泛素依赖型线粒体自噬

在寨卡病毒复制中的作用及机制研究

摘要

研究背景及目的：

寨卡病毒（Zika virus, ZIKV）是一种蚊媒RNA病毒，属于黄病毒科黄病毒属，被世界卫生组织（World Health Organization, WHO）列为引发全球卫生紧急事件的病原体。目前没有被批准用于ZIKV预防或治疗的药物。泛素依赖型线粒体自噬是一种线粒体质量控制机制，通过选择性降解受损线粒体来维持细胞内环境稳态，其过程包括线粒体自噬启动和线粒体自噬受体介导的自噬溶酶体形成。许多病毒通过劫持或拮抗线粒体自噬来促进自身感染，但目前线粒体自噬在ZIKV感染及复制中的作用尚不明确。

本研究聚焦线粒体自噬与ZIKV病毒感染，首先探究了ZIKV感染对线粒体的影响，发现ZIKV病毒感染导致线粒体损伤；在此基础上，深入探究了线粒体损伤后线粒体自噬激活和线粒体自噬受体—视神经蛋白（Optineurin, OPTN）对ZIKV复制的影响及其作用机制。该结果有助于为治疗ZIKV感染提供新的靶点。

研究方法：

1.对GEO数据库中ZIKV感染导致小头畸形的婴儿大脑皮层样本的RNA-seq数据进行重分析，筛选出ZIKV感染和未感染的差异表达基因，进一步进行基因本体论（Gene ontology, GO）通路分析，确定ZIKV感染是否对线粒体相关生物过程产生影响。在ZIKV感染的A549细胞中，对线粒体进行荧光染色，利用共聚焦显微镜和透射电镜观察分析ZIKV感染对线粒体形态和功能的影响。

2. 在ZIKV感染的A549细胞中，使用线粒体解偶联剂氯硝柳胺（Niclosamide, NIC）激活线粒体自噬，或采用U0126-EtOH抑制线粒体自噬，检测细胞中泛素磷酸化水平、自噬激活水平和ZIKV复制水平的变化，获得线粒体自噬激活对ZIKV复制的作用。进一步开展体内实验，采用1日龄BALB/c小鼠颅内注射ZIKV的方式构建ZIKV诱导小头症的小鼠模型，在该模型中检测NIC处理对脑组织坏死以及ZIKV在血液和脑组织中复制的作用，并检测NIC对泛素依赖型线粒体自噬通路相关分子的影响，从而在体内验证NIC激活的线粒体自噬对ZIKV复制的作用。为确定线粒体自噬激活抑制ZIKV复制的关键分子，在A549细胞中敲减PTEN诱导激酶1（PTEN induced kinase 1, PINK1），在RNA和蛋白水平检测其对ZIKV复制和NIC诱导的抗病毒作用的影响。

3.在A549细胞和U251-MG细胞中检测ZIKV感染对线粒体自噬受体OPTN表达水平的影响；检测过表达或敲减OPTN对ZIKV复制的影响，从而探究OPTN在ZIKV复制中的作用。为探究OPTN在ZIKV复制中的作用机制，在A549、U251-MG和293T细胞中过表达OPTN，通过Western blotting、Real-time PCR和双荧光报告基因检测等手段检测OPTN高表达对Ⅰ型干扰素（Interferon, IFN）表达及其下游通路中STAT1磷酸化水平、干扰素刺激反应元件（Interferon stimulated response element, ISRE）活性、干扰素刺激基因（Interferon stimulated genes, ISGs）表达水平的影响。

研究结果：

1. ZIKV感染影响包括线粒体自噬在内的线粒体相关生物过程，导致线粒体碎裂、活性降低、功能受损。经重分析，RNA-seq共筛选出1569个ZIKV感染和未感染大脑皮层样本的差异表达基因，其中上调基因1326个，下调基因243个。多种差异表达基因富集在包括线粒体自噬在内的线粒体相关生物过程中。激光共聚焦和透射电镜观察发现，与未感染细胞相比，ZIKV感染的细胞中线粒体碎裂、活性降低、功能受损。

2. 体外实验显示，线粒体自噬抑制ZIKV复制。NIC激活泛素依赖型线粒体自噬后，以剂量依赖的方式抑制ZIKV复制；U0126-EtOH抑制线粒体自噬后，促进ZIKV复制，并挽救NIC对ZIKV复制的抑制作用，提示NIC诱导的线粒体自噬能够在体外抑制ZIKV复制。

3. 在小鼠ZIKV感染模型中验证了线粒体自噬能够在体内抑制ZIKV复制。动物实验表明，NIC治疗在体内抑制ZIKV复制，缓解小头症；这种抑制作用是通过线粒体自噬来实现的，表现为NIC促进PINK1和Parkin易位到线粒体外膜，挽救被ZIKV感染抑制的PINK1自磷酸化，激活泛素在Ser65位点的磷酸化。

4. PINK1是线粒体自噬抑制ZIKV复制的关键靶点。敲减PINK1促进ZIKV复制，并逆转NIC的抗ZIKV复制作用。提示PINK1是线粒体自噬抑制ZIKV复制的关键靶点。

5. 过表达OPTN在RNA和蛋白水平均抑制ZIKV复制，促进IFN-α的抗ZIKV作用；过表达OPTN诱导IFN-α 、IFN-β表达增加，从而激活JAK/STAT信号通路，促进STAT1磷酸化，进而增强ISRE活性，诱导抗病毒ISGs表达增加，最终发挥抑制ZIKV复制的作用。

结论：

1. ZIKV感染影响线粒体动力学，导致线粒体碎裂、活性降低、功能受损。

2. 线粒体自噬抑制ZIKV复制，其中PINK1是发挥该抑制作用的关键靶点。

3.ZIKV感染也可通过促进线粒体自噬受体OPTN的表达，激活JAK/STAT信号通路来启动宿主抗ZIKV的防御机制。

The role and the underlying mechanism of ubiquitin-dependent mitophagy in Zika virus replication

Abstract

Background and Aims:

Zika virus (ZIKV) is a mosquito-borne RNA virus belonging to the genus Flavivirus of the Flaviviridae family. It was once declared as a global health emergency by the World Health Organization (WHO). There are currently no vaccines nor approved drugs for the prevention or treatment of ZIKV infection. Ubiquitin-dependent mitophagy is a mitochondrial quality control mechanism that maintains intracellular environmental homeostasis, through selective degradation of damaged mitochondria. The process includes the initiation of mitophagy, and the formation of autophagy lysosomes mediated by mitophagy receptors. Many viruses promote their infection by either hijacking or antagonizing mitophagy. However, the role of mitophagy in ZIKV infection remains unclear. Therefore, exploring the mechanism of mitophagy in ZIKV replication is helpful to provide new targets for the treatment of ZIKV infection.

This study aimed to investigate the role of the mitophagy pathway in ZIKV infection. Firstly, we investigated the effect of ZIKV infection on mitochondria, confirming that ZIKV infection leads to mitochondrial damage. Next, we explored the effects and mechanism of mitophagy activation and the mitophagy receptor-optineurin (OPTN) on ZIKV replication.

Methods:

1. We reanalyzed the RNA-seq data of cerebral cortex samples of infants with microcephaly caused by ZIKV infection from GEO datasets and screened for differentially expressed genes between ZIKV-infected and uninfected tissues, and further Gene Ontology (GO) pathway analysis was performed to determine the effect of ZIKV infection on mitochondria-related biological processes. Mitochondria were stained with fluorescence in A549 cells, and the effects of ZIKV infection on mitochondrial morphology and functions were observed and analyzed by laser confocal and transmission electron microscopy (TEM).

2. Mitophagy was activated by niclosamide (NIC), a mitochondrial uncoupling agent, and inhibited by U0126-EtOH in ZIKV-infected A549 cells. Ubiquitin phosphorylation levels, autophagy activation levels, and ZIKV replication levels were detected to investigate the role of mitophagy activation on ZIKV replication. Furthermore, we constructed a ZIKV-induced microcephaly mouse model by intracranial injection of ZIKV into one-day-old BALB/c mice for in vivo experiments. In this model, the effects of NIC treatment on brain tissue necrosis and ZIKV replication in both blood and brain tissue were detected. Simultaneously, the effects of NIC on mitophagy pathway-related molecules, such as LC3B, PINK1, and Parkin were also detected to verify the role of NIC-induced mitophagy activation in ZIKV replication. To identify the key molecules involved in the inhibitory effect of mitophagy activation on ZIKV replication, PINK1 was knocked down in A549 cells, and its effect on ZIKV replication and NIC-induced antiviral effect was detected at RNA and protein levels.

3. The effect of ZIKV infection on the expression level of mitophagy receptor OPTN was detected in both A549 cells and U251-MG cells. We also detected the effect of overexpression or knockdown of OPTN on ZIKV replication, to investigate the role of OPTN in ZIKV replication. To further explore the mechanism of OPTN in ZIKV replication, the effect of OPTN overexpression on type I interferon (IFN) expression and its downstream pathway including phosphorylated STAT1 level, interferon stimulated response element (ISRE) activity, and interferon stimulated genes (ISGs) expression were detected by Western blotting, dual-luciferase reporter assays and Real-time PCR respectively in A549, U251-MG, and 293T cells.

Results:

1. ZIKV infection affected mitochondria-related biological processes including mitophagy, leading to fragmentation, decreased activity and functional impairment of mitochondria. By reanalysis, a total of 1569 differentially expressed genes (DEGs) were identified from RNA-seq dataset of ZIKV-infected and uninfected cerebral cortex samples, including 1326 up-regulated genes and 243 down-regulated genes. Multiple DEGs were enriched in mitochondria-related biological processes including mitophagy. Laser confocal and TEM observations revealed that mitochondria were fragmented, less active, and functionally impaired in ZIKV-infected cells compared to uninfected cells.

2. Mitophagy inhibited ZIKV replication in vitro. In vitro experiments showed that NIC activated ubiquitin-dependent mitophagy and inhibited ZIKV replication in a dose-dependent manner; U0126-EtOH inhibited mitophagy and promoted ZIKV replication, rescuing the inhibitory effect of NIC on ZIKV replication, suggesting that NIC-induced mitophagy suppressed ZIKV replication in vitro.

3. Mitophagy inhibited ZIKV replication in vivo. Data from ZIKV-infected mouse model showed that NIC treatment inhibited ZIKV replication and alleviated microcephaly in vivo, and this inhibitory effect was mediated by mitophagy activation as shown by the translocation of PINK1 and Parkin to the outer mitochondrial membrane, restored ZIKV-induced inhibition of PINK1 autophosphorylation and increased ubiquitin phosphorylation level at the Ser65 in mouse brain tissue.

   4. PINK1 was the key target for the mitophagy-mediated inhibition of ZIKV. Knockdown of PINK1 promoted ZIKV replication and reversed the anti-ZIKV replication effect of NIC suggesting PINK1 is a key target of mitophagy to inhibit ZIKV replication.

5. ZIKV infection increased mitophagy receptor OPTN expression leading to the activation of JAK/STAT signaling to inhibit ZIKV replication. Overexpression of OPTN inhibited ZIKV replication at both RNA and protein levels, and promoted the anti-ZIKV effect of IFN-α. Overexpression of OPTN stimulated the expression of IFN-α and IFN-β, which activated the JAK/STAT signaling pathway as shown by increased STAT1 phosphorylation level and enhanced ISRE activity. This, in turn, stimulated expression of ISGs to exert the inhibitory effect on ZIKV replication.

Conclusion

1. ZIKV infection affects mitochondrial dynamics leading to fragmentation, decreased activity and functional impairment of mitochondria.

2. Mitophagy inhibits ZIKV replication and PINK1 is a key target for the inhibitory effect.

3. ZIKV infection increases the expression level of mitophagy receptor OPTN leading to the activation of JAK/STAT signaling to initiate the host anti-ZIKV mechanism.