Inhibition of influenza A virus replication by RNA interference targeted against the PB1 subunit of the RNA polymerase gene
Inhibition of influenza A virus replication by RNA interference targeted against the PB1 subunit of the RNA polymerase gene
复制标题
通过针对 RNA 聚合酶基因 PB1 亚基的 RNA 干扰抑制甲型流感病毒复制
DOI：
10.1007/s00705-011-1087-8
复制
发表时间：
2011-11-01
影响因子：
2.7
通讯作者：
Li, Mingyuan
中科院分区：
文献类型：
--
作者：
Li, Wanyi;Yang, Xiaofan;Li, Mingyuan
上个世纪流感大流行对人类健康造成了巨大威胁。然而，目前的疫苗接种策略和抗病毒药物提供的保护有限。RNA干扰（RNAi）是抑制流感病毒复制的有效手段。PB 1是流感病毒RNA聚合酶的关键蛋白亚基。编码该蛋白的基因PB 1在不同亚型的IAV中高度保守，因此被选为本研究的靶标。该寡核苷酸PB 1-shRNA含有21-bp siRNA，其对应于PB 1线性vRNA的核苷酸1，632至1，652，末端掺入BamHI或EcoRI限制性酶切位点。将PB 1-shRNA寡核苷酸定向克隆到RNAi就绪的pSIREN穿梭载体中。通过限制性内切酶消化证实了所得pSIREN/PB 1质粒的正确结构。用pSIREN/PB 1转染Madin-Darby犬肾（MDCK）细胞，随后用MOI为0.1的IAV（A/PR/8/34，H1N1）感染。48小时后测定细胞培养上清液中的病毒滴度，发现相对于对照，病毒生长被抑制超过50倍。此外，用脂质体包封的pSIREN/PB 1接种鸡胚和小鼠，然后用A/PR/8/34病毒攻击。结果表明，在鸡胚尿囊液中病毒复制至少抑制100倍，实验小鼠的存活率在50%至100%之间。本研究证明重组质粒pSIREN/PB 1表达的PB 1-shRNA在体内外均能抑制甲型流感病毒的复制。这些观察结果为开发一种新的有效的流感感染治疗方法提供了基础。
Influenza (flu) pandemics have posed a great threat to human health in the last century. However, current vaccination strategies and antiviral drugs provide limited protection. RNA interference (RNAi) is an effective means of suppressing influenza virus replication. PB1 is the critical protein subunit of the influenza virus RNA polymerase. The gene encoding this protein,PB1, is highly conserved among different subtypes of IAV and was therefore chosen as the target in this study. The oligonucleotide,PB1-shRNA, contains a 21-bp siRNA corresponding to nucleotides 1,632 to 1,652 ofPB1linear vRNA withBamHIorEcoRIrestriction enzyme sites incorporated at the ends. ThePB1-shRNA oligonucleotide was directionally cloned into the RNAi-ready pSIREN-shuttle vector. The correct structure of the resulting pSIREN/PB1plasmid was confirmed by restriction endonuclease digestion. Madin-Darby canine kidney (MDCK) cells were transfected with pSIREN/PB1and subsequently infected with IAV at an MOI of 0.1 (A/PR/8/34, H1N1). The virus titer in cell culture supernatants was determined 48 hours later, and it was found that virus growth was inhibited by more than 50-fold relative to controls. Furthermore, embryonated eggs and mice were inoculated with liposome-encapsulated pSIREN/PB1and then challenged with the A/PR/8/34 virus. The results showed at least a 100-fold inhibition in virus replication in egg allantoic fluid and a survival rate of between 50% and 100% in experimental mice. This study demonstrates thatPB1-shRNA expressed by the recombinant plasmid pSIREN/PB1inhibits influenza A virus replication both in vitro and in vivo. These observations provide a foundation for the development of a new and efficient treatment of influenza infections.