Varjak Margus, Maringer Kevin, Watson Mick, Sreenu Vattipally B, Fredericks Anthony C, Pondeville Emilie, Donald Claire L, Sterk Jelle, Kean Joy, Vazeille Marie, Failloux Anna-Bella, Kohl Alain, Schnettler Esther
MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland.
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
mSphere. 2017 May 3;2(3). doi: 10.1128/mSphere.00144-17. eCollection 2017 May-Jun.
The small interfering RNA (siRNA) pathway is a major antiviral response in mosquitoes; however, another RNA interference pathway, the PIWI-interacting RNA (piRNA) pathway, has been suggested to be antiviral in mosquitoes. Piwi4 has been reported to be a key mediator of this response in mosquitoes, but it is not involved in the production of virus-specific piRNAs. Here, we show that Piwi4 associates with members of the antiviral exogenous siRNA pathway (Ago2 and Dcr2), as well as with proteins of the piRNA pathway (Ago3, Piwi5, and Piwi6) in an -derived cell line, Aag2. Analysis of small RNAs captured by Piwi4 revealed that it is predominantly associated with virus-specific siRNAs in Semliki Forest virus-infected cells and, to a lesser extent, with viral piRNAs. By using a Dcr2 knockout cell line, we showed directly that Ago2 lost its antiviral activity, as it was no longer bound to siRNAs, but Piwi4 retained its antiviral activity in the absence of the siRNA pathway. These results demonstrate a complex interaction between the siRNA and piRNA pathways in and identify Piwi4 as a noncanonical PIWI protein that interacts with members of the siRNA and piRNA pathways, and its antiviral activities may be independent of either pathway. Mosquitoes transmit several pathogenic viruses, for example, the chikungunya and Zika viruses. In mosquito cells, virus replication intermediates in the form of double-stranded RNA are cleaved by Dcr2 into 21-nucleotide-long siRNAs, which in turn are used by Ago2 to target the virus genome. A different class of virus-derived small RNAs, PIWI-interacting RNAs (piRNAs), have also been found in infected insect cells. These piRNAs are longer and are produced in a Dcr2-independent manner. The only known antiviral protein in the PIWI family is Piwi4, which is not involved in piRNA production. It is associated with key proteins of the siRNA and piRNA pathways, although its antiviral function is independent of their actions.
小干扰RNA（siRNA）途径是蚊子主要的抗病毒反应；然而，另一种RNA干扰途径，即PIWI相互作用RNA（piRNA）途径，也被认为在蚊子中具有抗病毒作用。据报道，Piwi4是蚊子这种反应的关键调节因子，但它不参与病毒特异性piRNA的产生。在这里，我们表明，在源自蚊子的细胞系Aag2中，Piwi4与抗病毒外源性siRNA途径的成员（Ago2和Dcr2）以及piRNA途径的蛋白质（Ago3、Piwi5和Piwi6）相关联。对Piwi4捕获的小RNA的分析表明，在感染了塞姆利基森林病毒的细胞中，它主要与病毒特异性siRNA相关联，在较小程度上与病毒piRNA相关联。通过使用Dcr2基因敲除细胞系，我们直接表明，Ago2失去了其抗病毒活性，因为它不再与siRNA结合，但在没有siRNA途径的情况下，Piwi4仍保留其抗病毒活性。这些结果证明了在蚊子中siRNA和piRNA途径之间存在复杂的相互作用，并确定Piwi4是一种非典型的PIWI蛋白，它与siRNA和piRNA途径的成员相互作用，其抗病毒活性可能独立于这两种途径。蚊子传播几种致病病毒，例如基孔肯雅病毒和寨卡病毒。在蚊子细胞中，双链RNA形式的病毒复制中间体被Dcr2切割成21个核苷酸长的siRNA，这些siRNA反过来被Ago2用于靶向病毒基因组。在受感染的昆虫细胞中还发现了另一类病毒衍生的小RNA，即PIWI相互作用RNA（piRNA）。这些piRNA更长，并且以不依赖Dcr2的方式产生。PIWI家族中唯一已知的抗病毒蛋白是Piwi4，它不参与piRNA的产生。它与siRNA和piRNA途径的关键蛋白相关联，尽管其抗病毒功能独立于它们的作用。
