Dengue virus (DENV) requires clathrin-mediated endocytosis for its entry into the cells where the adaptor protein complex (AP) is vital for the clathrin-coated vesicle formation. The role of AP-2 was previously examined in the early stages of DENV infection; however, the role of AP-2 in the late stage of DENV infection was not determined. The µ1 subunit of AP-2 (AP2M1) is one of the most important cytoplasmic carrier domains in clathrin-mediated endocytosis and the phosphorylation of this subunit by the kinase enzyme, AP-2 associated protein kinase 1 (AAK1), stimulates clathrin and supports the cell surface receptor incorporation. In the present study, we primarily aimed to investigate the role of AP2M1 by gene silencing approach as well as using naked DENV RNA transfection into AP2M1 knockdown cells. Secondarily, an inhibitor of AAK1, sunitinib was used to investigate whether AAK1 could influence the virus production in DENV-infected Huh7 cells. The knockdown of AP2M1 in the DENV-infected Huh7 cells displayed a reduction in the viral titer at 24 h post-infection. Furthermore, experiments were conducted to bypass the DENV internalization using a naked DENV RNA transfection into the AP2M1 knockdown cells. Higher intracellular DENV RNA, DENV E protein, and intracellular virion were observed, whereas the extracellular virion production was comparably less than that of control. Treatment with sunitinib in DENV-infected Huh7 cells was able to reduce extracellular virion production and was consistent with all four serotypes of DENV. Therefore, our findings demonstrate the role of AP2M1 in the exocytosis step of DENV replication leading to infectious DENV production and the efficacy of sunitinib in suppressing virus production during the infection with different serotypes of DENV.

中文翻译：

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衔接子蛋白复合物2的µ1亚基的抑制减少了登革热病毒的释放。

登革热病毒（DENV）需要网格蛋白介导的内吞作用才能进入细胞，在那里衔接蛋白复合物（AP）对于网格蛋白涂层的囊泡形成至关重要。以前曾在DENV感染的早期阶段检查过AP-2的作用。然而，尚未确定AP-2在DENV感染后期的作用。AP-2的µ1亚基（AP2M1）是网格蛋白介导的内吞作用中最重要的细胞质载体结构域之一，该亚基被激酶AP-2相关蛋白激酶1（AAK1）磷酸化，刺激了网格蛋白并支持细胞表面受体的结合。在本研究中，我们主要旨在通过基因沉默方法以及将裸DENV RNA转染到AP2M1敲低细胞中来研究AP2M1的作用。其次，是AAK1的抑制剂 舒尼替尼用于研究AAK1是否会影响DENV感染的Huh7细胞中的病毒产生。DENV感染的Huh7细胞中AP2M1的敲低表明感染后24 h病毒滴度降低。此外，进行了实验以使用裸DENV RNA转染到AP2M1敲低细胞中绕过DENV内部化。观察到较高的细胞内DENV RNA，DENV E蛋白和细胞内病毒体，而细胞外病毒体的产生却比对照少。在DENV感染的Huh7细胞中用舒尼替尼治疗能够减少细胞外病毒体的产生，并且与DENV的所有四种血清型一致。因此，