siRNA targeting the leader sequence of SARS-CoV inhibits virus replication.
siRNA targeting the leader sequence of SARS-CoV inhibits virus replication.
复制标题
靶向SARS-COV领导者序列的siRNA抑制病毒复制。
DOI：
10.1038/sj.gt.3302479
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发表时间：
2005-05
期刊：
影响因子：
5.1
通讯作者：
Chang Z
中科院分区：
文献类型：
--
作者：
Li T;Zhang Y;Fu L;Yu C;Li X;Li Y;Zhang X;Rong Z;Wang Y;Ning H;Liang R;Chen W;Babiuk LA;Chang Z
据报道，SARS-CoV（SARS 相关冠状病毒）是冠状病毒家族中的一种新型病毒成员，是导致严重急性呼吸系统综合症的原因。冠状病毒的复制是通过一种独特的机制发生的，当从基因组开始转录时，利用转录本中的前导序列。因此，我们克隆了SARS-CoV（BJ01）的Leader序列，该序列与SARS-CoV（HKU-39849）中鉴定的Leader序列相同，并构建了针对该Leader序列的特异性siRNA。使用与克隆的 SARS-CoV 前导序列融合的 EGFP 和 RFP 报告基因，我们证明针对前导序列的 siRNA 降低了 293T 细胞中报告基因的 mRNA 丰度和蛋白表达水平。通过在 Vero E6 细胞中稳定表达 siRNA，我们提供的数据表明，通过 RT-PCR 和 Northern blot 分析，siRNA 可以有效且特异性地降低 SARS-CoV 基因的 mRNA 丰度。我们的数据表明，针对前导序列的 siRNA 通过沉默基因表达来抑制 Vero E6 细胞中 SARS-CoV 的复制。我们通过瞬时转染实验进一步证明，针对Leader序列的siRNA对SARS-CoV复制的抑制作用比针对Spike基因或反义寡脱氧核苷酸的siRNA强得多。该报告提供的证据表明，使用 siRNA 靶向前导序列可能是抑制 SARS-CoV 复制的有力工具。
SARS-CoV (the SARS-Associated Coronavirus) was reported as a novel virus member in the coronavirus family, which was the cause of severe acute respiratory syndrome. Coronavirus replication occurs through a unique mechanism employing Leader sequence in the transcripts when initiating transcription from the genome. Therefore, we cloned the Leader sequence from SARS-CoV(BJ01), which is identical to that identified from SARS-CoV(HKU-39849), and constructed specific siRNA targeting the Leader sequence. Using EGFP and RFP reporter genes fused with the cloned SARS-CoV Leader sequence, we demonstrated that the siRNA targeting the Leader sequence decreased the mRNA abundance and protein expression levels of the reporter genes in 293T cells. By stably expressing the siRNA in Vero E6 cells, we provided data that the siRNA could effectively and specifically decrease the mRNA abundance of SARS-CoV genes as analyzed by RT-PCR and Northern blot. Our data indicated that the siRNA targeting the Leader sequence inhibited the replication of SARS-CoV in Vero E6 cells by silencing gene expression. We further demonstrated, via transient transfection experiments, that the siRNA targeting the Leader sequence had a much stronger inhibitory effect on SARS-CoV replication than the siRNAs targeting the Spike gene or the antisense oligodeoxynucleotides did. This report provides evidence that targeting Leader sequence using siRNA could be a powerful tool in inhibiting SARS-CoV replication.
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