Short 5' UTRs serve as a marker for mRNA translation inhibition by the IFIT2-IFIT3 antiviral complex

Recognition of non-self nucleic acids, including cytoplasmic dsDNA, dsRNA, or mRNAs lacking proper 5' cap structures, is critical for the induction of innate immunity against viruses. Here, we demonstrate that short 5' untranslated regions (UTRs), a characteristic of many viral mRNAs, can also serve as molecular pattern for innate immune recognition via the interferon-induced proteins, IFIT2 and IFIT3. The IFIT2-IFIT3 heterodimer, formed through an intricate domain swap structure, mediates viral mRNA 5' end recognition, translation inhibition, and ultimately antiviral activity. Critically, 5' UTR lengths <50 nucleotides are necessary and sufficient to sensitize an mRNA to translation inhibition by the IFIT2-IFIT3 complex. Thus, diverse viruses whose mRNAs contain short 5' UTRs, such as vesicular stomatitis virus and parainfluenza virus 3, are sensitive to IFIT2-IFIT3-mediated antiviral activity. Our work reveals a new size-restricted pattern of nucleic acid innate immune recognition for the selective repression of viral replication. Overall design: eCLIP was performed as previously described (Blue SB, et al. Nature Protocols 2022), using antibodies against FLAG (M2 / F1804, Sigma) and HA (HA.11 / 901502, Biolegend) for 293T experiments, and IFIT2 (PA3-845, ThermoFisher) and IFIT3 (ABF1048, Millipore) for MEF experiments. Most experiments were performed in biological duplicate, except for uninfected 293T samples (which were single replicates).

识别非自身核酸，包括细胞质dsDNA（双链DNA）、dsRNA（双链RNA）或缺乏正确5'帽结构的mRNA，对于诱导抗病毒先天免疫至关重要。本研究证实，许多病毒mRNA所具有的短5'非翻译区（5' untranslated regions, UTRs），同样可作为先天免疫识别的分子模式，其识别过程依赖于干扰素诱导蛋白IFIT2和IFIT3。通过复杂的结构域交换结构形成的IFIT2-IFIT3异二聚体，可介导病毒mRNA的5'端识别、翻译抑制，并最终发挥抗病毒活性。至关重要的是，长度小于50个核苷酸的5' UTR，是使mRNA对IFIT2-IFIT3复合物介导的翻译抑制产生敏感性的充要条件。因此，众多携带短5' UTR的病毒——如水疱性口炎病毒与副流感病毒3型——均对IFIT2-IFIT3介导的抗病毒作用敏感。本研究揭示了一种受长度约束的新型核酸先天免疫识别模式，可实现对病毒复制的选择性抑制。实验设计：本研究按照此前报道的方法（Blue SB等，"Nature Protocols" 2022）开展增强交联免疫沉淀（eCLIP）实验。其中，针对293T细胞的实验使用针对FLAG标签（M2 / F1804，Sigma）与HA标签（HA.11 / 901502，Biolegend）的抗体；针对小鼠胚胎成纤维细胞（MEF）的实验则使用IFIT2抗体（PA3-845，ThermoFisher）与IFIT3抗体（ABF1048，Millipore）。绝大多数实验设置了生物学重复，仅未感染的293T样本为单次重复。