Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea.
Chonnam National University Veterinary Teaching Hospital, Gwangju, Republic of Korea.
J Virol. 2019 Feb 5;93(4). doi: 10.1128/JVI.01773-18. Print 2019 Feb 15.
The genus belongs to the family , and its members are common causative agents of severe acute gastroenteritis in both humans and animals. Some caliciviruses are known to use either terminal sialic acids or histo-blood group antigens as attachment factors and/or cell surface proteins, such as CD300lf, CD300ld, and junctional adhesion molecule 1 of tight junctions (TJs), as receptors. However, the roles of TJs and their proteins in sapovirus entry have not been examined. In this study, we found that porcine sapovirus (PSaV) significantly decreased transepithelial electrical resistance and increased paracellular permeability early in infection of LLC-PK cells, suggesting that PSaV dissociates TJs of cells. This led to the interaction between PSaV particles and occludin, which traveled in a complex into late endosomes via Rab5- and Rab7-dependent trafficking. Inhibition of occludin using small interfering RNA (siRNA), a specific antibody, or a dominant-negative mutant significantly blocked the entry of PSaV. Transient expression of occludin in nonpermissive Chinese hamster ovary (CHO) cells conferred susceptibility to PSaV, but only for a limited time. Although claudin-1, another TJ protein, neither directly interacted nor was internalized with PSaV particles, it facilitated PSaV entry and replication in the LLC-PK cells. We conclude that PSaV particles enter LLC-PK cells by binding to occludin as a coreceptor in PSaV-dissociated TJs. PSaV and occludin then form a complex that moves to late endosomes via Rab5- and Rab7-dependent trafficking. In addition, claudin-1 in the TJs opened by PSaV infection facilitates PSaV entry and infection as an entry factor. Sapoviruses (SaVs) cause severe acute gastroenteritis in humans and animals. Although they replicate in intestinal epithelial cells, which are tightly sealed by apical-junctional complexes, such as tight junctions (TJs), the mechanisms by which SaVs hijack TJs and their proteins for successful entry and infection remain largely unknown. Here, we demonstrate that porcine SaVs (PSaVs) induce early dissociation of TJs, allowing them to bind to the TJ protein occludin as a functional coreceptor. PSaVs then travel in a complex with occludin into late endosomes through Rab5- and Rab7-dependent trafficking. Claudin-1, another TJ protein, does not directly interact with PSaV but facilitates the entry of PSaV into cells as an entry factor. This work contributes to our understanding of the entry of SaV and other caliciviruses into cells and may aid in the development of efficient and affordable drugs to treat SaV infections.
该属属于科，其成员是人类和动物严重急性胃肠炎的常见病原体。一些杯状病毒已知使用末端唾液酸或组织血型抗原作为附着因子和/或细胞表面蛋白，如紧密连接 (TJ) 的 CD300lf、CD300ld 和连接黏附分子 1 (JAM1)，作为受体。然而，TJ 及其蛋白在诺如病毒进入中的作用尚未被检测到。在这项研究中，我们发现猪诺如病毒 (PSaV) 在感染 LLC-PK 细胞早期显著降低了上皮细胞间电阻并增加了细胞旁通透性，表明 PSaV 分离了细胞的 TJ。这导致 PSaV 颗粒与紧密连接蛋白 Occludin 相互作用，通过 Rab5 和 Rab7 依赖性运输进入晚期内体。使用小干扰 RNA (siRNA)、特异性抗体或显性负突变体抑制 Occludin 显著阻止了 PSaV 的进入。在非允许性中国仓鼠卵巢 (CHO) 细胞中转瞬表达 Occludin 赋予了 PSaV 的易感性，但仅在有限的时间内。尽管另一种 TJ 蛋白 Claudin-1 既不直接相互作用也不被 PSaV 颗粒内化，但它促进了 PSaV 在 LLC-PK 细胞中的进入和复制。我们得出结论，PSaV 颗粒通过与 Occludin 作为 PSaV 分离 TJ 中的核心受体结合进入 LLC-PK 细胞。PSaV 和 Occludin 然后形成一个复合物，通过 Rab5 和 Rab7 依赖性运输进入晚期内体。此外，PSaV 感染打开的 TJ 中的 Claudin-1 作为进入因子促进 PSaV 的进入和感染。诺如病毒 (SaVs) 会导致人类和动物患严重急性胃肠炎。尽管它们在肠道上皮细胞中复制，这些细胞被顶端连接复合物（如紧密连接 (TJ)）紧密密封，但 SaVs 劫持 TJ 及其蛋白以成功进入和感染的机制在很大程度上仍不清楚。在这里，我们证明了猪诺如病毒 (PSaVs) 诱导 TJ 的早期解离，使其能够与 TJ 蛋白 Occludin 结合作为功能性核心受体。然后，PSaV 与 Occludin 一起通过 Rab5 和 Rab7 依赖性运输进入晚期内体。另一种 TJ 蛋白 Claudin-1 不直接与 PSaV 相互作用，但作为进入因子促进 PSaV 进入细胞。这项工作有助于我们理解 SaV 和其他杯状病毒进入细胞的机制，并可能有助于开发有效且经济实惠的药物来治疗 SaV 感染。
