Direct interactions between bidensovirus BmDNV‐Z proteins and midgut proteins from the virus target Bombyx mori
Direct interactions between bidensovirus BmDNV‐Z proteins and midgut proteins from the virus target Bombyx mori
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DOI：
10.1111/febs.12088
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发表时间：
2013-02
期刊：
影响因子：
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通讯作者：
Yan-Yuan Bao;Li-Bo Chen;Wenjuan Wu;Dong Zhao;Ying Wang;Xia Qin;Chuan-Xi Zhang
中科院分区：
文献类型：
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作者：
Yan-Yuan Bao;Li-Bo Chen;Wenjuan Wu;Dong Zhao;Ying Wang;Xia Qin;Chuan-Xi Zhang
在本研究中，我们旨在通过酵母双杂交 (Y2H) 系统鉴定家蚕中肠和双链病毒 BmDNV-Z 之间的蛋白质-蛋白质相互作用。为了实现这一目标，我们构建了表达宿主基因的 Gal4 激活域融合文库和表达 BmDNV-Z 基因的 Gal4 DNA 结合域融合诱饵载体。 Y2H 测定揭示了宿主和病毒蛋白之间的 15 种潜在相互作用。为了验证相互作用，我们修改并重建了一对双分子荧光互补（BiFC）载体，并实现了候选宿主基因和病毒基因在昆虫培养细胞中的共表达。 BiFC 测定证实了相互作用的特异性，包括 B. mori 35 kDa 蛋白酶和由 VD1-ORF2 和 VD2-ORF1 编码的两个 BmDNV-Z 蛋白； B. mori transgelin 和 VD2-ORF3 编码的 BmDNV-Z 蛋白；以及体外由 VD2-ORF3 编码的 B. mori 丝氨酸蛋白酶前体和 BmDNV-Z。我们的研究结果表明，特定的宿主中肠蛋白参与了家蚕和 BmDNV-Z 之间的相互作用，这将有助于我们了解 BmDNV-Z 感染的分子机制。
In this study we aimed to identify the protein–protein interactions between Bombyx mori midgut and the bidensovirus BmDNV‐Z via a yeast two‐hybrid (Y2H) system. To achieve this we constructed a Gal4 activation domain fusion library that expresses the host genes and Gal4 DNA binding domain fusion bait vectors that express BmDNV‐Z genes. Y2H assay revealed 15 potential interactions between host and viral proteins. To verify the interactions, we modified and reconstructed a pair of bimolecular fluorescence complementation (BiFC) vectors and achieved the co‐expressions of the candidate host genes and viral genes in insect culture cells. The BiFC assay confirmed the specificity of the interactions including B. mori 35 kDa protease and two BmDNV‐Z proteins encoded by VD1‐ORF2 and VD2‐ORF1; B. mori transgelin and BmDNV‐Z protein encoded by VD2‐ORF3; and B. mori serine protease precursor and BmDNV‐Z encoded by VD2‐ORF3 in vitro. Our findings revealed that the specific host midgut proteins are involved in the interactions between B. mori and BmDNV‐Z, which will facilitate our understanding of the molecular mechanisms of BmDNV‐Z infection.