Xiangyang No.1 People's HospitalHubei University of Medicine, Xiangyang, Hubei Province, China.
Department of Clinical Laboratory, Xiangyang No.1 People's Hospital, Hubei University of Medicine, Xiangyang, China.
Virol J. 2021 Nov 18;18(1):224. doi: 10.1186/s12985-021-01697-8.
The JEV genome is a positive-sense RNA with a highly structured capped 5'UTR, 3'UTR and a large open reading frame. 3'UTR is the untranslated region of flavivirus and has various important functions during viral replication, such as translation, replication and encapsidation. During viral replication, the 3'UTR interacts with viral proteins and host proteins and is required for viral RNA replication and translocation.
The expression level of FUBP3 was knocked down by siRNA and Flag-tagged FUBP3 overexpression plasmid was constructed for overexpression. BHK-21 cells were cultured and infected with JEV to investigate the functional role of FUBP3 in the viral infection cycle. Subcellular localization of FUBP3 and viral replication complexes was observed by dual immunofluorescence staining.
Four host proteins were specifically associated with the 3'UTR of JEV, and FUBP3 was selected to further investigate its potential functional role in the JEV infection cycle. Knockdown of FUBP3 protein resulted in a significant decrease in JEV viral titer, whereas ectopic overexpression of FUBP3 resulted in increased JE viral infectivity. In cells stably knocked down for FUBP3 and then infected with JEV, we found almost no detectable viral NS5 protein. In contrast, when cells stably knocking-down of FUBP3 overexpressed FUBP3, we found a significant increase in viral RNA production over time compared to controls. We also demonstrated that FUBP3 re-localized in the cytoplasm after infection with JEV and co-localized with viral proteins. Exogenous overexpression of FUBP3 was also shown to be located in the JE replication complex and to assist viral replication after JEV infection.
The overall results suggest that FUBP3 regulates RNA replication of JEV and promotes subsequent viral translation and viral particle production.
JEV 基因组是一条正链 RNA，具有高度结构化的帽状 5'UTR、3'UTR 和一个大的开放阅读框。3'UTR 是黄病毒的非翻译区，在病毒复制过程中具有多种重要功能，如翻译、复制和包装。在病毒复制过程中，3'UTR 与病毒蛋白和宿主蛋白相互作用，是病毒 RNA 复制和转位所必需的。
通过 siRNA 敲低 FUBP3 的表达水平，并构建 Flag 标记的 FUBP3 过表达质粒进行过表达。培养 BHK-21 细胞并用 JEV 感染，以研究 FUBP3 在病毒感染周期中的功能作用。通过双免疫荧光染色观察 FUBP3 和病毒复制复合物的亚细胞定位。
四种宿主蛋白与 JEV 的 3'UTR 特异性结合，选择 FUBP3 进一步研究其在 JEV 感染周期中的潜在功能作用。FUBP3 蛋白的敲低导致 JEV 病毒滴度显著降低，而过表达 FUBP3 导致 JE 病毒感染性增加。在稳定敲低 FUBP3 后再感染 JEV 的细胞中，我们发现几乎检测不到可检测的病毒 NS5 蛋白。相比之下，当稳定敲低 FUBP3 的细胞过表达 FUBP3 时，与对照相比，我们发现病毒 RNA 产生随时间显著增加。我们还表明，感染 JEV 后，FUBP3 在细胞质中重新定位，并与病毒蛋白共定位。外源性过表达的 FUBP3 也被证明位于 JE 复制复合物中，并在 JEV 感染后协助病毒复制。
总体结果表明，FUBP3 调节 JEV 的 RNA 复制，并促进随后的病毒翻译和病毒粒子产生。
