家蚕ADP/ATP转运酶(BmANT)抑制BmNPV增殖作用机制研究

Inhibition of BmNPV proliferation by Bombyx mori ADP/ATP transportase (BmANT)

摘要:

[目的]家蚕核型多角体病毒（Bombyx mori nucleopolyhedrovirus，BmNPV）隶属于杆状病毒，需要借助宿主细胞能量代谢进行自身增殖复制。家蚕ADP/ATP转运酶（Bombyx mori ADP/ATP translocase，BmANT）是线粒体转运蛋白，在BmNPV感染条件下和家蚕热休克蛋白60（heat shock protein 60，BmHSP60）具有直接的相互作用。因此，鉴定Bmant基因在BmNPV感染过程中的功能特征，有助于解析杆状病毒劫持宿主细胞因子促进自身增殖复制机制，完善杆状病毒和宿主相互作用网络。[方法]通过结构域预测BmANT蛋白的结构特征，荧光定量PCR分析Bmant基因在BmNPV感染后的变化特征；并过表达BmANT检测其对病毒DNA复制和病毒蛋白表达变化影响；进一步在转录水平分析Bmant和Bmhsp60基因的调控关系；最后通过流式细胞术等技术鉴定Bmant和Bmhsp60基因共同调控BmNPV增殖复制的机制。[结果]SMART软件预测显示BmANT包含3个线粒体载体结构域，BmNPV感染24 h后Bmant基因持续下调表达。过表达Bmant基因能够显著抑制BmNPV DNA的复制和VP39蛋白表达。荧光定量PCR分析显示Bmant和Bmhsp60基因具有相互拮抗作用，能够相互抑制转录。Bmant和Bmhsp60共同过表达分析显示，BmANT和BmHSP60共同作用BmNPV能够抑制病毒的增殖复制。[结论]结果表明，BmANT是一个线粒体载体蛋白，具有显著的抗病毒作用，能够下调Bmhsp60基因表达，并抑制BmNPV增殖复制。

关键词:家蚕;家蚕核型多角体病毒;BmANT;BmHSP60;抗病毒

Abstract:

[Objective] Bombyx mori nucleopolyhedrovirus (BmNPV) belongs to the family Baculoviridae. It replicates and proliferates using its host cell's energy metabolism. Bombyx mori ADP/ATP translocase (BmANT) is a transporter that interacts with the host energy metabolism factor, heat shock protein 60 (BmHSP60), during BmNPV infection. To determine which of the functional characteristics of the Bmant gene affect the process of BmNPV infection, we here analyze the mechanism by which the baculovirus hijacks host cytokines to promote viral proliferation and replication. [Methods] The structural characteristics of the Bmant gene were predicted, and qRT-PCR was performed to analyze the changes in the Bmant gene after BmNPV infection. The effect of this gene on viral DNA replication and viral protein expression was analyzed using overexpression of the Bmant gene. We further analyzed the relationship between the Bmant and Bmhsp60 genes in transcriptional regulation. The mechanism by which Bmant and Bmhsp60 regulates the proliferation and replication of BmNPV was identified using flow cytometry and Western blotting analysis. [Results] SMART software predicted that BmANT protein contained three mitochondrial vector domains, and the Bmant gene down-regulated expression 24 h after BmNPV infection. Overexpression of the Bmant gene significantly inhibited BmNPV DNA replication and VP39 protein expression. QRT-PCR analysis showed the Bmant and Bmhsp60 genes to have mutual antagonism and to be capable of inhibiting each other's transcription. Co-transfection analysis of the Bmant and Bmhsp60 genes showed that the Bmant gene can significantly inhibit the action of the Bmhsp60 gene and ultimately inhibit the proliferation and replication of BmNPV. [Conclusion] The results indicated that the Bmant gene is a mitochondrial carrier protein with significant antiviral activity that is capable of down-regulating the expression of Bmhsp60 and inhibiting BmNPV proliferation and replication.

Key words:Bombyx mori;BmNPV;BmANT;BmHSP60;antiviral