Generation of Bi-Reporter-Expressing Tri-Segmented Arenavirus.
Generation of Bi-Reporter-Expressing Tri-Segmented Arenavirus.
复制标题
DOI：
10.1007/978-1-0716-2453-1_17
复制
发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Martinez-Sobrido, Luis
中科院分区：
文献类型：
--
作者：
Ye, Chengjin;Martinez-Sobrido, Luis
反向遗传系统提供了一个强大的工具，以产生重组沙粒病毒表达报告，以促进沙粒病毒生命周期的研究和抗病毒对策的发现。质粒编码的病毒核糖核蛋白组分启动质粒驱动的全长病毒基因组的转录和复制，从而产生感染性病毒。因此，这种方法对于产生表达报告基因的重组沙粒病毒是理想的，可以用作病毒复制的有效替代品。将小的病毒片段(S)分裂为两个病毒片段（S1和S2），每个片段编码一个报告基因，可以拯救重组三片段沙粒病毒。双报告基因表达的重组三片段沙粒病毒是研究沙粒病毒生物学的一个很好的工具，包括沙粒病毒感染的预防和治疗对策的鉴定和表征。在本章中，我们描述了重组沙粒病毒的生成和体外表征的详细方案，重组沙粒病毒含有三段基因组，表达基于家族原型成员淋巴细胞性脉络丛脑膜炎病毒（LCMV）的两个报告基因。类似的实验方法可用于为沙粒病毒家族的其他成员生成双报告基因表达的三片段重组病毒。
Reverse genetics systems provide a powerful tool to generate recombinant arenavirus expressing reporters to facilitate the investigation of the arenavirus life cycle and also for the discovery of antiviral counter-measures. The plasmid-encoded viral ribonucleoprotein components initiate the transcription and replication of a plasmid-driven full-length viral genome, resulting in infectious virus. Thereby, this approach is ideal for the generation of recombinant arenaviruses expressing reporter genes that can be used as valid surrogates for virus replication. By splitting the small viral segment (S) into two viral segments (S1 and S2), each of them encoding a reporter gene, recombinant tri-segmented arenavirus can be rescued. Bi-reporter-expressing recombinant tri-segmented arenaviruses represent an excellent tool to study the biology of arenaviruses, including the identification and characterization of both prophylactic and therapeutic countermeasures for the treatment of arenaviral infections. In this chapter, we describe a detailed protocol on the generation and in vitro characterization of recombinant arenaviruses containing a tri-segment genome expressing two reporter genes based on the prototype member in the family, lymphocytic choriomeningitis virus (LCMV). Similar experimental approaches can be used for the generation of bi-reporter-expressing tri-segment recombinant viruses for other members in the arenavirus family.