Coronavirus hijacks STX18-ATG14 axis-regulated lipophagy to evade an anti-viral effect

ABSTRACT

ATG14 is a core subunit of the class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) for macroautophagy/autophagy initiation and also binds to the STX17 to promote autophagosome-lysosome fusion. Our recent work found that ATG14 also targets lipid droplets (LDs) and interacts with mammalian Atg8-family proteins (ATG8s) to mediate lipophagy (selective autophagic degradation of lipid droplets). We also demonstrated that STX18 (syntaxin 18) acts as a negative regulator that disrupts the interactions of ATG14-ATG8s and the formation of the PtdIns3K-C1 through binding to ATG14. Furthermore, we found that knockdown of STX18 induces LD-associated anti-viral protein RSAD2/Viperin degradation dependent on ATG14-mediated lipophagy. Additionally, coronavirus M protein hijacks STX18 to induce lipophagy and degrade RSAD2, facilitating virus production. In summary, our findings reveal new roles of ATG14 in lipid metabolism and viral replication as an autophagic receptor.

ATG14 is a core component of the autophagic initiation class III PtdIns3K complex I, which consists of PIK3C3/VPS34, BECN1, PIK3R4/p150, NRBF2 and ATG14, and is responsible for the initiation of autophagy by generating phosphatidylinositol-3-phosphate (PtdIns3P). In addition, during final maturation of autophagy, oligomeric ATG14 directly binds to the STX17-SNAP29-VAMP8 complex to promote membrane tethering and autophagosome-lysosome fusion. Beside the roles in early and late stages of autophagy, the gain and loss of ATG14 result in a fall and increase in triglyceride levels in the liver and serum of mice, indicating that ATG14 May function in lipid metabolism while the precise mechanism(s) remains unknown.

LDs are dynamic intracellular organelles that are filled with neutral lipids, which can be selectively degraded by an autophagic mechanism, a process called lipophagy. Several proteins have been demonstrated to regulate lipophagy, whereas the molecular mechanisms and specific receptors linking LDs to phagophores are unclear.

In order to determine the roles of ATG14 in lipophagy, we first examined the colocalization of LDs and ATG14 [1]. ATG14 localizes to LDs when exposed to oleic acid but fails to target to LDs under steady state conditions. Interestingly, overexpression of ATG14 leads to a decrease in the count of LDs and triglyceride storage, and this reduction can be reversed by chloroquine or bafilomycin A₁ treatment, suggesting that ATG14 targets LDs and triggers lipophagy.

During selective autophagy, unique cargo is engulfed by phagophores through the direct interaction of phagophore-associated ATG8s with selective receptors via MAP1LC3/LC3-interacting regions (LIRs). We confirmed that ATG14 interacts with LC3A, LC3C, GABARAP, and GABARAPL2. Additionally, we generated a point mutant ATG14[LIRm] to break the LIR, and found that ATG14[LIRm] neither binds to ATG8s, nor recruits LC3 to LDs. Moreover, only ATG14 rescues the reduced LDs content and triglyceride storage, but not ATG14[LIRm] in atg14 knockout cells, suggesting that the direct interactions between LD-localized ATG14 and ATG8s are required for lipophagy.

We then focused on the regulatory mechanism by which ATG14 functions as an autophagic receptor in lipophagy. Based on siRNA screening, we focused on STX18. The interaction between ATG14 and STX18 decreases under serum starvation, and depletion of STX18 induces lipophagy, which is dependent on ATG14. Furthermore, STX18 associates with LDs. These results indicate that STX18 functions as a negative regulator for ATG14-mediated lipophagy.

Mechanistically, we found that knockdown of STX18 enhances the ATG14-LC3C interaction, whereas overexpression of STX18 significantly disrupts the interactions between ATG14 and LC3A, LC3C, GABARAP, or GABARAPL2. These data suggest that STX18 negatively regulates ATG14-ATG8s interactions. Additionally, we also found that STX18 overexpression remarkably reduces the interactions between the ATG14-BECN1-PIK3C3/VPS34 components, whereas depletion of STX18 enhances PtdIns3P production, indicating that STX18 inhibits the formation of the PtdIns3K-C1. Furthermore, we purified PtdIns3KC3-C1 from HEK293F cells and confirmed that STX18 inhibits the assembly of the complexes in vitro. Based on these results, we propose that STX18 regulates lipophagy by disrupting the interactions of ATG14-ATG8s and the formation of the PtdIns3K-C1.

The LD monolayer also accommodates a variety of anti-microbial and anti-viral proteins that contribute to the innate immune response. RSAD2/Viperin, through its N-terminal amphipathic alpha-helix, localizes to LDs and mediates anti-viral responses. Our data showed that STX18 depletion or ATG14 overexpression-induced lipophagy lead to the degradation of RSAD2. Importantly, ATG14[LIRm] fails to rescue the degradation of RSAD2 caused by STX18 depletion in atg14 KO cells. Overall, our results demonstrate that RSAD2 can be degraded by the STX18-ATG14 axis-regulated lipophagy.

Interestingly, we found that RSAD2 knockdown enhances SARS-CoV-2 replication. We then wondered whether SARS-CoV-2 could exploit the STX18-ATG14 axis-regulated lipophagy to degrade RSAD2 and promote viral replication. Based on our previous IP/MS, STX18 is on the candidate list for the SARS-CoV-2 M interactome. Moreover, M protein interacts with STX18, which leads to an increased association of ATG14 with LC3 and the formation of PtdIns3K-C1, thereby triggering lipophagy for RSAD2 degradation. We also found that STX18 overexpression and ATG14 depletion significantly decrease SARS-CoV-2 replication. Altogether, our data suggest that SARS-CoV-2 M protein binds to STX18 and subverts the STX18-ATG14 interaction to induce lipophagy and degrade RSAD2, thereby facilitating virus production.

In conclusion, our study reveals a new role of ATG14 in lipophagy and coronavirus replication. We found that ATG14 targets LDs and interacts with ATG8s to mediate lipophagy. Our study led to the discovery of a novel lipophagy regulator, STX18, that binds to ATG14 and disrupts the interactions of ATG14-ATG8s and the formation of PtdIns3K-C1. Importantly, SARS-CoV-2 hijacks STX18 by viral protein M, and subverts the interaction of STX18 with ATG14, thereby promoting the formation of PtdIns3K-C1 and ATG14-ATG8s interaction, resulting in the induction of ATG14-mediated lipophagy to degrade RSAD2 to evade the anti-viral effect (Figure 1).

Figure 1.

Proposed model for STX18-ATG14 axis regulation of lipophagy. In wild-type cells, STX18 interacts with ATG14 and disrupts the interactions of ATG14 with LC3 and subverts the formation of PtdIns3K-C1 to inhibit lipophagy. In coronavirus-infected cells, viral M protein binds to STX18 and subverts the STX18-ATG14 interaction to induce lipophagy and degrade the LD-associated anti-viral protein RSAD2.

Funding Statement

This work was supported by the National Natural Science Foundation of China (32370809, U22A20337).

Disclosure statement

No potential conflict of interest was reported by the author(s).