Chu J J H, Leong P W H, Ng M L
Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, Singapore 117597, Singapore.
Virology. 2006 Jun 5;349(2):463-75. doi: 10.1016/j.virol.2006.01.022. Epub 2006 Feb 21.
The initial interaction between mosquito-borne flavivirus West Nile and mosquito cells is poorly characterized. This study analyzed the endocytic and the associated signaling pathway that mediate the infectious entry of West Nile virus (WNV) into mosquito cell line (C6/36). Pretreatment of C6/36 cells with pharmacological drugs that blocks clathrin-mediated endocytosis significantly inhibited virus entry. Furthermore, the transfection of functional blocking antibody against clathrin molecules and the overexpression of dominant-negative mutants of Eps15 in C6/36 cells caused a marked reduction in WNV internalization. WNV was shown to activate focal adhesion kinase (FAK) to facilitate the endocytosis of virus but not the mitogen-activated protein kinases (ERK1 and ERK2). Subsequent to the internalization of WNV, the virus particles are translocated along the endosomal pathway as revealed by double-immunofluorescence assays with anti-WNV envelope protein and cellular markers for early and late endosomes. Specific inhibitor for protein kinase C (PKC) was shown to be highly effective in blocking WNV entry by inhibiting endosomal sorting event. The disruption of the microtubule network using nocodazole also drastically affects the entry process of WNV but not the disruption of actin filaments by cytochalasin D. Finally, a low-pH-dependent step is required for WNV infection as revealed by the resistance of C6/36 cells to WNV infection in the presence of lysosomotropic agents.
蚊媒黄病毒西尼罗河病毒与蚊子细胞之间的初始相互作用目前了解甚少。本研究分析了介导西尼罗河病毒（WNV）进入蚊子细胞系（C6/36）的内吞作用及相关信号通路。用阻断网格蛋白介导的内吞作用的药物预处理C6/36细胞，可显著抑制病毒进入。此外，在C6/36细胞中转染针对网格蛋白分子的功能性阻断抗体以及过表达Eps15的显性负性突变体，会导致WNV内化显著减少。WNV可激活粘着斑激酶（FAK）以促进病毒的内吞作用，但不激活丝裂原活化蛋白激酶（ERK1和ERK2）。WNV内化后，通过用抗WNV包膜蛋白以及早期和晚期内体的细胞标志物进行双重免疫荧光测定发现，病毒颗粒沿内体途径转运。蛋白激酶C（PKC）的特异性抑制剂通过抑制内体分选事件，在阻断WNV进入方面显示出高效性。用诺考达唑破坏微管网络也会极大地影响WNV的进入过程，但用细胞松弛素D破坏肌动蛋白丝则不会。最后，如在溶酶体促渗剂存在的情况下C6/36细胞对WNV感染具有抗性所显示的，WNV感染需要一个低pH依赖性步骤。
