Hass Meike, Gölnitz Uta, Müller Stefanie, Becker-Ziaja Beate, Günther Stephan
Department of Virology, Bernhard-Nocht-Institute of Tropical Medicine, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Germany.
J Virol. 2004 Dec;78(24):13793-803. doi: 10.1128/JVI.78.24.13793-13803.2004.
Lassa virus is endemic to West Africa and causes hemorrhagic fever in humans. To facilitate the functional analysis of this virus, a replicon system was developed based on Lassa virus strain AV. Genomic and antigenomic minigenomes (MG) were constructed consisting of the intergenic region of S RNA and a reporter gene (Renilla luciferase) in antisense orientation, flanked by the 5' and 3' untranslated regions of S RNA. MGs were expressed under the control of the T7 promoter. Nucleoprotein (NP), L protein, and Z protein were expressed from plasmids containing the T7 promoter and internal ribosomal entry site. Transfection of cells stably expressing T7 RNA polymerase (BSR T7/5) with MG in the form of DNA or RNA and plasmids for the expression of NP and L protein resulted in high levels of Renilla luciferase expression. The replicon system was optimized with respect to the ratio of the transfected constructs and by modifying the 5' end of the MG. Maximum activity was observed 24 to 36 h after transfection with a signal-to-noise ratio of 2 to 3 log units. Northern blot analysis provided evidence for replication and transcription of the MG. Z protein downregulated replicon activity close to background levels. Treatment with ribavirin and alpha interferon inhibited replicon activity, suggesting that both act on the level of RNA replication, transcription, or ribonucleoprotein assembly. In conclusion, this study describes the first replicon system for a highly pathogenic arenavirus. It is a tool for investigating the mechanisms of replication and transcription of Lassa virus and may facilitate the testing of antivirals outside a biosafety level 4 laboratory.
拉沙病毒在西非流行，可导致人类出血热。为便于对该病毒进行功能分析，基于拉沙病毒AV株开发了一种复制子系统。构建了基因组和反基因组微型基因组（MG），其由S RNA的基因间隔区和一个反义方向的报告基因（海肾荧光素酶）组成，两侧为S RNA的5'和3'非翻译区。MG在T7启动子的控制下表达。核蛋白（NP）、L蛋白和Z蛋白从含有T7启动子和内部核糖体进入位点的质粒中表达。用DNA或RNA形式的MG以及用于表达NP和L蛋白的质粒转染稳定表达T7 RNA聚合酶的细胞（BSR T7/5），导致海肾荧光素酶高水平表达。通过优化转染构建体的比例并修饰MG的5'末端对复制子系统进行了优化。转染后24至36小时观察到最大活性，信噪比为2至3个对数单位。Northern印迹分析为MG的复制和转录提供了证据。Z蛋白将复制子活性下调至接近背景水平。用利巴韦林和α干扰素处理可抑制复制子活性，表明二者均作用于RNA复制、转录或核糖核蛋白组装水平。总之，本研究描述了首个针对高致病性沙粒病毒的复制子系统。它是研究拉沙病毒复制和转录机制的工具，可能有助于在生物安全4级实验室之外进行抗病毒药物测试。
