The C-terminal PARP domain of the long ZAP isoform contributes essential effector functions for CpG-directed antiviral activity
The C-terminal PARP domain of the long ZAP isoform contributes essential effector functions for CpG-directed antiviral activity
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DOI：
10.1101/2021.06.22.449398
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发表时间：
2021-06
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通讯作者：
Dorota Kmieć;Maria-José Lista-Brotos;Mattia Ficarelli;Chad M. Swanson;S. Neil
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作者：
Dorota Kmieć;Maria-José Lista-Brotos;Mattia Ficarelli;Chad M. Swanson;S. Neil
锌指抗病毒蛋白（ZAP）是一种广泛的病毒复制抑制剂。其最具特征的功能是结合病毒RNA中存在的CpG二核苷酸，并通过招募TRIM 25、KHNYN和其他细胞RNA降解机制，靶向它们进行降解或阻止它们的翻译。ZAP的活性需要选择性结合含CpG的RNA的N-末端RNA结合结构域。然而，对其余结构域的功能贡献知之甚少。使用ZAP敏感和ZAP不敏感的人类免疫缺陷病毒I型（HIV-1），我们表明，催化失活的聚ADP核糖聚合酶（PARP）结构域的长ZAP亚型（ZAP-L）是必需的CpG特异性病毒限制。C-末端CaaX盒中介导S-法尼基化的关键半胱氨酸的突变以及PARP结构域内的非活性催化位点三联体在较小程度上破坏了ZAP-L的活性。在ZAP-S中加入CaaX盒部分恢复了抗病毒活性，这解释了为什么ZAP-S缺乏CpG依赖性抗病毒活性，尽管RNA结合结构域保守。共聚焦显微镜证实了CaaX基序介导的ZAP-L定位于囊泡结构，并增强了与细胞内膜的物理联系。重要的是，PARP结构域和CaaX盒一起调节ZAP-L及其辅因子TRIM 25和KHNYN之间的相互作用，这意味着其适当的亚细胞定位是建立抗病毒复合物所必需的。通过抑制严重急性呼吸综合征冠状病毒2（SARS-CoV-2）复制，进一步证实了PARP结构域和CaaX盒对ZAP-L的CpG导向的抗病毒活性的重要贡献。因此，ZAP-L在细胞内膜上的区室化在ZAP-L介导的抗病毒活性中提供了必要的效应子功能。细胞内在的抗病毒因子，如锌指抗病毒蛋白（ZAP），提供了对抗病毒病原体的第一道防线。ZAP通过选择性结合富含CpG二核苷酸的RNA发挥作用，这些RNA在某些病毒中比在脊椎动物宿主中更常见，导致其降解。在这里，我们表明，目标这些外来元素的能力不仅取决于ZAP的N-末端RNA结合结构域，但在中央和C-末端区域的其他决定因素也调节这一过程。PARP结构域及其相关的CaaX盒对于ZAP的CpG特异性活性至关重要，并且是与辅因子TRIM 25和KHNYN最佳结合所需的。此外，CaaX盒，已知介导的翻译后修饰的疏水性S-法呢基基团，引起ZAP从细胞质的重新定位，并增加其与细胞内膜的关联。ZAP分布的这种变化对于抑制ZAP致敏的HIV-1和SARS-CoV-2都是必不可少的。我们的工作揭示了CpG RNA结合结构域外的决定簇如何协助ZAP的抗病毒活性，并强调了S-法尼基化和膜缔合在这一过程中的作用。
The zinc finger antiviral protein (ZAP) is a broad inhibitor of virus replication. Its best-characterized function is to bind CpG dinucleotides present in viral RNA and, through the recruitment of TRIM25, KHNYN and other cellular RNA degradation machinery, target them for degradation or prevent their translation. ZAP’s activity requires the N-terminal RNA binding domain that selectively binds CpG-containing RNA. However, much less is known about the functional contribution of the remaining domains. Using ZAP-sensitive and ZAP-insensitive human immunodeficiency virus type I (HIV-1), we show that the catalytically inactive poly-ADP-ribose polymerase (PARP) domain of the long ZAP isoform (ZAP-L) is essential for CpG-specific viral restriction. Mutation of a crucial cysteine in the C-terminal CaaX box that mediates S-farnesylation and, to a lesser extent, the inactive catalytic site triad within the PARP domain, disrupted the activity of ZAP-L. Addition of the CaaX box to ZAP-S partly restored antiviral activity, explaining why ZAP-S lacks CpG-dependent antiviral activity despite conservation of the RNA-binding domain. Confocal microscopy confirmed the CaaX motif mediated localization of ZAP-L to vesicular structures and enhanced physical association with intracellular membranes. Importantly, the PARP domain and CaaX box together modulate the interaction between ZAP-L and its cofactors TRIM25 and KHNYN, implying that its proper subcellular localisation is required to establish an antiviral complex. The essential contribution of the PARP domain and CaaX box to ZAP-L’s CpG-directed antiviral activity was further confirmed by inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Thus, compartmentalization of ZAP-L on intracellular membranes provides an essential effector function in the ZAP-L-mediated antiviral activity. Author summary Cell-intrinsic antiviral factors, such as the zinc-finger antiviral protein (ZAP), provide a first line of defence against viral pathogens. ZAP acts by selectively binding CpG dinucleotide-rich RNAs, which are more common in some viruses than their vertebrate hosts, leading to their degradation. Here, we show that the ability to target these foreign elements is not only dependent on ZAP’s N-terminal RNA-binding domain, but additional determinants in the central and C-terminal regions also regulate this process. The PARP domain and its associated CaaX box, are crucial for ZAP’s CpG-specific activity and required for optimal binding to cofactors TRIM25 and KHNYN. Furthermore, a CaaX box, known to mediate post-translational modification by a hydrophobic S-farnesyl group, caused re-localization of ZAP from the cytoplasm and increased its association with intracellular membranes. This change in ZAP’s distribution was essential for inhibition of both a ZAP-sensitized HIV-1 and SARS-CoV-2. Our work unveils how the determinants outside the CpG RNA-binding domain assist ZAP’s antiviral activity and highlights the role of S-farnesylation and membrane association in this process.