T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus
T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus
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DOI：
10.1099/vir.0.2008/002097-0
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发表时间：
2008-09-01
影响因子：
3.8
通讯作者：
Weber, Friedemann
中科院分区：
文献类型：
--
作者：
Habjan, Matthias;Penski, Nicola;Weber, Friedemann
裂谷热病毒（RVFV）是非洲人类和家养动物中大规模和反复暴发急性发热性疾病的原因。它属于布尼亚病毒科，白蛉病毒属，其负链RNA基因组由三个片段组成。在这里，我们报告了两个不同的系统来拯救RVFV野生型菌株ZH 548的建立和表征。第一个系统基于BHK-21细胞克隆BSR-T7/5，其稳定表达T7 RNA聚合酶（T7 pol）。野生型RVFV的拯救用三个T7 pol驱动的cDNA质粒实现，所述cDNA质粒代表反基因组意义上的病毒RNA区段。第二个系统涉及用三个RNA pol I驱动的病毒片段质粒和两个RNA pol II驱动的支持质粒转染的293 T细胞，以表达病毒聚合酶组分L和N。已知T7 pol转录物的5'三磷酸基团通过细胞内RNA受体RIG-1强烈激活抗病毒干扰素系统。尽管如此，T7 pol和pol I/II系统具有相似的效率。这甚至是真正的拯救RVFV突变缺乏干扰素拮抗剂非结构蛋白。进一步的实验表明，BHK-21和BSR-T7/5细胞对T7 pol转录物的无反应性最可能是由于RIG-I途径的缺陷。我们的反向遗传学系统现在使我们能够操纵RVFV的基因组并研究其毒力机制。此外，BHK衍生的细胞系具有受损的RIG-I途径的发现可以解释它们适合于繁殖和拯救多种病毒。
Rift Valley fever virus (RVFV) is responsible for large and recurrent outbreaks of acute febrile illness among humans and domesticated animals in Africa. It belongs to the family Bunyaviridae, genus Phlebovirus, and its negative-stranded RNA genome consists of three segments. Here, we report the establishment and characterization of two different systems to rescue the RVFV wild-type strain ZH548. The first system is based on the BHK-21 cell clone BSR-T7/5, which stably expresses T7 RNA polymerase (T7 pol). Rescue of wild-type RVFV was achieved with three T7 pol-driven cDNA plasmids representing the viral RNA segments in the antigenomic sense. The second system involves 293T cells transfected with three RNA pol I-driven plasmids for the viral segments and two RNA pol II-driven support plasmids to express the viral polymerase components L and N. It is known that the 5' triphosphate group of T7 pol transcripts strongly activates the antiviral interferon system via the intracellular RNA receptor RIG-I Nonetheless, both the T7 pol and the pol I/II system were of similar efficiency. This was even true for the rescue of a RVFV mutant lacking the interferon antagonist nonstructural proteins. Further experiments demonstrated that the unresponsiveness of BHK-21 and BSR-T7/5 cells to T7 pol transcripts is most probably due to a deficiency in the RIG-I pathway. Our reverse genetics systems now enable us to manipulate the genome of RVFV and study its virulence mechanisms. Moreover, the finding that BHK-derived cell lines have a compromised RIG-I pathway may explain their suitability for propagating and rescuing a wide variety of viruses.