Xianghu Laboratory, Hangzhou, China.
State Key Laboratory for Animal Disease Control and Prevention, South China Agricultural University, Guangzhou, China.
J Virol. 2024 Apr 16;98(4):e0013924. doi: 10.1128/jvi.00139-24. Epub 2024 Mar 19.
Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel porcine enteric coronavirus, and the broad interspecies infection of SADS-CoV poses a potential threat to human health. This study provides experimental evidence to dissect the roles of distinct domains within the SADS-CoV spike S1 subunit in cellular entry. Specifically, we expressed the S1 and its subdomains, S1 and S1. Cell binding and invasion inhibition assays revealed a preference for the S1 subdomain in binding to the receptors on the cell surface, and this unknown receptor is not utilized by the porcine epidemic diarrhea virus. Nanoparticle display demonstrated hemagglutination of erythrocytes from pigs, humans, and mice, linking the S1 subdomain to the binding of sialic acid (Sia) involved in virus attachment. We successfully rescued GFP-labeled SADS-CoV (rSADS-GFP) from a recombinant cDNA clone to track viral infection. Antisera raised against S1, S1, or S1 contained highly potent neutralizing antibodies, with anti-S1 showing better efficiency in neutralizing rSADS-GFP infection compared to anti-S1. Furthermore, depletion of heparan sulfate (HS) by heparinase treatment or pre-incubation of rSADS-GFP with HS or constituent monosaccharides could inhibit SADS-CoV entry. Finally, we demonstrated that active furin cleavage of S glycoprotein and the presence of type II transmembrane serine protease (TMPRSS2) are essential for SADS-CoV infection. These combined observations suggest that the wide cell tropism of SADS-CoV may be related to the distribution of Sia or HS on the cell surface, whereas the S1 contains the main protein receptor binding site. Specific host proteases also play important roles in facilitating SADS-CoV entry.IMPORTANCESwine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel pathogen infecting piglet, and its unique genetic evolution characteristics and broad species tropism suggest the potential for cross-species transmission. The virus enters cells through its spike (S) glycoprotein. In this study, we identify the receptor binding domain on the C-terminal part of the S1 subunit (S1) of SADS-CoV, whereas the sugar-binding domain located at the S1 N-terminal part of S1 (S1). Sialic acid, heparan sulfate, and specific host proteases play essential roles in viral attachment and entry. The dissection of SADS-CoV S1 subunit's functional domains and identification of cellular entry cofactors will help to explore the receptors used by SADS-CoV, which may contribute to exploring the mechanisms behind cross-species transmission and host tropism.
猪急性腹泻综合征冠状病毒（SADS-CoV）是一种新型的猪肠道冠状病毒，其广泛的种间感染对人类健康构成潜在威胁。本研究提供了实验证据，解析了 SADS-CoV 刺突 S1 亚基内不同结构域在细胞进入中的作用。具体而言，我们表达了 S1 及其亚结构域 S1 和 S1。细胞结合和入侵抑制试验表明，S1 亚结构域优先与细胞表面受体结合，而这种未知受体不被猪流行性腹泻病毒利用。纳米颗粒显示可凝集猪、人和鼠的红细胞，将 S1 亚结构域与参与病毒附着的唾液酸（Sia）结合联系起来。我们成功地从重组 cDNA 克隆中拯救出 GFP 标记的 SADS-CoV（rSADS-GFP），以跟踪病毒感染。针对 S1、S1 或 S1 产生的抗血清含有高效的中和抗体，与针对 S1 的抗体相比，针对 S1 的抗体在中和 rSADS-GFP 感染方面效率更高。此外，肝素酶处理或 rSADS-GFP 与 HS 或其组成单糖预孵育可耗尽肝素硫酸酯（HS），从而抑制 SADS-CoV 的进入。最后，我们证明 S 糖蛋白的活性弗林裂解和 II 型跨膜丝氨酸蛋白酶（TMPRSS2）的存在对于 SADS-CoV 感染是必需的。这些综合观察结果表明，SADS-CoV 的广泛细胞嗜性可能与细胞表面 Sia 或 HS 的分布有关，而 S1 包含主要的蛋白受体结合位点。特定的宿主蛋白酶在促进 SADS-CoV 进入中也起着重要作用。重要性猪急性腹泻综合征冠状病毒（SADS-CoV）是一种新型病原体，感染仔猪，其独特的遗传进化特征和广泛的物种嗜性表明存在跨种传播的潜力。该病毒通过其刺突（S）糖蛋白进入细胞。在这项研究中，我们确定了 SADS-CoV S1 亚基（S1）C 末端部分上的受体结合域，而 S1 的 S1 氨基末端部分上的糖结合域（S1）。唾液酸、肝素硫酸酯和特定的宿主蛋白酶在病毒附着和进入中起着重要作用。解析 SADS-CoV S1 亚基的功能域并确定细胞进入辅助因子将有助于探索 SADS-CoV 使用的受体，这可能有助于探索跨种传播和宿主嗜性的机制。
