Multiple membrane trafficking networks operate in the eukaryotic cell and are hijacked by viruses to establish infection. Recent studied have highlighted that viruses can exploit distinct pathways depending on the cell type. Japanese encephalitis virus (JEV), a neurotropic flavivirus, can infect neuronal cells through a clathrin-independent endocytic mechanism. To further characterize the membrane trafficking requirements for JEV infection of neuronal cells, we have performed a RNA interference-based study targeting 136 proteins in the human cell line IMR-32. Through quantitative RT-PCR and plaque assays we have validated that JEV infection in neuronal cells was independent of clathrin, and identified host-factors that were crucial for establishment of infection. Several of these proteins were involved in regulation of actin filament organization such as RHOA, RAC1, proteins of the ARP2/3 complex and N-WASP family, LIMK1, PAK1 and ROCK2. The small molecule inhibitors of ARP2/3 complex, CK-548 and of the N-WASP, Wiskostatin inhibited virus replication highlighting the important roles of these proteins in the virus life-cycle. We also identified ATG12, BECN1, VAPA, VAPB and VCP proteins as crucial host-factors for JEV replication across epithelial and neuronal cell lineages.

多个膜运输网络在真核细胞中运行，并被病毒劫持以建立感染。最近的研究表明，病毒可以根据细胞类型利用不同的途径。日本脑炎病毒（JEV），一种神经性黄病毒可以通过不依赖网格蛋白的内吞机制感染神经元细胞。为了进一步表征JEV感染神经元细胞的膜运输需求，我们进行了一项基于RNA干扰的研究，靶向人细胞系IMR-32中的136种蛋白质。通过定量RT-PCR和噬菌斑测定，我们已验证神经元细胞中的JEV感染独立于网格蛋白，并鉴定了对感染建立至关重要的宿主因子。这些蛋白质中的几种参与肌动蛋白丝组织的调节，例如RHOA，RAC1，ARP2 / 3复合物和N-WASP家族的蛋白质，LIMK1，PAK1和ROCK2。ARP2 / 3复合物的小分子抑制剂CK-548和N-WASP的小分子抑制剂Wiskostatin抑制病毒复制，突显了这些蛋白在病毒生命周期中的重要作用。