Departamento de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Cantoblanco, 28049 Madrid, Spain.
Viruses. 2020 Jul 7;12(7):734. doi: 10.3390/v12070734.
Endocytosis is a pathway used by viruses to enter cells that can be classified based on the proteins involved, such as dynamin, clathrin or caveolin. Although the entry of herpes simplex type 1 (HSV-1) by endocytosis has been documented in different cell types, its dependence on clathrin has not been described whereas its dependence on dynamin has been shown according to the cell line used. The present work shows how clathrin-mediated endocytosis (CME) is one way that HSV-1 infects the human oligodendroglial (HOG) cell line. Partial dynamin inhibition using dynasore revealed a relationship between decrease of infection and dynamin inhibition, measured by viral titration and immunoblot. Co-localization between dynamin and HSV-1 was verified by immunofluorescence at the moment of viral entry into the cell. Inhibition by chlorpromazine revealed that viral progeny also decreased when clathrin was partially inhibited in our cell line. RT-qPCR of immediately early viral genes, specific entry assays and electron microscopy all confirmed clathrin's participation in HSV-1 entry into HOG cells. In contrast, caveolin entry assays showed no effect on the entry of this virus. Therefore, our results suggest the participation of dynamin and clathrin during endocytosis of HSV-1 in HOG cells.
内吞作用是病毒进入细胞的一种途径,可以根据涉及的蛋白质进行分类,例如网格蛋白、衔接蛋白或窖蛋白。虽然已在不同的细胞类型中记录到单纯疱疹病毒 1(HSV-1)通过内吞作用进入细胞,但尚未描述其对网格蛋白的依赖性,而根据所用的细胞系已表明其对衔接蛋白的依赖性。本研究表明了网格蛋白介导的内吞作用(CME)是 HSV-1 感染人少突胶质细胞(HOG)细胞系的一种方式。使用 dynasore 部分抑制衔接蛋白发现,病毒滴定和免疫印迹测量的感染减少与衔接蛋白抑制之间存在关联。在病毒进入细胞的那一刻,通过免疫荧光证实了衔接蛋白和 HSV-1 之间的共定位。氯丙嗪的抑制作用表明,当我们的细胞系中网格蛋白部分受到抑制时,病毒子代也会减少。对即刻早期病毒基因的 RT-qPCR、特定进入测定和电子显微镜都证实了网格蛋白参与了 HSV-1 进入 HOG 细胞。相比之下,窖蛋白进入测定显示该病毒的进入不受影响。因此,我们的结果表明,网格蛋白和衔接蛋白在 HOG 细胞中 HSV-1 的内吞作用中发挥作用。