Yeoh Zoe C, Meagher Jennifer L, Kang Chia-Yu, Bieniasz Paul D, Smith Janet L, Ohi Melanie D
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109.
Department Biological Chemistry, University of Michigan, Ann Arbor, MI 48109.
Proc Natl Acad Sci U S A. 2024 Dec 24;121(52):e2415048121. doi: 10.1073/pnas.2415048121. Epub 2024 Dec 18.
Detecting viral infection is a key role of the innate immune system. The genomes of some RNA viruses have a high CpG dinucleotide content relative to most vertebrate cell RNAs, making CpGs a molecular marker of infection. The human zinc-finger antiviral protein (ZAP) recognizes CpG, mediates clearance of the foreign CpG-rich RNA, and causes attenuation of CpG-rich RNA viruses. While ZAP binds RNA, it lacks enzymatic activity that might be responsible for RNA degradation and thus requires interacting cofactors for its function. One of these cofactors, KHNYN, has a predicted nuclease domain. Using biochemical approaches, we found that the KHNYN NYN domain is a single-stranded RNA ribonuclease that does not have sequence specificity and digests RNA with or without CpG dinucleotides equivalently in vitro. We show that unlike most KH domains, the KHNYN KH domain does not bind RNA. Indeed, a crystal structure of the KH region revealed a double-KH domain with a negatively charged surface that accounts for the lack of RNA binding. Rather, the KHNYN C-terminal domain (CTD) interacts with the ZAP RNA-binding domain (RBD) to provide target RNA specificity. We define a minimal complex composed of the ZAP RBD and the KHNYN NYN-CTD and use a fluorescence polarization assay to propose a model for how this complex interacts with a CpG dinucleotide-containing RNA. In the context of the cell, this module would represent the minimum ZAP and KHNYN domains required for CpG-recognition and ribonuclease activity essential for attenuation of viruses with clusters of CpG dinucleotides.
检测病毒感染是先天免疫系统的一项关键作用。相对于大多数脊椎动物细胞RNA而言，一些RNA病毒的基因组具有较高的CpG二核苷酸含量，使得CpG成为感染的分子标志物。人类锌指抗病毒蛋白（ZAP）识别CpG，介导富含外来CpG的RNA的清除，并导致富含CpG的RNA病毒的衰减。虽然ZAP结合RNA，但其缺乏可能负责RNA降解的酶活性，因此需要相互作用的辅助因子来发挥其功能。这些辅助因子之一，KHNYN，具有一个预测的核酸酶结构域。通过生化方法，我们发现KHNYN NYN结构域是一种单链RNA核糖核酸酶，它不具有序列特异性，并且在体外对含有或不含有CpG二核苷酸的RNA进行同等程度的消化。我们表明，与大多数KH结构域不同，KHNYN KH结构域不结合RNA。实际上，KH区域的晶体结构揭示了一个具有带负电荷表面的双KH结构域，这解释了其缺乏RNA结合的原因。相反，KHNYN C末端结构域（CTD）与ZAP RNA结合结构域（RBD）相互作用以提供靶RNA特异性。我们定义了一个由ZAP RBD和KHNYN NYN-CTD组成的最小复合物，并使用荧光偏振测定法提出了一个关于该复合物如何与含CpG二核苷酸的RNA相互作用的模型。在细胞环境中，该模块将代表识别CpG和核糖核酸酶活性所需的最小ZAP和KHNYN结构域，这对于具有CpG二核苷酸簇的病毒的衰减至关重要。
