The BECN1-USP19 axis plays a role in the crosstalk between autophagy and antiviral immune responses

ABSTRACT

Macroautophagy/autophagy is a conserved intracellular degradation system that traffics substrates including protein aggregates, defunct or disused organelles and invading pathogens to lysosomes via double-membrane vesicles called autophagosomes. BECN1/Beclin 1 functions as a key protein in autophagy initiation and progression; however, the role of BECN1 in innate immunity has not been fully investigated. Recently, we have found that USP19 affects the ubiquitination of BECN1, hence promoting the formation of autophagosomes and inhibiting DDX58/RIG-I-mediated type I interferon signaling.

Ubiquitination is an essential post-translational modification process, which is involved in the regulation of many cellular activities, including autophagy. To gain insights into how ubiquitination affects the roles of autophagy-related (ATG) proteins in autophagy, we performed a screen by using small interfering RNAs (siRNAs) targeting human deubiquitinating enzyme (DUB) genes, and identified USP19 as a positive regulator of autophagy initiation and progression. USP19 associates with the BECN1 complex members, but not with ATG8 subfamily members or cargo receptors. Notably, starvation can enhance the association between USP19 and BECN1. By detecting the protein abundance, we found that USP19 specifically stabilizes BECN1 in an enzyme activity-dependent manner. In addition, we found that USP19 rescues the proteasomal degradation of BECN1 (Fig. 1). Although USP13 has been implicated in the regulation of BECN1 stability, USP19 appears to function in BECN1 stability in USP13 knockout (KO) cells. Moreover, we analyzed the 7 different types of poly-ubiquitination of BECN1 and found that USP19 markedly removed the K11-linked ubiquitination of BECN1, but had no appreciable effect on the ubiquitination of BECN1 having other linkages.

Figure 1.

A working model of USP19-mediated BECN1 stabilization in the regulation of autophagy and type I IFN signaling. USP19 cleaves the K11-linked ubiquitin chains on BECN1 to promote autophagy, while blocks DDX58-MAVS association to suppress type I IFN signaling, thus keeping a balance between autophagy and antiviral responses.

In order to discover the detailed mechanism underlying BECN1 ubiquitination, we generated a series of mutant constructs by substituting lysine residues with arginines (R) with a focus on key lysine residues that are conserved in the orthologs of BECN1. We found that USP19 loses the ability to stabilize the K437R BECN1 mutant (BECN1^K437R), and that this mutant displays an impaired K11-linked ubiquitination when compared with wild-type (WT) BECN1. We next investigated whether K11-linked polyubiquitin chains on K437 serve as a degradation signal for BECN1, and observed that the degradation rate of the BECN1^K437R mutant is decreased compared to that of WT BECN1. To test whether USP19 mediated autophagic flux by BECN1 stabilization, we transfected BECN1 KO cells with WT BECN1 or a version expressing the K437R mutant, and found that the BECN1^K437R mutant dramatically enhances the formation of LC3 puncta and accumulation of LC3-II.

Several autophagy-related factors such as ATG12–ATG5, ATG9 and ULK1 have autophagy-independent immune functions, and play important roles in antiviral immunity. We further determined whether USP19-mediated autophagy is involved in the regulation of type I interferon (IFN) signaling and observed that USP19 specifically inhibits the DDX58-mediated, but not the MB21D1/cGAS-mediated type I IFN signaling pathway. To confirm the inhibitory effect of USP19 on type I IFN activation and antiviral immune responses, we used several different RNA viruses to infect human cells, and found that USP19 suppresses type I IFN signaling and antiviral immune responses. To determine whether the inhibition of type I IFN signaling by USP19 is dependent on the autophagy process, we examined the activation of type I IFN signaling in BECN1, ATG5 and SQSTM1 KO cells. We found that USP19 cannot inhibit the activation of type I IFN signaling only in BECN1 KO cells, but not in ATG5 or SQSTM1 KO cells, suggesting that the inhibition of type I IFN signaling by USP19 is associated with BECN1 in an autophagy-independent manner.

We next evaluated the potential role of BECN1 in type I IFN signaling and found that BECN1 itself is a novel negative regulator of DDX58-mediated type I IFN signaling (Fig. 1). BECN1 appears to target MAVS by binding to its CARD domain. We then checked the association between DDX58 and MAVS relative to the existence of USP19 and BECN1. We observed that BECN1-USP19 axis inhibits DDX58-MAVS interaction. It is previously reported that the ATG12–ATG5 conjugate suppresses type I IFN signaling by association with DDX58 and MAVS. Therefore, we next studied whether BECN1 inhibits DDX58-mediated signaling in an ATG5-independent manner, and found that this was indeed the case. BECN1 and ATG12–ATG5 affect antiviral immunity independently, leading to the integration of autophagy and type I IFN signaling and broadening the multilayer regulation of the crosstalk (Fig. 1).

In summary, our study provides evidence that in mammalian cells, BECN1-USP19 is not only involved in the regulation of autophagic flux, but also associated with type I IFN signaling. The non-autophagic role of BECN1 in type I IFN signaling increases our understanding of the non-canonical roles of autophagy-related genes. Biallelic deletion of Becn1 results in the lethality of mice, thus, the in vivo function of BECN1 in innate immunity remains largely unknown. In our study, we generated BECN1 KO cells and validated its negative regulation of antiviral immune responses. In previous work, BECN1 was reported to interact with MB21D1 for inhibiting its enzyme activity, therefore negatively regulating MB21D1-mediated type I IFN signaling. Even though BECN1 serves as a negative regulator in both DDX58 and MB21D1-mediated type I IFN signaling, generation of tissue specific Becn1 KO mice to reveal the in vivo role of BECN1 in antiviral responses is needed. Therefore, further study should be carried out in this field in order to develop strategies for the treatment and prevention of virus infection.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by National Key Basic Research Program of China (2015CB859800 and 2014CB910800), National Natural Science Foundation of China (31370869, 31522018), Guangdong Natural Science Funds for Distinguished Young Scholar (S2013050014772), Guangdong Innovative Research Team Program (NO. 201001Y0104687244 and 2011Y035), the Fundamental Research Funds for the Central Universities (15lgjc02), and the Training Program for Outstanding Young Teachers in Higher Education institutions of Guangdong Province (YQ2015001). R.-F.W was in part supported by grants (CA090327 and CA101795, CA121191) from NCI, NIH.