Exo70促进牛疱疹病毒1型释放的分子机制研究

Exo70是Exocyst复合体关键亚基，通过调控分泌泡的胞内运输及胞膜锚定调节物质分泌，尚无其调控病毒释放机制的报道。牛疱疹病毒1型（BoHV-1）经分泌泡方式释放，但机制不清楚。我们前期研究发现，Exo70基因调控BoHV-1分泌泡胞内运输及胞膜锚定，促进病毒释放。本研究拟过表达及沉默Exocyst复合体各亚基基因，探讨Exo70是单独或者以Exocyst复合体方式发挥作用；突变Exo70与胞膜PI(4,5)P2结合位点，检测病毒蛋白gB细胞膜定位变化，分析其促进病毒分泌泡锚定细胞膜机制；过表达和沉默Rab11a及Exo70，分析Rab11a在后者促进病毒胞内运输中作用；过表达及沉默STUB1等候选E3泛素连接酶，检测Exo70蛋白泛素化变化，揭示Exo70降解机制。本研究拟以Exo70与病毒分泌泡胞内运输及细胞膜锚定的关系为切入点，探讨Exo70促进BoHV-1释放的分子机制。

Exo70 is a key subunit of exocyst complex, which regulates secretion of substances via regulating intracellular transport and membrane anchoring of secretory vesicles, whereas the molecular mechanism that Exo70 regulates viral releases remains unclear. Although bovine herpesvirus 1 (BoHV-1) is released via secretory vesicles, its mechanism is unknown. Our previous study showed that Exo70 gene regulated the intracellular transport and cellular membrane anchoring of BoHV-1 secretory vesicles and promoted the virus release. In this study, overexpression and silencing of different subunit genes of exocyst complex will be performed, and then whether Exo70 functions alone or in the manner of exocyst complex will be explored. After mutation of the binding sites between Exo70 and PI (4,5) P2, the cellular membrane location of viral gB protein will be detected, and the mechanism of Exo70 promoting viral secretory vesicle anchoring cell membrane will be analyzed. Rab11a and Exo70 will be respectively overexpressed or silenced, the role of Rab11a in the process of Exo70 promoting viral intracellular transport will be determined. After overexpression or silencing of E3 ubiquitin ligase candidate genes including STUB1, the ubiquitination of Exo70 protein will be detected to reveal the mechanism of Exo70 degradation. A new perspective from the relationship between Exo70 and intracellular transport as well as membrane anchoring of viral secretory vesicles is to investigate, which will further explore the molecular mechanism of Exo70 promoting the release of BoHV-1.