Mechanism and Function of Mitophagy Regulation by HHV-8 Viral Interferon Regulatory Factor-1

摘要 人类疱疹病毒8型(HHV-8)是卡波西肉瘤、原发性渗出性淋巴瘤(PEL)和 多中心Castleman病，通常与获得性免疫缺陷综合征有关。HHV-8 除潜伏期外，裂解复制对于维持宿主内的病毒载量以及相关的 发病机制。最近的证据表明，HHV-8编码的病毒干扰素调节因子1(vIRF-1)在 抑制病毒感染诱导的细胞凋亡和先天免疫在促进病毒复制中的重要作用 宿主细胞。我们最近首次发现，vIRF-1通过一种新的机制定位于线粒体。 病毒复制和线粒体定位的vIRF-1是病毒复制成功的关键。然而， 线粒体定位的vIRF-1对HHV-8生物学和发病机制的功能意义尚不清楚 下定决心。我们的初步结果显示，溶解的vIRF-1阳性的PEL线粒体含量减少。 细胞，但通过药物抑制自噬和有丝分裂而恢复。此外，使用重组HHV- 8编码vIRF-1，线粒体靶向受损，vIRF-1衍生的多肽对应于 线粒体靶向区域，我们证明了线粒体靶向vIRF-1在 HHV-8复制过程中线粒体含量和细胞凋亡的调节。我们的进一步研究表明，vIRF- 1与自噬相关蛋白(GEC1和EF-Tu)相互作用，并受到翻译后修饰的影响 (caspase-5介导的切割和K63连锁的多泛素化)对HHV-8复制和线粒体的影响 损坏。此外，Mdivi-1抑制有丝分裂吞噬增强了病毒诱导的MAVS介导的细胞凋亡 复制并抑制HHV-8的生产性复制。基于这些发现，我们假设vIRF-1可以 感知和应对HHV-8复制引起的线粒体损伤，促进功能障碍的清除 通过激活线粒体的有丝分裂，从而抑制线粒体介导的抗病毒反应和 推广HHV-8复制。因此，干扰vIRF-1激活的有丝分裂吞噬作用的方法可能导致 新型抗病毒药物的开发。为了进一步明确vIRF-1的机制和功能意义- 在HHV-8复制中激活有丝分裂吞噬，我们建议：1)阐明vIRF-1的分子机制(S)- HHV-8复制激活的丝裂原吞噬作用，2)确定其对丝裂原吞噬的调节意义 线粒体定位的vIRF-1的新的翻译后修饰，以及3)决定功能 VIRF-1激活的有丝分裂吞噬在MAV调节和HHV-8生物学中的意义。圆满完成 在这一应用中的研究将描绘涉及vIRF-1激活的分子相互作用和过程 丝裂原吞噬，并为抑制有丝分裂抑制的抗病毒和治疗潜力提供证据。总体而言，这 该提案将确定一种通过线粒体实现病毒-宿主相互作用的新范例，并可能提供一种 为未来针对vIRF-1吞噬相关相互作用的抗病毒药物的开发奠定基础。

ABSTRACT Human herpesvirus 8 (HHV-8) is a causal agent for Kaposi’s sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman’s disease that are often associated with acquired immunodeficiency syndrome. HHV-8 lytic replication, in addition to latency, is important for maintaining viral load within the host as well as for related pathogenesis. Recent evidence shows that HHV-8-encoded viral interferon regulatory factor 1 (vIRF-1) plays an important role in promoting virus replication by suppressing apoptosis and innate immunity elicited by infection of host cells. We recently found, for the first time, that vIRF-1 localizes to mitochondria via a novel mechanism upon virus replication and mitochondria-localized vIRF-1 is essential for successful virus replication. However, the functional significance of mitochondria-localized vIRF-1 to HHV-8 biology and pathogenesis remains to be determined. Our preliminary results showed that mitochondrial content is reduced in lytic vIRF-1-positive PEL cells but restored by pharmacological inhibition of autophagy and mitophagy. Moreover, using recombinant HHV- 8 encoding vIRF-1 with impaired mitochondria targeting and a vIRF-1-derived peptide corresponding to the mitochondrial targeting region, we demonstrated that mitochondria targeting of vIRF-1 plays an important role in regulation of mitochondria content and apoptosis during HHV-8 replication. Our further studies revealed that vIRF- 1 interacts with autophagy-related proteins (GEC1 and EF-Tu) and is subject to posttranslational modifications (caspase-5-mediated cleavage and K63-linked polyubiquitination) upon HHV-8 replication and mitochondrial damage. Moreover, inhibition of mitophagy by Mdivi-1 enhances MAVS-mediated apoptosis induced by virus replication and inhibits HHV-8 productive replication. Based on these findings, we hypothesize that vIRF-1 can sense and respond to mitochondria damage induced by HHV-8 replication and promote removal of dysfunctional mitochondria by activating mitophagy, resulting in inhibition of mitochondria-mediated antiviral responses and promotion of HHV-8 replication. Therefore, an approach to interfere with vIRF-1-activated mitophagy could lead to the development of novel antiviral agents. To further define the mechanisms and functional significance of vIRF-1- activated mitophagy in HHV-8 replication, we propose to: 1) delineate the molecular mechanism(s) of vIRF-1- mediated mitophagy activated by HHV-8 replication, 2) determine the significance to mitophagy regulation of novel posttranslational modifications of mitochondria-localized vIRF-1, and 3) determine the functional significance of vIRF-1-activated mitophagy in MAVS regulation and HHV-8 biology. Successful completion of the studies in this application will delineate the molecular interactions and processes involved in vIRF-1-acitvated mitophagy and provide evidence for the antiviral and therapeutic potentials of mitophagy inhibition. Overall, this proposal will identify a novel paradigm of virus-host interaction via mitochondria and could potentially provide a basis for future development of antiviral agents targeting mitophagy-associated interactions of vIRF-1.