Sequences of oligonucleotides used in this study.

Recently, viruses have been shown to regulate selective autophagy for productive infections. For instance, human herpesvirus 8 (HHV-8), also known as Kaposi’s sarcoma-associated herpesvirus (KSHV), activates selective autophagy of mitochondria, termed mitophagy, thereby inhibiting antiviral innate immune responses during lytic infection in host cells. We previously demonstrated that HHV-8 viral interferon regulatory factor 1 (vIRF-1) plays a crucial role in lytic replication-activated mitophagy by interacting with cellular mitophagic proteins, including NIX and TUFM. However, the precise molecular mechanisms by which these interactions lead to mitophagy activation remain to be determined. Here, we show that vIRF-1 binds directly to mammalian autophagy-related gene 8 (ATG8) proteins, preferentially GABARAPL1 in infected cells, in an LC3-interacting region (LIR)-independent manner. Accordingly, we identified key residues in vIRF-1 and GABARAPL1 required for mutual interaction and demonstrated that the interaction is essential for mitophagy activation and HHV-8 productive replication. Interestingly, the mitophagy receptor NIX promotes vIRF-1-GABARAPL1 interaction, and NIX/vIRF-1-induced mitophagy is significantly inhibited in GABARAPL1-deficient cells. Moreover, a vIRF-1 variant defective in GABARAPL1 binding substantially loses the ability to induce vIRF-1/NIX-induced mitophagy. These results suggest that NIX supports vIRF-1 activity as a mitophagy mediator. In addition, we found that NIX promotes vIRF-1 aggregation and stabilizes aggregated vIRF-1. Together, these findings indicate that vIRF-1 plays a role as a viral mitophagy mediator that can be activated by a cellular mitophagy receptor.近年来已有研究证实，病毒可通过调控选择性自噬以支持自身的增殖性感染。例如，人类疱疹病毒8型（Human Herpesvirus 8, HHV-8），又称卡波西肉瘤相关疱疹病毒（Kaposi’s Sarcoma-Associated Herpesvirus, KSHV），可激活线粒体选择性自噬（即线粒体自噬，mitophagy），从而在宿主细胞的裂解性感染过程中抑制抗病毒先天免疫应答。我们此前的研究证实，HHV-8编码的病毒干扰素调节因子1（viral Interferon Regulatory Factor 1, vIRF-1）可通过与细胞线粒体自噬蛋白（包括NIX与TUFM）相互作用，在裂解复制激活的线粒体自噬过程中发挥关键调控作用。然而，这些相互作用介导线粒体自噬激活的确切分子机制仍有待阐明。本研究中，我们发现vIRF-1可直接结合哺乳动物自噬相关基因8（Autophagy-Related Gene 8, ATG8）家族蛋白，在感染细胞中优先结合GABARAPL1，且该结合过程不依赖于LC3互作区域（LC3-Interacting Region, LIR）。据此，我们鉴定出vIRF-1与GABARAPL1相互结合所需的关键氨基酸残基，并证实该相互作用对于线粒体自噬激活及HHV-8的增殖性复制至关重要。有趣的是，线粒体自噬受体NIX可促进vIRF-1与GABARAPL1的相互结合，且在GABARAPL1缺陷细胞中，NIX/vIRF-1诱导的线粒体自噬会受到显著抑制。此外，无法结合GABARAPL1的vIRF-1突变体，其介导vIRF-1/NIX诱导的线粒体自噬的能力会大幅丧失。上述结果表明，NIX可增强vIRF-1作为线粒体自噬介质的活性。此外，我们还发现NIX可促进vIRF-1的聚集并稳定聚集状态的vIRF-1。综上，本研究结果揭示，vIRF-1可作为一种病毒源性线粒体自噬介质，且其活性可被细胞线粒体自噬受体激活。