昆虫杆状病毒的潜伏感染以及超感染排斥机制

Baculovirus latent infection has been frequently observed in insect populations, which has important effects on the baculovirus epizootics and insect population size. However, the molecular mechanism has not been clarified. In our previous work, Spodoptera exigua nucleopolyhedrovirus (SeMNPV) latently infected Se301 cells P8-Se301-C1 were established, and the cells show the traits of superinfection exclusion when infected with homologous virus SeMNPV. By using the SeMNPV-infected P8-Se301-C1 cells as superinfection exclusion model system, the aims of this project are to investigate the superinfection exclusion mechanism of insect cells to baculovirus. We first use chloroquine, actinomycin D and aphidicolin to block the baculovirus infection in the early stages, so to study the timing of superinfection exclusion upon homologous virus infection. Then, by using RNA-Seq technique, we are going to illustrate the transcriptomes of the P8-Se301-C1 cells superinfected with SeMNPV, to identify the biological pathways that particularly processed by latent infection, and to reveal the immune responses of the latently infected cells to superinfection exclusion by homologous baculovirus infection. Finally, by the method of RNA interference, we will determine the functions of the differentially expressed host genes associated with supinfection exclusion in the P8-Se301-C1 cells. This study would be fundamental to reveal the interactions between the baculovirus and latently infected insect cells, and the molecular mechanism of baculovirus latent infection and its activation. Moreover, it will provide important theoretical basis for better application of baculovirus as pesticide in biocontrol of agricultural and forestry insects.

杆状病毒的潜伏感染具有普遍性，但其分子机理尚不清楚。Spodoptera exigua nucleopolyhedrovirus（SeMNPV）是危害全球农作物的杂食性害虫——甜菜夜蛾特异且高致病力的天然病原物。申请人于2009年报道了SeMNPV潜伏感染细胞株P8-Se301-C1的建立及该细胞株对同源病毒的超感染具排斥现象。本项目拟（1）利用氯喹、放线菌素D和芽栖菌素处理P8-Se301-C1细胞，将SeMNPV的复制分别阻断于胞吞、RNA转录和DNA复制等阶段，探索超感染排斥的关键节点；（2）利用RNA-Seq技术解析SeMNPV超感染P8-Se301-C1细胞的转录组，阐明相关的生物学通路；（3）通过RNAi技术沉默上述通路部分基因的表达，发现克服超感染排斥的关键基因。本项目研究结果将为提高昆虫病毒的致病性，调控病毒流行病，综合管理农田害虫具有重要意义。

杆状病毒的潜伏感染具有普遍性，但其分子机理尚不清楚。SeMNPV是危害全球农作物的杂食性害虫甜菜夜蛾特异且高致病力的天然病原物。先前我们建立SeMNPV潜伏感染细胞株P8-Se301-C1 (C1)，对同源病毒的超感染具排斥现象。本项目通过药物阻断、RNA-Seq和RNAi技术等，研究差异表达宿主细胞的基因功能及宿主细胞克服杆状病毒超感染排斥机制。结果如下：1.RNA-Seq测序表明，与SeMNPV感染6 h的Se301细胞相比，超感染的C1细胞中包括1781个上调和1194个下调基因。GO富集分析到1093个差异表达基因。KEGG分析表明其中的363个差异表达基因被注释到114条通路中，主要为胞外物质的相互作用和代谢途径通路。2.通过氯喹处理、4°C孵育和间接固定相酶联免疫法明确C1超感染排斥发生在病毒吸附阶段。抗体封闭SeMNPV F蛋白位点，Se301细胞病毒吸附量下降。利用SeMNPV F蛋白替换AcMNPV GP64，C1细胞对重组AcMNPV的吸附降低。表明F蛋白参与SeMNPV和AcMNPV感染Se301细胞的吸附阶段，并在C1细胞超感染排斥中起关键作用。3.筛选获得细胞受体相关的糖基化蛋白基因Senpc1。利用NPC1抑制剂处理和RNAi干扰Se301细胞中Senpc1转录表达，细胞表面SeMNPV吸附量下降，表明SeNPC1蛋白参与SeMNPV感染Se301细胞的吸附阶段。生物信息学和酵母双杂实验表明F蛋白与SeNPC1蛋白存在互作。推测Senpc1表达下调降低SeMNPV对细胞的吸附，导致C1对SeMNPV吸附阶段的排斥。4.C1细胞中钙网蛋白crt基因转录水平较Se301细胞下调。通过RNAi干扰Se301细胞Secrt转录后，细胞增殖和SeMNPNV病毒DNA复制下调，但不影响子代病毒产量。表明Secrt调控Se301细胞增殖，参与SeMNPV感染细胞的复制阶段。5.C1细胞中线粒体能量代谢、活性氧及Sehsp60转录水平较Se301细胞增高。RNAi干扰C1细胞中Sehsp60转录水平，胞内能量代谢及活性氧水平下调，SeMNPV病毒DNA复制增加。表明Sehsp60影响潜伏感染甜菜夜蛾细胞线粒体的能量代谢及氧化应激水平，参与SeMNPV感染C1细胞的病毒DNA复制。研究结果为提高昆虫病毒的致病性，调控病毒流行病，综合管理农田害具重要意义。