杆状病毒核心基因ac78的缺失影响多粒包埋型病毒粒子形成的分子机制研究

Baculoviruses are a very diverse group of insect-specific viruses with rod-shaped, enveloped, double-stranded DNA genomes that specifically infect insect species mainly from the orders Lepidoptera, Hymenoptera, and Diptera. ac78 of the archetype of the Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is a recently identified baculovirus core gene. Our previous studies showed that the deletion of ac78 severely affected the morphogenesis of multiple nucleocapsid-enveloped occlusion-derived virions (M-ODVs). Bioinformatics analysis showed that Ac78 contains a putative cytochrome C oxidase N-terminal region, which has been shown to be important in the M-ODV morphogenesis. Here, we hypothesize that Ac78 may have cytochrome C oxidase activity; Ac78may work together with the baculovirus sulfhydryl oxidase P33 in a redox pathway for protein disulfide bond formation, which may be related to the formation of M-ODVs..In this study, we will be working on the role of ac78 in the formation of M-ODVs using various molecular biology, immunohistochemistry, immunoprecipitation techniques and methods. To investigate whether Ac78 has cytochrome C oxidase activity, Ac78 will be prokaryotic expressed in Escherichia coli and purified, and the cytochrome C oxidase activity of Ac78 will be assayed using a Purified Cytochrome C Oxidase Activity Assay Kit. To detect the interrelation of Ac78 with baculovirus sulfhydryl oxidase P33, two p33 repaired viruses will be constructed, in which the p33 gene with its native promoter and ORF plus a FLAG tag fused in-frame to the C terminus of p33 ORF is inserted into the polh locus of ac78 deletion bacmid bAc78KO and AcMNPV bacmid bMON14272, respectively. Sf9 cells will be transfected with these recombinant viruses and be collected at the designated times post transfection. The sulfhydryl oxidase activity of P33 in Sf9 cells will be detected, and the reductive degree of P33 sulfhydryl will be analyzed by Western blotting. Using immunoprecipitation and co-immunoprecipitation, we will investigate the baculovirus proteins which have interaction with Ac78 or P33, respectively. Subsequently, these protein repaired viruses will be constructed on the basis of p33 deletion bacmid bAc92KO, bAc78KO and bMON14272, respectively. To investigate whether AcMNPV has a redox pathway for protein disulfide bond formation which is related to the formation of M-ODVs, these recombinant viruses will be used to study the redox relationship of these proteins with Ac78 or P33..In summary, this study will further investigate the molecular mechanism for the effects of deletion of ac78 on the the formation of M-ODVs. This research will be important for revealing the replication and infection mechanism of baculovirus. It also will provide important theoretical basis for the better use of baculovirus to control pests and construct improved genetic engineering baculovirus.

苜蓿丫纹夜蛾核多角体病毒（AcMNPV）ac78是最近被发现的杆状病毒核心基因，缺失该基因的病毒在感染的细胞中多粒包埋型病毒（M-ODV）的产生显著减少。生物信息学分析显示Ac78可能具有细胞色素C氧化酶活性。本项目拟以ac78为研究对象，应用分子生物学、免疫组织化学、免疫沉淀等技术和手段，原核表达并纯化Ac78，检测Ac78的酶活性；在野生型和ac78缺失型bacmid中分别构建p33补回型重组病毒，利用这些重组病毒分析Ac78与杆状病毒巯基氧化酶P33的关系；利用免疫沉淀和免疫共沉淀技术，确定Ac78和P33通过与病毒中的哪些蛋白相互作用来行使功能，探讨AcMNPV中与M-ODV形成相关的可能的二硫键形成还原通路。深入研究Ac78参与M-ODV形成的分子机制对揭示杆状病毒的复制与侵染机制具有重要意义，也为更好地利用杆状病毒防治害虫和构建遗传改良基因工程杆状病毒提供重要的理论依据。

苜蓿丫纹夜蛾核多角体病毒(AcMNPV) ac78是杆状病毒核心基因，我们前期研究发现，缺失该基因的病毒在感染的细胞中多粒包埋型病毒粒子M-ODV的产生显著减少，且不能在敏感宿主的中肠中建立初始感染；生物信息学分析表明，Ac78含有一个可能的细胞色素C氧化酶N端功能区，并且该区域已被证明在感染性芽生型病毒粒子BV和M-ODV的形态发生中具有重要作。推测Ac78具有细胞色素C氧化酶活性，并以氧化酶的形式参与M-ODV的形态发生。本项目通过构建Ac78的原核表达质粒，在大肠杆菌中成功获得可溶性表达，纯化Ac78，使用纯化细胞色素C氧化酶活性测定试剂盒检测Ac78的氧化酶活性，结果呈阴性，表明Ac78可能不具有细胞色素C氧化酶活性。对Ac78关键氨基酸位点的研究发现，Ac78 D4、P6、P20、D27、L78、Y82、V85和I86对于感染性BV和M-ODV的有效产生不是必需的，而R9和K22对于感染性BV和M-ODV的有效产生是必需的。在野生型AcMNPV bacmid (bMON14272)和ac78缺失型bacmid (bAc78KO)中分别补回一份C端带FLAG标签且在自身启动子控制下的p33，成功构建了重组病毒vAcWT:ac92FLAG和vAc78KO:ac92FLAG，测定比较两者中带FLAG标签的P33的巯基氧化酶活性，平均kcat值分别为81/min和86/min，并无明显差异，表明Ac78可能不影响P33的巯基氧化酶活性。此外，在研究过程中发现，AcMNPV orf111 (ac111)参与了AcMNPV的口服感染过程，并且该过程具有宿主依赖性。. 综上所述，本项目深入研究了Ac78的作用机制，解析了Ac78关键氨基酸的功能，对揭示杆状病毒的复制与侵染机制具有重要意义，也为更好地利用杆状病毒防治害虫和构建遗传改良基因工程杆状病毒提供重要的理论依据。