烟芽夜蛾囊泡病毒3h株编码的3H-117蛋白能干扰AcMNPV的口服活性

3H-117 encoded by Heliothis virescens ascovirus 3h interferes the oral activity of AcMNPV

摘要:

【目的】囊泡病毒是一类体液传播的昆虫病毒，具有特殊的致病历程和良好的生防应用潜力。烟芽夜蛾囊泡病毒3h株（Heliothis virescens ascovirus 3h，HvAV-3h）是我国分离的第一株囊泡病毒，其编码的3H-117蛋白被报道为HvAV-3h病毒粒子的结构蛋白。【方法】为了进一步探究3h-117基因的功能，本研究以Bac-to-Bac昆虫表达系统为研究平台，将3h-117插入到苜蓿银纹夜蛾核型多角体病毒（Autographa californica multiple nuclepolyhedrovirus，AcMNPV）的基因组中，成功构建了具有口服活性的重组病毒AcMNPV-117。【结果】通过与野生型病毒（AcMNPV-Egfp）的比较，证实在感染Sf9细胞72-96 h后，AcMNPV-117的出芽型病毒粒子产量以及病毒DNA的拷贝数显著降低，且AcMNPV-117的多角体明显增大。进一步的生测结果表明，AcMNPV-117对三龄甜菜夜蛾（Spodoptera exigua）的90%致死浓度明显高于AcMNPV-Egfp。此外，被病毒感染的甜菜夜蛾幼虫生长相对健康幼虫明显减缓，且试虫的日均取食量也因病毒的感染而被抑制。共聚焦结果表明，3H-117与AcMNPV-117感染后宿主细胞核骨架的聚集过程相关，提示3H-117在囊泡病毒感染过程中可能具有瓦解宿主细胞核的功能。【结论】本研究的结果进一步验证了3h-117的功能，为囊泡病毒的分子生物学研究奠定了基础。

Abstract:

[Objective] Ascoviruses are humor transmitted insect viruses that have unique pathogenicity and are competent to develop into bioinsecticides. Heliothis virescens ascovirus 3h (HvAV-3h) is the first ascovirus strain that isolated in China, and the 3H-117 protein encoded by HvAV-3h are reported as a structural protein of HvAV-3h.[Methods] In order to further illustrate the function of 3h-117, the Bac-to-Bac insect expression system was employed to construct a recombinant Autographa californica multiple nuclepolyhedrovirus (AcMNPV) by inserting 3h-117 into the genome of AcMNPV.[Results] Compared with the wild type control AcMNPV (AcMNPV-Egfp), the generated recombinant baculoviurs (AcMNPV-117) had a significantly reduced budded virus production and viral DNA copies in the infected Sf9 cells during 72-96 hpi, and an enlarged occlusion bodies was found in AcMNPV-117. Further bioassays showed that the 90% lethal dosage of AcMNPV-117 against the third instar Spodoptera exigua are significantly higher than the 90% lethal dosage of AcMNPV-Egfp. Furthermore, the growth of the tested S. eixuga larvae infected with AcMNPVs was slower than that of the healthy larval growth, and the average daily food intake of the tested larvae was also inhibited by the baculovirus infection. The confocal observation showed that 3H-117 is associated with the nuclear actin polymerization in AcMNPV-117 infected Sf9 cells, which indicated that the function of 3H-117 in the infectious procedures of ascovirus is associated to the disintegration of host cell nucleus.[Conclusion] The results obtained in this study confirmed the function of 3h-117, which laid a foundation for the molecular biology research of ascovirus.