PDF(Pubmed)
Abstract：
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, S1A and S1B. Cell binding and invasion inhibition assays revealed a preference for the S1B 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 S1A 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, S1A, or S1B contained highly potent neutralizing antibodies, with anti-S1B showing better efficiency in neutralizing rSADS-GFP infection compared to anti-S1A. 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 S1B 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 (S1B) of SADS-CoV, whereas the sugar-binding domain located at the S1 N-terminal part of S1 (S1A). 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.
摘要：
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,SADS-CoV的广泛种间感染对人类健康构成潜在威胁。这项研究提供了实验证据来剖析SADS-CoV尖峰S1亚基内不同结构域在细胞进入中的作用。具体来说,我们表达了S1及其子域，S1A和S1B。细胞结合和侵袭抑制试验揭示了在结合细胞表面上的受体时对S1B亚结构域的偏好,这种未知的受体并没有被猪流行性腹泻病毒利用。纳米颗粒显示显示来自猪的红细胞的血凝，人类,和老鼠，将S1A亚结构域与参与病毒附着的唾液酸(Sia)的结合连接。我们成功地从重组cDNA克隆中拯救了GFP标记的SADS-CoV（rSADS-GFP）以追踪病毒感染。抗血清针对S1，S1A，或S1B含有高效中和抗体，与抗S1A相比，抗S1B在中和rSADS-GFP感染方面表现出更好的效率。此外，通过肝素酶处理或rSADS-GFP与HS或组成单糖预孵育来消耗硫酸乙酰肝素（HS）可以抑制SADS-CoV进入。最后,我们证明，S糖蛋白的弗林蛋白酶活性裂解和II型跨膜丝氨酸蛋白酶（TMPRSS2）的存在对于SADS-CoV感染至关重要。这些综合观察表明，SADS-CoV的广泛细胞嗜性可能与Sia或HS在细胞表面的分布有关，而S1B包含主要的蛋白受体结合位点。特定宿主蛋白酶在促进SADS-CoV进入中也起重要作用。重要猪急性腹泻综合征冠状病毒(SADS-CoV)是一种感染仔猪的新型病原体,其独特的遗传进化特征和广泛的物种嗜性表明了跨物种传播的潜力。病毒通过其刺突(S)糖蛋白进入细胞。在这项研究中,我们鉴定了SADS-CoV的S1亚基（S1B）的C末端部分的受体结合域，而糖结合域位于S1的S1N末端部分（S1A）。唾液酸,硫酸乙酰肝素，和特定的宿主蛋白酶在病毒附着和进入中起着必不可少的作用。SADS-CoVS1亚基的功能结构域的解剖和细胞进入辅因子的鉴定将有助于探索SADS-CoV使用的受体，这可能有助于探索跨物种传播和宿主嗜性背后的机制。
