Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Department of Laboratory Medicine, Linkou Chang Gung Memorial Hospital, Taoyuan, Taiwan.
Methods. 2020 Nov 1;183:4-12. doi: 10.1016/j.ymeth.2019.08.011. Epub 2019 Sep 5.
There is conclusive evidential support for the existence of virus-derived small RNA (vsRNA) in mammals. Two types of vsRNA have been reported from picornaviruses. The first is virus-derived short-interfering RNA (vsiRNA) that is processed from viral double-stranded RNA intermediates during RNA replication. The other is small RNA derived from the highly base-paired single-stranded genomic region, e.g. the internal ribosome entry site (IRES) of picornaviruses. vsiRNA interacts with the Argonaute protein to control viral RNA replication through the process of RNA interference. However, the function of structure-based vsRNA is largely unknown. We previously identified vsRNA1 generated from the enterovirus-A71 (EV-A71) IRES region by the endogenous enzyme Dicer. Exogenous vsRNA1 can inhibit IRES activity both in vivo and in vitro, hence viral replication is inhibited. Here we describe key methods used to characterize vsRNA, including annotation by next-generation sequencing, abundance measurement by Northern blotting, determination of Dicer-dependence by gel-shift assay and in vitro cleavage assay, and the inhibitory effect on IRES activity via in vitro translation assay.
有确凿的证据支持哺乳动物中存在病毒衍生的小 RNA (vsRNA)。从小 RNA 病毒中已经报道了两种类型的 vsRNA。第一种是病毒衍生的短干扰 RNA (vsiRNA)，它是在 RNA 复制过程中从病毒双链 RNA 中间体加工而来的。另一种是从小 RNA 衍生而来的高度碱基配对的单链基因组区域，例如小 RNA 病毒的内部核糖体进入位点 (IRES)。vsiRNA 通过 RNA 干扰与 Argonaute 蛋白相互作用来控制病毒 RNA 复制。然而，结构基础的 vsRNA 的功能在很大程度上是未知的。我们之前通过内源性酶 Dicer 从肠道病毒-A71 (EV-A71) IRES 区鉴定出 vsRNA1。外源性的 vsRNA1 可以在体内和体外抑制 IRES 活性，从而抑制病毒复制。在这里，我们描述了用于表征 vsRNA 的关键方法，包括通过下一代测序进行注释、通过 Northern 印迹进行丰度测量、通过凝胶迁移分析和体外切割分析确定 Dicer 依赖性、以及通过体外翻译测定对 IRES 活性的抑制作用。
