P33介导的杆状病毒二硫键形成通路中底物蛋白的研究

Disulfide bonds play important roles to the protein function. Some large DNA viruses encode their own disulfide bond formation pathway for viral replication. Cellular and viral disulfide bond formation pathways are generally made of multiple proteins including sulfhydryl oxidase, shuttle protein(s), and substrate(s). Recently, a new sulfhydryl oxidase P33 was identified in baculovirus. P33 encodes a falvin adenine dinucleotide (FAD)-linked sulfhydryl oxidase related to the cellular Erv enzyme family. It is required for the replication of baculovirus and has been proved to have sulfhudryl oxidase activity in vitro. So far sulfhudryl oxidases have been identified in six viral families: poxviridae, asfarviridae, iridovirdae, mimiviridae, phycodnaviridae and baculoviridae. Among these viruses, baculovirus is the only one that replicates in the nucleus, while the others all replicates in the cytoplasm. Structural analysis showed that P33 is a highly divergent member of the Erv flavoenzyme family with a complex quaternary structural arrangement which has not been previously seen in cellular or viral enzymes of the family. Therefore, it is predicted that baculovirus may encode a novel disulfide bond formation pathway. However, so far, there has not been any report of P33 being involved in disulfide bond formation of baculoviral proteins, and no substrate proteins have been identified. In this project, we plan to identify the substrate(s) of the potential P33 mediated baculoviral disulfide bond formation pathway by using the prototype baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) with following studies: 1) systematically screening all the conserved structural proteins of AcMNPV with bioinformatics, biochemistry and proteomics methods to identify the potential substrates with disulfide bond(s). 2) Using different techniques to identify proteins which interact with P33, and then select proteins with potential to be substrates or shuttle proteins. 3) Constructing a recombinant AcMNPV in which the expression of P33 is regulated by an inducer. Then the redox status of the conserved cysteins in the above candidates identified by 1) and 2) will be studied under the conditions with and without the expression of P33. With these methods, we should be able to show the evidence whether P33 is involved in the disulfide bond formation of baculoviral proteins, identify the substrate protein(s), and prove that baculovirus do encode its own disulfide bond formation pathway. The results will extend the knowledge of disulfide bond formation pathways in nature, promote the future studies of the molecular mechanism of this novel disulfide bond formation pathway, as well as improve our understanding of the widely used baculovirus expression system.

二硫键在蛋白质的生命活动中发挥着重要作用。一些大的DNA病毒因复制的需要编码了自身的二硫键形成通路。二硫键形成通路一般由巯基氧化酶、传递蛋白和底物蛋白所组成。 最近在杆状病毒中新发现了一个巯基氧化酶P33，它是病毒复制的必需蛋白，在体外具有巯基氧化酶活性。以前报道的含巯基氧化酶的病毒都在细胞质中复制,而杆状病毒是核内复制的病毒, P33的结构也有别于其它巯基氧化酶，预示着杆状病毒可能编码一套全新的二硫键形成通路。但是，目前尚无证据证明P33是否介导了,以及介导了哪些杆状病毒蛋白质的二硫键形成。本项目拟1）系统揭示杆状病毒保守结构蛋白中哪些存在二硫键；2）利用蛋白相互作用等技术发现与P33相互作用的病毒蛋白；3）通过对比P33存在及缺失时上述蛋白质二硫键形成的差异，最终揭示P33介导形成二硫键的底物蛋白，从而确证杆状病毒存在依赖于P33的二硫键形成通路，为今后深入研究其分子机制奠定基础。

二硫键在蛋白的折叠和装配过程中发挥着重要作用，许多病毒蛋白含有分子间或分子内二硫键，且它们与病毒的入侵、感染的装配密切相关。杆状病毒含有一个保守的巯基氧化酶P33，预示其可能自身编码一套二硫键形成通路。本项目的研究目标是证实 P33介导杆状病毒蛋白二硫键的形成，并揭示通路中的底物蛋白，为深入研究杆状病毒二硫键形成通路奠定基础。研究内容包括：1）揭示杆状病毒巯基氧化酶P33的结构与功能的关系的内在联系；2）通过系统性筛选P33可能的候选底物蛋白；3）利用p33缺失型重组病毒，最终确定 P33的底物蛋白。.通过4年的研究，我们取得了下列重要结果：1）深入揭示了P33的结构与功能：鉴定了棉铃虫核多角体病毒HaP33的功能（Kuang et al., 2016, Virologica Sinica）；解析了苜蓿银纹夜蛾核型多角体病毒P33 (AcP33)野生型及5种突变体的蛋白晶体结构，发现了三个重要的结构保守性区域对巯基氧化酶活性及病毒的形态发生至关重要(Kuang et al., 2017, Journal of Virology) 。2）对P33介导的二硫键通路中的底物蛋白进行了系统性鉴定和筛选：筛选出VP91，PIF5等7种候选底物蛋白。3）首次揭示PIF5是P33的底物蛋白：证实了杆状病毒口服感染因子PIF5分子内二硫键的形成是由P33介导形成的；揭示了PIF5三对分子内二硫键对杆状病毒的口服感染是必需的(Zhang et al., revised to Jounral of Virology)。.上述研究深入揭示了P33的结构与功能的关系，首次发现了P33的底物蛋白PIF5，确证了P33介导的杆状病毒二硫键形成通路的存在，为今后深入揭示该系统的分子机制奠定了坚实的基础。截至目前，共发表项目标注的SCI论文3篇，其中1篇发表在Journal of Virology；另外一篇已在Journal of Virology修回。.

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