HC对LXR受体的激动作用,因为LXR激动剂GW3965在高达5μMGW3965时对ARPE-19细胞的感染没有显著影响。我们探讨了内吞途径的作用,但确定不涉及网格蛋白依赖性和弗林蛋白依赖性的移植物。此外,20µM氯丙嗪,网格蛋白介导的内吞作用(CME)的抑制剂,也没有什么效果。相比之下,抗动力蛋白I/II抗体阻断了SARS-CoV-2尖峰假病毒的进入,朝代也是如此,动态蛋白GTP酶活性的非竞争性抑制剂。此外,抗caveolin-1抗体显著阻断ARPE-19的尖峰假型化慢病毒感染。然而,制霉菌素,一种典型的Caveolae依赖性内吞作用抑制剂,不影响感染,而吲哚美辛在48h时间点仅在10µM时抑制。最后,我们发现抗LDLR抗体阻断假病毒感染的程度与抗caveolin-1和抗动力蛋白I/II抗体相似,而LDLR特异性siRNA的转染导致峰值假型化慢病毒感染的减少,与乱序对照siRNA相比。因此,我们得出结论,ARPE-19细胞中的SARS-CoV-2尖峰假病毒粒子感染是一个主要由LDLR介导的动力蛋白依赖性过程。
Here, we present evidence that caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. Firstly, we found that, while Angiotensin-converting enzyme 2 (ACE2) is expressed in these cells, blocking ACE2 by antibody treatment did not prevent infection by SARS-CoV-2 spike pseudovirions, nor did antibody blockade of extracellular vimentin and other cholesterol-rich lipid raft proteins. Next, we implicated the role of cholesterol homeostasis in infection by showing that incubating cells with different cyclodextrins and oxysterol 25-hydroxycholesterol (25-HC) inhibits pseudovirion infection of ARPE-19. However, the effect of 25-HC is likely not via cholesterol biosynthesis, as incubation with lovastatin did not appreciably affect infection. Additionally, is it not likely to be an agonistic effect of 25-HC on LXR receptors, as the LXR agonist GW3965 had no significant effect on infection of ARPE-19 cells at up to 5 μM GW3965. We probed the role of endocytic pathways but determined that clathrin-dependent and flotillin-dependent rafts were not involved. Furthermore, 20 µM chlorpromazine, an inhibitor of clathrin-mediated endocytosis (CME), also had little effect. In contrast, anti-dynamin I/II antibodies blocked the entry of SARS-CoV-2 spike pseudovirions, as did dynasore, a noncompetitive inhibitor of dynamin GTPase activity. Additionally, anti-caveolin-1 antibodies significantly blocked spike pseudotyped lentiviral infection of ARPE-19. However, nystatin, a classic inhibitor of caveolae-dependent endocytosis, did not affect infection while indomethacin inhibited only at 10 µM at the 48 h time point. Finally, we found that anti-LDLR antibodies block pseudovirion infection to a similar degree as anti-caveolin-1 and anti-dynamin I/II antibodies, while transfection with LDLR-specific siRNA led to a decrease in spike pseudotyped lentiviral infection, compared to scrambled control siRNAs. Thus, we conclude that SARS-CoV-2 spike pseudovirion infection in ARPE-19 cells is a dynamin-dependent process that is primarily mediated by LDLR.