Das Soma, Laxminarayana Suhas Venkataramana, Chandra Nagasuma, Ravi Vasanthapuram, Desai Anita
Department of Neurovirology, National Institute of Mental Health and Neurosciences, Bangalore, India.
Virology. 2009 Mar 1;385(1):47-57. doi: 10.1016/j.virol.2008.10.025. Epub 2008 Dec 7.
Japanese encephalitis virus (JEV) envelope (E) protein has been shown to play a critical role in attachment to cells. However, the receptor interacting with envelope protein has not been conclusively identified. Using mouse neuroblastoma (Neuro2a) cells and purified JEV-E protein in 'Virus Overlay Protein Binding Assay' followed by MALDI-TOF analysis, we identified 'heat shock protein 70' (Hsp70) as a possible receptor for JEV. Indirect immunofluorescence and flow-cytometry analysis demonstrated localization of Hsp70 on Neuro2a cell surface. Co-immunoprecipitation followed by Western blot analysis reconfirmed the interaction between Hsp70 and JEV-E protein. Further, anti-Hsp70 polyclonal-antibodies were able to block JEV entry into Neuro2a cells. Additionally, using the bioinformatic tool - FTDOCK, docking between the proteins was performed. Amongst six interacting structural poses studied one pose involving RGD motif on JEV-E and leucine(539) on Hsp70 displayed stable interaction. These observations indicate that Hsp70 serves as putative receptor for JEV in Neuro2A cells.
日本脑炎病毒（JEV）包膜（E）蛋白已被证明在病毒与细胞的附着过程中起关键作用。然而，与包膜蛋白相互作用的受体尚未得到最终确定。在“病毒覆盖蛋白结合试验”中，我们使用小鼠神经母细胞瘤（Neuro2a）细胞和纯化的JEV-E蛋白，随后进行基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）分析，确定“热休克蛋白70”（Hsp70）可能是JEV的受体。间接免疫荧光和流式细胞术分析表明Hsp70定位于Neuro2a细胞表面。免疫共沉淀后进行蛋白质印迹分析再次证实了Hsp70与JEV-E蛋白之间的相互作用。此外，抗Hsp70多克隆抗体能够阻断JEV进入Neuro2a细胞。另外，使用生物信息学工具FTDOCK对这两种蛋白质进行对接。在所研究的六个相互作用结构构象中，其中一个构象涉及JEV-E上的RGD基序和Hsp70上的亮氨酸（539），显示出稳定的相互作用。这些观察结果表明，Hsp70在Neuro2A细胞中作为JEV的假定受体。
