Nonstructural protein Pns4 of rice dwarf virus is essential for viral infection in its insect vector.
Nonstructural protein Pns4 of rice dwarf virus is essential for viral infection in its insect vector.
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DOI：
10.1186/s12985-015-0438-6
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发表时间：
2015-12-09
期刊：
影响因子：
4.8
通讯作者：
Wei T
中科院分区：
文献类型：
--
作者：
Chen Q;Zhang L;Chen H;Xie L;Wei T
水稻矮缩病毒（Rice dwarf virus，RDV）是一种植物呼肠孤病毒，主要由绿色稻叶蝉（Nephotettix cincticeps）以持续繁殖方式传播。植物呼肠孤病毒被认为在称为病毒囊的细胞质结构内复制和组装。RDV的非结构蛋白Pns 4是一种磷蛋白，定位于病毒基质周围，在昆虫载体细胞中形成微管。然而，Pns 4微管在昆虫载体中病毒感染过程中的功能作用仍然未知。建立了针对RDV Pns 4基因的RNA干扰（RNAi）系统。体外合成Pns 4基因特异性双链RNA（dsRNA），通过转染法将其导入培养的叶蝉细胞或通过显微注射法将其导入昆虫体内。利用免疫荧光、Western blotting和RT-PCR等方法，分析了Pns 4基因合成的双链RNA（dsRNA）诱导的RNAi对病毒在细胞和昆虫载体中复制和传播的影响。在培养的叶蝉细胞中，由合成的来自Pns 4基因的dsRNA诱导的RNAi导致的Pns 4表达的敲低强烈抑制了微管的形成，从而阻止了病毒在昆虫载体细胞中的积累和有效的病毒感染。显微注射Pns 4基因dsRNA诱导的RNAi显著降低了N.五头蛇此外，它还强烈抑制微管和病毒囊的形成，防止病毒从完整昆虫载体的过滤室中的初始感染部位有效传播。RDV的Pns 4蛋白是病毒在昆虫载体中感染和复制所必需的。它可能直接参与病毒质在病毒侵染叶蝉载体过程中的复制和子代病毒粒子组装的功能作用。
Rice dwarf virus (RDV), a plant reovirus, is mainly transmitted by the green rice leafhopper, Nephotettix cincticeps, in a persistent-propagative manner. Plant reoviruses are thought to replicate and assemble within cytoplasmic structures called viroplasms. Nonstructural protein Pns4 of RDV, a phosphoprotein, is localized around the viroplasm matrix and forms minitubules in insect vector cells. However, the functional role of Pns4 minitubules during viral infection in insect vector is still unknown yet. RNA interference (RNAi) system targeting Pns4 gene of RDV was conducted. Double-stranded RNA (dsRNA) specific for Pns4 gene was synthesized in vitro, and introduced into cultured leafhopper cells by transfection or into insect body by microinjection. The effects of the knockdown of Pns4 expression due to RNAi induced by synthesized dsRNA from Pns4 gene on viral replication and spread in cultured cells and insect vector were analyzed using immunofluorescence, western blotting or RT-PCR assays. In cultured leafhopper cells, the knockdown of Pns4 expression due to RNAi induced by synthesized dsRNA from Pns4 gene strongly inhibited the formation of minitubules, preventing the accumulation of viroplasms and efficient viral infection in insect vector cells. RNAi induced by microinjection of dsRNA from Pns4 gene significantly reduced the viruliferous rate of N. cincticeps. Furthermore, it also strongly inhibited the formation of minitubules and viroplasms, preventing efficient viral spread from the initially infected site in the filter chamber of intact insect vector. Pns4 of RDV is essential for viral infection and replication in insect vector. It may directly participate in the functional role of viroplasm for viral replication and assembly of progeny virions during viral infection in leafhopper vector.