Viral envelope protein 53R gene highly specific silencing and iridovirus resistance in fish Cells by AmiRNA.
Viral envelope protein 53R gene highly specific silencing and iridovirus resistance in fish Cells by AmiRNA.
复制标题
AmiRNA 实现鱼细胞中病毒包膜蛋白 53R 基因高度特异性沉默和虹彩病毒抗性
DOI：
10.1371/journal.pone.0010308
复制
发表时间：
2010-04-23
期刊：
影响因子：
3.7
通讯作者：
Zhang QY
中科院分区：
文献类型：
--
作者：
Kim YS;Ke F;Lei XY;Zhu R;Zhang QY
背景囊膜蛋白53R是从虹彩病毒科青蛙Rana grylio病毒(RGV)中发现的，在病毒组装过程中起重要作用。虽然小发夹RNA(ShRNAs)对虹膜病毒主要衣壳蛋白(MCP)的抑制作用已被证明能在体外引起对病毒感染的抵抗，但利用RNA干扰(RNAi)抑制水生动物病毒包膜蛋白基因产物的研究尚未见报道。方法我们设计了针对病毒包膜蛋白基因RGV 53R的人工microRNAs(AmiRNAs)。通过将编码RGV 53R特异性miRNA的序列整合到前miRNA155(PSM155)载体中，该载体利用天然miR-155序列的骨架，可以在细胞内表达53R靶向的Pre-amiRNAs。在鱼类细胞系中，前亚胺核糖核酸可被类核糖核酸酶DICER加工成21-25个碱基的亚胺核糖核酸(AmIR-53R)。实时荧光定量聚合酶链式反应(Real-Time-PCR，RT-PCR)检测RGV感染后72h细胞内53R的表达水平，并用电子显微镜观察RGV感染前后细胞内RGV病毒颗粒的组装。结论AMIR-53R-1基因可沉默病毒囊膜蛋白RGV 53R，缺失病毒粒子组装，进一步鉴定出虹彩病毒囊膜蛋白基因的第一个amRNA，可在鱼类细胞中对病毒感染产生抵抗力。结果表明，针对RGV 53R基因36-57位的AmIR-53R-1能有效地抑制病毒感染(58%)。此外，电子显微镜观察发现病毒粒子组装缺陷或病毒粒子组装能力降低与AmIR-53R-1的表达密切相关。基于Mx基因的实时定量聚合酶链式反应，我们没有发现AMIR-53R-1激活干扰素的证据。
Background Envelope protein 53R was identified from frog Rana grylio virus (RGV), a member of the family Iridoviridae, and it plays an important role in the virus assembly. Although inhibition of iridovirus major capsid protein (MCP) by small hairpin RNAs (shRNAs) has been shown to cause resistance to viral infection in vitro, RNA interference (RNAi) to inhibit aquatic animal virus envelope protein gene product has not been reported. Methodology We devised artificial microRNAs (amiRNAs) that target a viral envelope protein gene RGV 53R. By incorporating sequences encoding amiRNAs specific to 53R of RGV into pre-miRNA155 (pSM155) vectors, which use the backbone of natural miR-155 sequence and could intracellularly express 53R-targeted pre-amiRNAs. The pre-amiRNAs could be processed by the RNase III-like enzyme Dicer into 21–25 nt amiRNAs (amiR-53Rs) in fish cell lines. The levels of 53R expression were analyzed through real-time PCR and RGV virions assembly were observed by electronic microscopy in fish cells transfected with or without amiR-53Rs at 72 h of RGV infection. Conclusion/Significance The results argue that viral envelope protein RGV 53R can be silenced and the virions assembly was deficient by amiR-53R-1, and further identified the first amiRNA of envelope protein gene from iridovirus that was able to cause resistance to virus infection in fish cells. The data demonstrate that the viral infection is efficiently suppressed (58%) by amiR-53R-1 targeting positon 36–57 of RGV 53R. Moreover, electron microscopic observations revealed virion assembly defect or reduced virions assembly capacity was closely correlated to expression of amiR-53R-1. Based on real time PCR of the Mx gene, we found no evidence of activation of IFN by amiR-53R-1.
登录
查看更多内容
新型干扰素诱导基因Gig2及其启动子的表达调控和功能表征
影响因子：
3.6
作者：
Jiang, Jun;Zhang, Yi-Bing;Gui, Jian-Fang
通讯作者：
Gui, Jian-Fang
影响因子：
4.7
作者：
Robertsen, B
通讯作者：
Robertsen, B
影响因子：
64.5
作者：
Zamore, PD;Tuschl, T;Bartel, DP
通讯作者：
Bartel, DP
影响因子：
1.4
作者：
Zhang, QY;Ruan, HM;Gui, HF
通讯作者：
Gui, HF
影响因子：
3.7
作者：
Sui HY;Zhao GY;Huang JD;Jin DY;Yuen KY;Zheng BJ
通讯作者：
Zheng BJ