Both α2,3- and α2,6-Linked Sialic Acids on O-Linked Glycoproteins Act as Functional Receptors for Porcine Sapovirus

Sapovirus, a member of the Caliciviridae family, is an important cause of acute gastroenteritis in humans and pigs. Currently, the porcine sapovirus (PSaV) Cowden strain remains the only cultivable member of the Sapovirus genus. While some caliciviruses are known to utilize carbohydrate receptors for entry and infection, a functional receptor for sapovirus is unknown. To characterize the functional receptor of the Cowden strain of PSaV, we undertook a comprehensive series of protein-ligand biochemical assays in mock and PSaV-infected cell culture and/or piglet intestinal tissue sections. PSaV revealed neither hemagglutination activity with red blood cells from any species nor binding activity to synthetic histo-blood group antigens, indicating that PSaV does not use histo-blood group antigens as receptors. Attachment and infection of PSaV were markedly blocked by sialic acid and Vibrio cholerae neuraminidase (NA), suggesting a role for α2,3-linked, α2,6-linked or α2,8-linked sialic acid in virus attachment. However, viral attachment and infection were only partially inhibited by treatment of cells with sialidase S (SS) or Maackia amurensis lectin (MAL), both specific for α2,3-linked sialic acid, or Sambucus nigra lectin (SNL), specific for α2,6-linked sialic acid. These results indicated that PSaV recognizes both α2,3- and α2,6-linked sialic acids for viral attachment and infection. Treatment of cells with proteases or with benzyl 4-O-β-D-galactopyranosyl-β-D-glucopyranoside (benzylGalNAc), which inhibits O-linked glycosylation, also reduced virus binding and infection, whereas inhibition of glycolipd synthesis or N-linked glycosylation had no such effect on virus binding or infection. These data suggest PSaV binds to cellular receptors that consist of α2,3- and α2,6-linked sialic acids on glycoproteins attached via O-linked glycosylation.

札幌病毒（Sapovirus）隶属于杯状病毒科（Caliciviridae），是引发人类与猪急性胃肠炎的重要病原体。目前，猪札幌病毒（porcine sapovirus, PSaV）考登株（Cowden strain）仍是札幌病毒属中唯一可培养的成员。已知部分杯状病毒可利用碳水化合物受体完成入侵与感染，但札幌病毒的功能性受体至今尚未明确。为解析猪札幌病毒考登株的功能性受体，我们针对模拟感染与猪札幌病毒感染的细胞培养体系及/或仔猪肠组织切片，开展了一系列全面的蛋白质-配体生化实验。
猪札幌病毒对任意物种的红细胞均未表现出血凝活性，亦无法结合合成组织血型抗原，表明其并未以组织血型抗原作为受体。猪札幌病毒的吸附与感染过程可被唾液酸及霍乱弧菌神经氨酸酶（neuraminidase, NA）显著阻断，提示α2,3-连接、α2,6-连接或α2,8-连接唾液酸可能参与病毒吸附过程。然而，仅针对α2,3-连接唾液酸的唾液苷酶S（sialidase S, SS）或朝鲜槐凝集素（Maackia amurensis lectin, MAL）处理，以及仅针对α2,6-连接唾液酸的接骨木凝集素（Sambucus nigra lectin, SNL）处理，仅能部分抑制病毒的吸附与感染。上述结果表明，猪札幌病毒可通过识别α2,3-与α2,6-连接唾液酸完成病毒吸附与感染。
对细胞采用蛋白酶处理，或采用抑制O-糖基化的试剂苄基4-O-β-D-半乳糖吡喃糖基-β-D-葡萄糖吡喃糖苷（benzylGalNAc）处理，同样可降低病毒结合与感染效率；而抑制糖脂合成或N-糖基化则对病毒结合与感染无显著影响。上述数据提示，猪札幌病毒的细胞受体为通过O-糖基化修饰结合于糖蛋白表面的α2,3-与α2,6-连接唾液酸。