Suppression of the Arboviruses Dengue and Chikungunya Using a Dual-Acting Group-I Intron Coupled with Conditional Expression of the Bax C-Terminal Domain.
Suppression of the Arboviruses Dengue and Chikungunya Using a Dual-Acting Group-I Intron Coupled with Conditional Expression of the Bax C-Terminal Domain.
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DOI：
10.1371/journal.pone.0139899
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Fraser MJ Jr
中科院分区：
文献类型：
--
作者：
Carter JR;Taylor S;Fraser TS;Kucharski CA;Dawson JL;Fraser MJ Jr
在南亚部分地区，登革热（DENV）和基孔肯雅热（CHIKV）的病媒和患者合并感染呈上升趋势，在世界其他地区（例如美国）也有可能发生这种情况。因此，我们设计了一种抗病毒方法，使用单个抗病毒I组内含子抑制蚊子细胞中两种虫媒病毒的复制。我们设计了内部、外部和指导序列的独特构型，其允许使用单个反式剪接I组内含子与两种病毒基因组中的保守靶序列进行同源识别和剪接，并检查了它们在与促凋亡3'外显子ΔN Bax偶联时抑制蚊子细胞中DENV和CHIKV感染的有效性。RT-PCR证明了这些内含子在转化的白纹伊蚊C6/36细胞中DENV或CHIKV靶位点下游的ΔN Bax序列的反式剪接中的效用，与病毒特异性靶向序列插入构建体的顺序无关。这种反式剪接反应形成DENV或CHIKV ΔN Bax RNA融合物，其导致凋亡性细胞死亡，如膜联蛋白V染色、半胱天冬酶和DNA片段化测定所证明。TCID 50-IFA分析证明了我们的抗虫媒病毒I组内含子方法对DENV和CHIKV感染的有效抑制。这代表了双作用I组内含子的第一次报道，并证明我们可以用单个独特配置的CHIKV/DENV双靶向I组内含子以序列特异性方式靶向DENV和CHIKV RNA，导致两种虫媒病毒的复制抑制，从而为多种虫媒病毒的转基因抑制提供有希望的单一抗病毒剂。
In portions of South Asia, vectors and patients co-infected with dengue (DENV) and chikungunya (CHIKV) are on the rise, with the potential for this occurrence in other regions of the world, for example the United States. Therefore, we engineered an antiviral approach that suppresses the replication of both arboviruses in mosquito cells using a single antiviral group I intron. We devised unique configurations of internal, external, and guide sequences that permit homologous recognition and splicing with conserved target sequences in the genomes of both viruses using a single trans-splicing Group I intron, and examined their effectiveness to suppress infections of DENV and CHIKV in mosquito cells when coupled with a proapoptotic 3' exon, ΔN Bax. RT-PCR demonstrated the utility of these introns in trans-splicing the ΔN Bax sequence downstream of either the DENV or CHIKV target site in transformed Aedes albopictus C6/36 cells, independent of the order in which the virus specific targeting sequences were inserted into the construct. This trans-splicing reaction forms DENV or CHIKV ΔN Bax RNA fusions that led to apoptotic cell death as evidenced by annexin V staining, caspase, and DNA fragmentation assays. TCID50-IFA analyses demonstrate effective suppression of DENV and CHIKV infections by our anti-arbovirus group I intron approach. This represents the first report of a dual-acting Group I intron, and demonstrates that we can target DENV and CHIKV RNAs in a sequence specific manner with a single, uniquely configured CHIKV/DENV dual targeting group I intron, leading to replication suppression of both arboviruses, and thus providing a promising single antiviral for the transgenic suppression of multiple arboviruses.