Development of an Insect Vector Cell Culture and RNA Interference System To Investigate the Functional Role of Fijivirus Replication Protein
Development of an Insect Vector Cell Culture and RNA Interference System To Investigate the Functional Role of Fijivirus Replication Protein
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开发昆虫载体细胞培养和 RNA 干扰系统以研究斐济病毒复制蛋白的功能作用
DOI：
10.1128/jvi.07121-11
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发表时间：
2012-05-01
影响因子：
5.4
通讯作者：
Wei, Taiyun
中科院分区：
文献类型：
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作者：
Jia, Dongsheng;Chen, Hongyan;Wei, Taiyun
建立了南方水稻黑条矮缩病毒（Southern rice black-streaked dwarf virus，SRBSDV）昆虫载体白背飞虱原代细胞体外培养体系，研究了该病毒的复制。通过透射电子显微镜和共聚焦显微镜观察发现，在病毒感染的培养昆虫载体细胞中，病毒囊泡（病毒复制的推定位点）含有非结构病毒蛋白P9-1、病毒RNA、外衣壳蛋白和病毒颗粒。P9-1表达后在非宿主昆虫细胞中形成病毒质样结构，表明在病毒感染细胞中观察到的病毒基质基本上由P9-1组成。在培养的昆虫载体细胞中，由来自P9-1基因的合成双链RNA（dsRNA）诱导的RNA干扰（RNAi）导致的P9-1表达的敲低强烈抑制病毒质形成和病毒感染。通过摄取dsRNA诱导的RNAi强烈地消除了病毒质的形成，阻止了病毒在完整载体昆虫体内的有效传播。这些结果表明，P9-1在病毒质的形成和病毒的复制过程中是必不可少的。该系统结合昆虫载体细胞培养和RNA干扰，可以进一步推进我们对斐济病毒复制蛋白生物活性的理解。
An in vitro culture system of primary cells from white-backed planthopper, an insect vector of Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus, was established to study replication of the virus. Viroplasms, putative sites of viral replication, contained the nonstructural viral protein P9-1, viral RNA, outer-capsid proteins, and viral particles in virus-infected cultured insect vector cells, as revealed by transmission electron and confocal microscopy. Formation of viroplasm-like structures in non-host insect cells upon expression of P9-1 suggested that the matrix of viroplasms observed in virus-infected cells was composed basically of P9-1. In cultured insect vector cells, knockdown of P9-1 expression due to RNA interference (RNAi) induced by synthesized double-stranded RNA (dsRNA) from the P9-1 gene strongly inhibited viroplasm formation and viral infection. RNAi induced by ingestion of dsRNA strongly abolished viroplasm formation, preventing efficient viral spread in the body of intact vector insects. All these results demonstrated that P9-1 was essential for viroplasm formation and viral replication. This system, combining insect vector cell culture and RNA interference, can further advance our understanding of the biological activities of fijivirus replication proteins.