The mitophagy receptor NIX induces vIRF-1 oligomerization and interaction with GABARAPL1 for the promotion of HHV-8 reactivation-induced mitophagy

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. Author summaryIn addition to their role in energy metabolism, mitochondria act as platforms for antiviral signaling that leads to apoptosis and type I interferon expression in response to virus infection. However, for their benefit, viruses have evolved strategies to attenuate mitochondria-mediated antiviral signaling, including selective autophagy of mitochondria (termed mitophagy) that eliminates dysfunctional or superfluous mitochondria. We previously demonstrated that mitochondria-localized viral interferon regulatory factor 1 (vIRF-1) plays a role in the activation of mitophagy during human herpesvirus 8 (HHV-8) via interaction with the mitophagy proteins NIX and TUFM, thereby inhibiting antiviral responses and contributing to productive replication. However, it remains to elucidate the precise molecular mechanisms and regulation of vIRF-1-mediated mitophagy, particularly interactions with the core autophagy machinery. In this report, we discover that vIRF-1 interacts selectively with GABARAPL1 among ATG8 proteins in a non-canonical manner in lytically HHV-8-infected cells, and the mitophagy receptor NIX promotes vIRF-1 oligomerization and interaction with the ATG8 protein. Our results reveal the presence of a functional complex of vIRF-1/NIX/GABARAPL1 for the promotion of mitophagy and also provide the first evidence of the post-translational regulation of viral mitophagic protein by cellular mitophagy receptor.