家蚕蛋白MyosinⅡ在BmNPV诱导宿主自噬以及病毒复制增殖中的作用机制研究

A huge amount of studies have shown that many mammalian RNA viruses can hijack the host autophagy machinery favoring viral replication. However, there are currently very few studies on the relationship of other animal viruses or DNA viruses with the host autophagy machinery. There are no reports about the autophagy machinery of the economic insect silkworm (Bombyx mori) and its relationship with the silkworm severe pathogen BmNPV (Bombyx mori nuclear polyhedrosis virus, a DNA virus)...Recently, production of aggregates and aggresomes by BmNPV infection in BmN was detected, thereby inducing autophagy pathway of host. Inhibition of autophagy by 3-MA can reduce significantly formation of viral occlusion body (OB), revealing that silkworm autophagy is indeed functioning in BmNPV infection. Moreover, our later research showed that BmMyosinⅡ was identified to participate in the formation of aggresome. ..Based on these findings, we will employ multiple techniques, including gene cloning, protein expression and purification, RNAi, Co-immunoprecipitation, Bac-to-Bac expression system, real-time fluorescence quantitative PCR, fluorescence microscopy, TALEN to further characterize the function of BmMyosinⅡ in the silkworm autophagy and the infection process of BmNPV. Our study will provide new insights into the important roles of autophagy machinery in the infection of DNA viruses, update our understanding of the interaction between virus and host, and facilitate the discovery of novel targets for the virus

最近大量研究发现哺乳动物RNA病毒利用宿主自噬途径来完成自己的复制增殖，但是对其它动物病毒或DNA病毒与自噬关系的报道则极少，也未见对经济昆虫家蚕自噬与其重大DNA病毒BmNPV关系的研究。目前实验证实BmNPV的侵染能够形成聚集体，并引起宿主自噬，自噬的抑制能够显著降低多角体病毒的产生，表明家蚕自噬与BmNPV侵染密切相关。我们的前期实验证实家蚕蛋白MyosinⅡ参与了BmNPV诱导的聚集体形成过程，在此基础上，我们拟综合应用基因克隆、蛋白表达纯化、Bac-to-Bac表达系统、免疫共沉淀，荧光定量PCR、荧光显微镜、RNAi、TALEN等方法，进一步研究BmMyosinⅡ在自噬及BmNPV感染中的功能，揭示自噬在DNA病毒侵染中的重要作用，增进对病毒与宿主相互作用的了解，有助于发现控制病毒播散的新靶点。

家蚕杆状病毒（BmNPV）既是一种重要的家蚕病害病原，对蚕业生产具有极大危害，也是一种重要的真核表达系统，可以用于蛋白制品的生产。目前对于哺乳动物RNA病毒利用宿主自噬途径完成自身复制增殖的研究较多，对于动物病毒或DNA病毒与自噬关系的报道则极少。本项目在前期预实验的基础上，研究发现BmNPV可以挟持宿主自噬途径来帮助病毒多角体和包涵体病毒的形成。干涉BmNMMYH的表达能够减弱自噬体的形成和自噬流，表明家蚕BmNMMY参与了病毒挟持宿主形成自噬的过程。而对BmNMMY互作蛋白的鉴定同样表明多个病毒蛋白参与了这一过程，为后续继续研究BmNPV与宿主自噬互作打下了基础。本研究对BmNPV侵染引起的自噬进行了初步分析，丰富了自噬在DNA病毒侵染中的作用研究素材，也为后期利用自噬途径对病毒扩散进行控制，以及调控杆状病毒表达系统表达外源蛋白打下基础。