Mears Harriet V, Emmott Edward, Chaudhry Yasmin, Hosmillo Myra, Goodfellow Ian G, Sweeney Trevor R
Division of Virology, Department of Pathology,, University of Cambridge Addenbrooke's Hospital Cambridge, Hills Road, Cambridge, CB29NJ, UK.
Office 332, Mugar Life Sciences Building 360 Huntington Ave, Northeastern University, Boston, MA, 02115-5000, USA.
Wellcome Open Res. 2019 May 15;4:82. doi: 10.12688/wellcomeopenres.15223.1. eCollection 2019.
Norovirus, also known as the winter vomiting bug, is the predominant cause of non-bacterial gastroenteritis worldwide. Disease control is predicated on a robust innate immune response during the early stages of infection. Double-stranded RNA intermediates generated during viral genome replication are recognised by host innate immune sensors in the cytoplasm, activating the strongly antiviral interferon gene programme. Ifit proteins (interferon induced proteins with tetratricopeptide repeats), which are highly expressed during the interferon response, have been shown to directly inhibit viral protein synthesis as well as regulate innate immune signalling pathways. Ifit1 is well-characterised to inhibit viral translation by sequestration of eukaryotic initiation factors or by directly binding to the 5' terminus of foreign RNA, particularly those with non-self cap structures. However, noroviruses have a viral protein, VPg, covalently linked to the 5' end of the genomic RNA, which acts as a cap substitute to recruit the translation initiation machinery. Ifit1 knockout RAW264.7 murine macrophage-like cells were generated using CRISPR-Cas9 gene editing. These cells were analysed for their ability to support murine norovirus infection, determined by virus yield, and respond to different immune stimuli, assayed by quantitative PCR. The effect of Ifit proteins on norovirus translation was also tested . Here, we show that VPg-dependent translation is completely refractory to Ifit1-mediated translation inhibition and Ifit1 cannot bind the 5' end of VPg-linked RNA. Nevertheless, knockout of Ifit1 promoted viral replication in murine norovirus infected cells. We then demonstrate that Ifit1 promoted interferon-beta expression following transfection of synthetic double-stranded RNA but had little effect on toll-like receptor 3 and 4 signalling. Ifit1 is an antiviral factor during norovirus infection but cannot directly inhibit viral translation. Instead, Ifit1 stimulates the antiviral state following cytoplasmic RNA sensing, contributing to restriction of norovirus replication.
诺如病毒，也被称为冬季呕吐病菌，是全球非细菌性肠胃炎的主要病因。疾病控制取决于感染早期强大的先天免疫反应。病毒基因组复制过程中产生的双链RNA中间体被细胞质中的宿主先天免疫传感器识别，从而激活强烈的抗病毒干扰素基因程序。干扰素诱导的含四肽重复序列蛋白（Ifit蛋白）在干扰素反应期间高度表达，已被证明可直接抑制病毒蛋白合成以及调节先天免疫信号通路。Ifit1通过隔离真核起始因子或直接结合外来RNA的5'末端，特别是那些具有非自身帽结构的RNA，来抑制病毒翻译，这一点已得到充分研究。然而，诺如病毒有一种病毒蛋白VPg，它与基因组RNA的5'末端共价连接，充当帽替代物以募集翻译起始机制。利用CRISPR - Cas9基因编辑技术构建了Ifit1基因敲除的RAW264.7鼠巨噬细胞样细胞。通过病毒产量测定这些细胞支持鼠诺如病毒感染的能力，并通过定量PCR检测其对不同免疫刺激的反应。还测试了Ifit蛋白对诺如病毒翻译的影响。在这里，我们表明依赖VPg的翻译对Ifit1介导的翻译抑制完全具有抗性，并且Ifit1不能结合VPg连接的RNA的5'末端。尽管如此，Ifit1基因敲除促进了鼠诺如病毒感染细胞中的病毒复制。然后我们证明，转染合成双链RNA后，Ifit1促进了干扰素 - β的表达，但对Toll样受体3和4信号传导影响很小。在诺如病毒感染期间，Ifit1是一种抗病毒因子，但不能直接抑制病毒翻译。相反，Ifit1在细胞质RNA感知后刺激抗病毒状态，有助于限制诺如病毒复制。
