Maeda Yuki, Shibutani Shusaku, Onishi Keiko, Iwata Hiroyuki
Laboratory of Veterinary Hygiene, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan.
Laboratory of Veterinary Hygiene, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan.
Virus Res. 2021 Sep;302:198492. doi: 10.1016/j.virusres.2021.198492. Epub 2021 Jun 24.
Ibaraki virus (IBAV) is the pathogen associated with Ibaraki disease. In a previous study, we suggested that IBAV enters hamster lung (HmLu-1) cells via endocytosis and subsequently escapes into the cytoplasm upon endosomal acidification. However, it is unclear which of the endocytic pathways IBAV utilizes. In this study, we aimed to further elucidate the pathway of IBAV entry into host cells. We found that IBAV replication was not suppressed by inhibitors of clathrin-mediated or caveolin-mediated endocytosis but was markedly suppressed by 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and cytochalasin D, both of which inhibit macropinocytosis. Monensin, which inhibits endosomal acidification, also suppressed IBAV replication. To assess the inhibitory effects of these reagents on endocytosis, dextran and transferrin were used as indicators of macropinocytosis and clathrin-mediated endocytic activity, respectively. Our data confirmed that EIPA and monensin inhibited dextran uptake, and cytochalasin D inhibited the uptake of both. Additionally, we confirmed that endosomal/lysosomal acidification was inhibited by monensin. These results suggest that the macropinocytosis pathway is the major route of IBAV entry and confirm that IBAV infection of HmLu-1 cells is dependent on endosomal acidification.
茨城县病毒（IBAV）是与茨城县疾病相关的病原体。在先前的一项研究中，我们提出IBAV通过内吞作用进入仓鼠肺（HmLu-1）细胞，随后在内体酸化时逃逸到细胞质中。然而，尚不清楚IBAV利用哪种内吞途径。在本研究中，我们旨在进一步阐明IBAV进入宿主细胞的途径。我们发现，网格蛋白介导的或小窝蛋白介导的内吞作用抑制剂不会抑制IBAV复制，但5-（N-乙基-N-异丙基）氨氯地平（EIPA）和细胞松弛素D可显著抑制IBAV复制，这两种物质均抑制巨胞饮作用。抑制内体酸化的莫能菌素也抑制了IBAV复制。为了评估这些试剂对内吞作用的抑制效果，分别使用葡聚糖和转铁蛋白作为巨胞饮作用和网格蛋白介导的内吞活性的指标。我们的数据证实，EIPA和莫能菌素抑制了葡聚糖摄取，细胞松弛素D抑制了两者的摄取。此外，并证实莫能菌素抑制了内体/溶酶体酸化。这些结果表明，巨胞饮作用途径是IBAV进入的主要途径，并证实HmLu-1细胞的IBAV感染依赖于内体酸化。
