Division of Avian Infectious Diseases, the State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
J Virol. 2021 Jun 10;95(13):e0228820. doi: 10.1128/JVI.02288-20.
The cellular entry pathways and the mechanisms of Newcastle disease virus (NDV) entry into cells are poorly characterized. In this study, we demonstrated that chicken interferon-induced transmembrane protein 1 (chIFITM1), which is located in the early endosomes, could limit the replication of NDV in chicken macrophage cell line HD11, suggesting the endocytic entry of NDV into chicken macrophages. Then, we presented a systematic study about the entry mechanism of NDV into chicken macrophages. First, we demonstrated that a low-pH condition and dynamin were required during NDV entry. However, NDV entry into chicken macrophages was independent of clathrin-mediated endocytosis. We also found that NDV entry was dependent on membrane cholesterol. The NDV entry and replication were significantly reduced by nystatin and phorbol 12-myristate 13-acetate treatment, overexpression of dominant-negative (DN) caveolin-1, or knockdown of caveolin-1, suggesting that NDV entry depends on caveola-mediated endocytosis. However, macropinocytosis did not play a role in NDV entry into chicken macrophages. In addition, we found that Rab5, rather than Rab7, was involved in the entry and traffic of NDV. The colocalization of NDV with Rab5 and early endosome suggested that NDV virion was transported to early endosomes in a Rab5-dependent manner after internalization. Of particular note, the caveola-mediated endocytosis was also utilized by NDV to enter primary chicken macrophages. Moreover, NDV entered different cell types using different pathways. Collectively, our findings demonstrate for the first time that NDV virion enters chicken macrophages via a pH-dependent, dynamin and caveola-mediated endocytosis pathway and that Rab5 is involved in the traffic and location of NDV. Although the pathogenesis of Newcastle disease virus (NDV) has been extensively studied, the detailed mechanism of NDV entry into host cells is largely unknown. Macrophages are the first-line defenders of host defense against infection of pathogens. Chicken macrophages are considered one of the main types of target cells during NDV infection. Here, we comprehensively investigated the entry mechanism of NDV in chicken macrophages. This is the first report to demonstrate that NDV enters chicken macrophages via a pH-dependent, dynamin and caveola-mediated endocytosis pathway that requires Rab5. The result is important for our understanding of the entry of NDV in chicken macrophages, which will further advance the knowledge of NDV pathogenesis and provide useful clues for the development of novel preventive or therapeutic strategies against NDV infection. In addition, this information will contribute to our further understanding of pathogenesis with regard to other members of the genus in the family.
新城疫病毒（NDV）进入细胞的细胞进入途径和机制尚未得到很好的描述。在这项研究中，我们证明了位于早期内体中的鸡干扰素诱导跨膜蛋白 1（chIFITM1）可以限制 NDV 在鸡巨噬细胞系 HD11 中的复制，表明 NDV 通过内吞作用进入鸡巨噬细胞。然后，我们对 NDV 进入鸡巨噬细胞的机制进行了系统研究。首先，我们证明 NDV 进入需要低 pH 值条件和胞吞作用所需的网格蛋白。然而，NDV 进入鸡巨噬细胞不依赖于网格蛋白介导的内吞作用。我们还发现 NDV 进入依赖于膜胆固醇。制霉菌素和佛波醇 12-肉豆蔻酸 13-乙酸酯处理、显性负（DN）小窝蛋白 1 的过表达或小窝蛋白 1 的敲低显著降低了 NDV 的进入和复制，表明 NDV 进入依赖于小窝介导的内吞作用。然而，巨胞饮作用在 NDV 进入鸡巨噬细胞中不起作用。此外，我们发现 Rab5 而不是 Rab7 参与了 NDV 的进入和运输。NDV 与 Rab5 和早期内体的共定位表明，NDV 病毒在内化后以 Rab5 依赖性方式被转运到早期内体。值得注意的是，小窝介导的内吞作用也被 NDV 用于进入原代鸡巨噬细胞。此外，NDV 通过不同的途径进入不同的细胞类型。总之，我们的研究结果首次表明，NDV 病毒通过 pH 依赖性、网格蛋白和小窝蛋白介导的内吞作用途径进入鸡巨噬细胞，Rab5 参与 NDV 的运输和定位。虽然新城疫病毒（NDV）的发病机制已得到广泛研究，但 NDV 进入宿主细胞的详细机制在很大程度上仍是未知的。巨噬细胞是宿主防御病原体感染的第一道防线。鸡巨噬细胞被认为是 NDV 感染时的主要靶细胞类型之一。在这里，我们全面研究了 NDV 在鸡巨噬细胞中的进入机制。这是首次报道 NDV 通过 pH 依赖性、网格蛋白和小窝蛋白介导的内吞作用途径进入鸡巨噬细胞，该途径需要 Rab5。该结果对于我们理解 NDV 在鸡巨噬细胞中的进入非常重要，这将进一步推进对 NDV 发病机制的认识，并为开发针对 NDV 感染的新型预防或治疗策略提供有用线索。此外，该信息将有助于我们进一步了解科中的其他属成员的发病机制。
