Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, California, United States of America.
Department of Chemistry and Biochemistry, University of California, Los Angeles, California, United States of America.
PLoS Pathog. 2024 Aug 29;20(8):e1011836. doi: 10.1371/journal.ppat.1011836. eCollection 2024 Aug.
The host interferon pathway upregulates intrinsic restriction factors in response to viral infection. Many of them block a diverse range of viruses, suggesting that their antiviral functions might have been shaped by multiple viral families during evolution. Host-virus conflicts have led to the rapid adaptation of host and viral proteins at their interaction hotspots. Hence, we can use evolutionary genetic analyses to elucidate antiviral mechanisms and domain functions of restriction factors. Zinc finger antiviral protein (ZAP) is a restriction factor against RNA viruses such as alphaviruses, in addition to other RNA, retro-, and DNA viruses, yet its precise antiviral mechanism is not fully characterized. Previously, an analysis of 13 primate ZAP orthologs identified three positively selected residues in the poly(ADP-ribose) polymerase-like domain. However, selective pressure from ancient alphaviruses and others likely drove ZAP adaptation in a wider representation of mammals. We performed positive selection analyses in 261 mammalian ZAP using more robust methods with complementary strengths and identified seven positively selected sites in all domains of the protein. We generated ZAP inducible cell lines in which the positively selected residues of ZAP are mutated and tested their effects on alphavirus replication and known ZAP activities. Interestingly, the mutant in the second WWE domain of ZAP (N658A) is dramatically better than wild-type ZAP at blocking replication of Sindbis virus and other ZAP-sensitive alphaviruses due to enhanced viral translation inhibition. The N658A mutant is adjacent to the previously reported poly(ADP-ribose) (PAR) binding pocket, but surprisingly has reduced binding to PAR. In summary, the second WWE domain is critical for engineering a more potent ZAP and fluctuations in PAR binding modulate ZAP antiviral activity. Our study has the potential to unravel the role of ADP-ribosylation in the host innate immune defense and viral evolutionary strategies that antagonize this post-translational modification.
宿主干扰素途径上调固有限制因子以响应病毒感染。其中许多因子可阻断多种病毒，表明它们的抗病毒功能可能在进化过程中受到多种病毒家族的影响。宿主-病毒的冲突导致宿主和病毒蛋白在其相互作用热点处迅速适应。因此，我们可以利用进化遗传分析来阐明限制因子的抗病毒机制和结构域功能。锌指抗病毒蛋白（ZAP）是一种针对 RNA 病毒（如甲病毒）的限制因子，此外还针对其他 RNA、逆转录和 DNA 病毒，但它的确切抗病毒机制尚未完全阐明。之前，对 13 种灵长类动物 ZAP 同源物的分析确定了多聚（ADP-核糖）聚合酶样结构域中的三个正选择残基。然而，来自古老的甲病毒和其他病毒的选择压力可能在更广泛的哺乳动物中推动了 ZAP 的适应。我们使用更强大的方法对 261 种哺乳动物 ZAP 进行了正选择分析，这些方法具有互补的优势，并鉴定了蛋白质所有结构域中的七个正选择位点。我们生成了可诱导 ZAP 的细胞系，其中突变了 ZAP 的正选择残基，并测试了它们对甲病毒复制和已知 ZAP 活性的影响。有趣的是，ZAP 的第二个 WWE 结构域中的突变体（N658A）在阻止辛德比斯病毒和其他 ZAP 敏感的甲病毒复制方面比野生型 ZAP 好得多，因为它增强了病毒翻译抑制。N658A 突变体位于先前报道的多聚（ADP-核糖）（PAR）结合口袋附近，但令人惊讶的是，它与 PAR 的结合减少。总之，第二个 WWE 结构域对于构建更有效的 ZAP 至关重要，而 PAR 结合的波动调节 ZAP 的抗病毒活性。我们的研究有可能揭示 ADP-核糖基化在宿主固有免疫防御和拮抗这种翻译后修饰的病毒进化策略中的作用。
