Successful Propagation of Flavivirus Infectious cDNAs by a Novel Method To Reduce the Cryptic Bacterial Promoter Activity of Virus Genomes
Successful Propagation of Flavivirus Infectious cDNAs by a Novel Method To Reduce the Cryptic Bacterial Promoter Activity of Virus Genomes
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DOI：
10.1128/jvi.01986-10
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发表时间：
2011-03-01
影响因子：
5.4
通讯作者：
Yueh, Andrew
中科院分区：
文献类型：
--
作者：
Pu, Szu-Yuan;Wu, Ren-Huang;Yueh, Andrew
反向遗传学是研究单链RNA病毒的有力工具。尽管已经做出了巨大的努力来改进用于构建黄病毒cDNA的方法，但是黄病毒cDNA在细菌中的毒性的原因仍然未知。在这里，我们进行了突变分析研究，以确定大肠杆菌启动子（ECP）的核苷酸（nt）1至3000的登革病毒2型（DENV 2）和日本脑炎病毒（JEV）基因组内的序列。使用含有DENV 2或JEV片段和空载体报告基因海肾荧光素酶的融合构建体，分别在DENV 2和JEV基因组的nt 1至3000内证明了8个和4个活性ECP。全长DENV 2和JEV cDNA通过插入突变而获得，所述突变降低它们在细菌中的ECP活性而不改变氨基酸序列。当BHK 21细胞用来自在登革基因组的prM-E-NS 1区域内具有多个沉默突变的DENV 2 cDNA克隆或在JEV基因组的nt 90处具有A至C突变的JEV cDNA克隆的体外转录RNA转染时，发生严重的细胞病变效应。在C6/36和BHK 21细胞中，来自DENV 2或JEV cDNA克隆的病毒粒子表现出与其亲本病毒相似的感染性。通过将沉默突变引入DENV 2感染性cDNA或亚基因组DENV 2复制子克隆的核心基因的中心部分（nt 160至243），揭示了病毒复制所必需的顺式作用元件。这种构建DENV 2和JEV感染性克隆的新策略可应用于其他黄病毒或致病性RNA病毒，以促进病毒学、病毒致病性和疫苗开发的研究。
Reverse genetics is a powerful tool to study single-stranded RNA viruses. Despite tremendous efforts having been made to improve the methodology for constructing flavivirus cDNAs, the cause of toxicity of flavivirus cDNAs in bacteria remains unknown. Here we performed mutational analysis studies to identify Escherichia coli promoter (ECP) sequences within nucleotides (nt) 1 to 3000 of the dengue virus type 2 (DENV2) and Japanese encephalitis virus (JEV) genomes. Eight and four active ECPs were demonstrated within nt 1 to 3000 of the DENV2 and JEV genomes, respectively, using fusion constructs containing DENV2 or JEV segments and empty vector reporter gene Renilla luciferase. Full-length DENV2 and JEV cDNAs were obtained by inserting mutations reducing their ECP activity in bacteria without altering amino acid sequences. A severe cytopathic effect occurred when BHK21 cells were transfected with in vitro-transcribed RNAs from either a DENV2 cDNA clone with multiple silent mutations within the prM-E-NS1 region of dengue genome or a JEV cDNA clone with an A-to-C mutation at nt 90 of the JEV genome. The virions derived from the DENV2 or JEV cDNA clone exhibited infectivities similar to those of their parental viruses in C6/36 and BHK21 cells. A cis-acting element essential for virus replication was revealed by introducing silent mutations into the central portion (nt 160 to 243) of the core gene of DENV2 infectious cDNA or a subgenomic DENV2 replicon clone. This novel strategy of constructing DENV2 and JEV infectious clones could be applied to other flaviviruses or pathogenic RNA viruses to facilitate research in virology, viral pathogenesis, and vaccine development.