A Nuclear Export Signal in KHNYN Required for Its Antiviral Activity Evolved as ZAP Emerged in Tetrapods.
A Nuclear Export Signal in KHNYN Required for Its Antiviral Activity Evolved as ZAP Emerged in Tetrapods.
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DOI：
10.1128/jvi.00872-22
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发表时间：
2023-01-31
影响因子：
5.4
通讯作者：
中科院分区：
文献类型：
--
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锌指抗病毒蛋白（ZAP）通过直接结合细胞质病毒RNA中的CpG二核苷酸来抑制蛋白质合成并靶向RNA进行降解，从而抑制病毒复制。 ZAP 在四足动物中进化，在爬行动物、鸟类和哺乳动物中存在明显的直系同源物。当 ZAP 出现时，其他蛋白质可能已经进化成为其抗病毒活性的辅助因子。 KHNYN 是一种假定的核糖核酸内切酶，是 ZAP 限制逆转录病毒所必需的。为了确定 ZAP 出现后的进化路径，我们将哺乳动物和爬行动物中的 KHNYN 直系同源物与不编码 ZAP 的鱼类中的 KHNYN 直系同源物进行了比较。这鉴定出 KHNYN 中的残基在编码 ZAP 的物种中高度保守，包括 CUBAN 结构域中的几个残基。 CUBAN 结构域与 NEDD8 和 Cullin-RING E3 泛素连接酶相互作用。 CUBAN 结构域的删除降低了 KHNYN 的抗病毒活性，增加了蛋白质表达并增加了核定位。然而，CUBAN 结构域-NEDD8 相互作用所需的残基突变增加了 KHNYN 丰度，但不影响其抗病毒活性或细胞质定位，表明 Cullin 介导的降解可能控制其稳态，并且蛋白质周转的调节与其抗病毒活性是分开的。相比之下，CUBAN 结构域中的 C 端残基形成了 CRM1 依赖性核输出信号 (NES)，这是其抗病毒活性所必需的。 NES 的缺失或突变增加了 KHNYN 的核定位并减少了其与 ZAP 的相互作用。该 NES 的最后 2 个位置不存在于鱼类 KHNYN 直向同源物中，我们假设它们的进化允许 KHNYN 充当 ZAP 辅助因子。重要性 干扰素系统是抑制病毒和其他病原体的先天免疫反应的一部分。该系统大约在 5 亿年前出现在早期脊椎动物中。从那时起，一些基因已经进化成为抗病毒干扰素刺激基因（ISG），而另一些基因则进化为它们编码的蛋白质可以与ISG编码的蛋白质相互作用并促进其活性。然而，这仍然没有得到很好的描述。 ZAP 是一种在四足动物进化过程中产生的 ISG，可抑制病毒复制。由于 KHNYN 与 ZAP 相互作用，并且是其对抗逆转录病毒的抗病毒活性所必需的，因此我们进行了进化分析，以确定 KHNYN 中的特定氨基酸在 ZAP 出现后如何进化。这确定了在四足动物中进化的核输出信号，并且是 KHNYN 在细胞中运输并与 ZAP 相互作用所必需的。总体而言，KHNYN 中的特定残基经过进化，使其能够充当 ZAP 抗病毒活性的辅助因子。
The zinc finger antiviral protein (ZAP) inhibits viral replication by directly binding CpG dinucleotides in cytoplasmic viral RNA to inhibit protein synthesis and target the RNA for degradation. ZAP evolved in tetrapods and there are clear orthologs in reptiles, birds, and mammals. When ZAP emerged, other proteins may have evolved to become cofactors for its antiviral activity. KHNYN is a putative endoribonuclease that is required for ZAP to restrict retroviruses. To determine its evolutionary path after ZAP emerged, we compared KHNYN orthologs in mammals and reptiles to those in fish, which do not encode ZAP. This identified residues in KHNYN that are highly conserved in species that encode ZAP, including several in the CUBAN domain. The CUBAN domain interacts with NEDD8 and Cullin-RING E3 ubiquitin ligases. Deletion of the CUBAN domain decreased KHNYN antiviral activity, increased protein expression and increased nuclear localization. However, mutation of residues required for the CUBAN domain-NEDD8 interaction increased KHNYN abundance but did not affect its antiviral activity or cytoplasmic localization, indicating that Cullin-mediated degradation may control its homeostasis and regulation of protein turnover is separable from its antiviral activity. By contrast, the C-terminal residues in the CUBAN domain form a CRM1-dependent nuclear export signal (NES) that is required for its antiviral activity. Deletion or mutation of the NES increased KHNYN nuclear localization and decreased its interaction with ZAP. The final 2 positions of this NES are not present in fish KHNYN orthologs and we hypothesize their evolution allowed KHNYN to act as a ZAP cofactor. IMPORTANCE The interferon system is part of the innate immune response that inhibits viruses and other pathogens. This system emerged approximately 500 million years ago in early vertebrates. Since then, some genes have evolved to become antiviral interferon-stimulated genes (ISGs) while others evolved so their encoded protein could interact with proteins encoded by ISGs and contribute to their activity. However, this remains poorly characterized. ZAP is an ISG that arose during tetrapod evolution and inhibits viral replication. Because KHNYN interacts with ZAP and is required for its antiviral activity against retroviruses, we conducted an evolutionary analysis to determine how specific amino acids in KHNYN evolved after ZAP emerged. This identified a nuclear export signal that evolved in tetrapods and is required for KHNYN to traffic in the cell and interact with ZAP. Overall, specific residues in KHNYN evolved to allow it to act as a cofactor for ZAP antiviral activity.
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