Introducing a cleavable signal peptide enhances the packaging efficiency of lentiviral vectors pseudotyped with Japanese encephalitis virus envelope proteins
Introducing a cleavable signal peptide enhances the packaging efficiency of lentiviral vectors pseudotyped with Japanese encephalitis virus envelope proteins
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DOI：
10.1016/j.virusres.2016.12.007
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发表时间：
2017-02-02
期刊：
影响因子：
5
通讯作者：
Wen, Xintian
中科院分区：
文献类型：
--
作者：
Liu, Hanyang;Wu, Rui;Wen, Xintian
假型病毒的使用为研究乙型脑炎病毒的特性提供了便利。信号肽是膜靶向和膜插入的关键决定因素，可影响假型病毒的包装。在这项研究中，我们产生了三个假型的慢病毒载体与JEV包膜蛋白，共表达强信号肽从水泡性口炎病毒（VSV）-G（VSVMEpv）或弱信号肽的JEV（SPMEpv），或病毒没有信号肽在前面的JEV prM/E（MEpv）。Western blot结果表明，JEV E蛋白和HIV p24蛋白存在于3种假型JEV E慢病毒载体的同一颗粒中。电子显微镜显示三种假型的基于JEV-E的慢病毒载体的直径为120-180 nm。实时定量逆转录聚合酶链反应（RT-PCR）结果显示，VSVMEpv的滴度是MEpv的17倍，而SPMEpv的滴度是MEpv的6倍。包含信号肽增强了基于假型JEV-E的慢病毒载体的包装效率。有一个强信号肽帮助它们产生更多的病毒颗粒。绿色荧光蛋白和荧光素酶的表达表明，VSVMEpv、SPMEpv和MEpv的转导滴度或相对荧光单位无显著差异。我们认为，信号肽不影响假型JEV-E的慢病毒载体的感染性。此外，我们的研究结果表明，假型JEV对BHK-21细胞表现出优先嗜性，支持亲本JEV表现出的模拟功能。因此，我们的研究提供了一种具有成本效益的方法来产生假型JEV-E为基础的慢病毒载体，这可能是一个有效的模型，以调查一些JEV的感染特性。(C)2016爱思唯尔B. V.保留所有权利。
Research into the properties of Japanese encephalitis virus (JEV) has been facilitated by use of pseudo typed viruses. The signal peptide is a key determinant for membrane targeting and membrane insertion, which could affect packaging of pseudotyped viruses. In this study, we generated three lentiviral vectors pseudotyped with JEV envelope proteins that co-express either a strong signal peptide from vesicular stomatitis virus (VSV)-G (VSVMEpv) or a weak signal peptide of JEV (SPMEpv), or a virus without a signal peptide in front of the JEV prM/E (MEpv). Western blot demonstrated that JEV E protein and HIV p24 were present in the same particles of the three pseudotyped JEV-E based lentiviral vectors. Electron microscopy revealed that the three pseudotyped JEV-E based lentiviral vectors were 120-180 nm in diameter. Real-time quantitative reverse transcriptase polymerase chain reaction showed that the titer of VSVMEpv was 17-fold higher than that of MEpv, while the titer of SPMEpv was six-fold higher than that of MEpv. Inclusion of a signal peptide enhanced packaging efficiency of pseudotyped JEV-E based lentiviral vectors. With a strong signal peptide helping they generate a higher number of viral particles. Green fluorescent protein and luciferase expression showed that the transduction titer or relative fluorescence units of VSVMEpv, SPMEpv and MEpv were not significantly different. We suggest that the signal peptide does not influence the infectivity of pseudotyped JEV-E based lentiviral vectors. In addition, our findings indicated that pseudotyped JEVs show preferential tropism for BHK-21 cells, supporting the mimic function displayed by parental JEV. Therefore, our study provided a cost-effective method to generate pseudotyped JEV-E based lentiviral vectors, which may represent a valid model to investigate some of the infectious properties of JEV. (C) 2016 Elsevier B.V. All rights reserved.