ATG4B antagonizes antiviral immunity by GABARAP-directed autophagic degradation of TBK1.

ABBREVIATIONS: Baf A1: bafilomycin A1; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; IFN: interferon; IKBKE/IKKi: inhibitor of nuclear factor kappa B kinase subunit epsilon; IRF3: interferon regulatory factor 3; ISG: interferon-stimulated gene; ISRE: IFN-stimulated response element; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; PAMPs: pathogen-associated molecule patterns; RIGI/DDX58: RNA sensor RIG-I; SeV: Sendai virus; siRNA: small interfering RNA; TBK1: TANK binding kinase 1; WT: wild-type; VSV: vesicular stomatitis virus.

Molecular mechanisms and cellular functions of liquid-liquid phase separation during antiviral immune responses.

Spatiotemporal separation of cellular components is vital to ensure biochemical processes. Membrane-bound organelles such as mitochondria and nuclei play a major role in isolating intracellular components, while membraneless organelles (MLOs) are accumulatively uncovered via liquid-liquid phase separation (LLPS) to mediate cellular spatiotemporal organization. MLOs orchestrate various key cellular processes, including protein localization, supramolecular assembly, gene expression, and signal transduction. During viral infection, LLPS not only participates in viral replication but also contributes to host antiviral immune responses. Therefore, a more comprehensive understanding of the roles of LLPS in virus infection may open up new avenues for treating viral infectious diseases. In this review, we focus on the antiviral defense mechanisms of LLPS in innate immunity and discuss the involvement of LLPS during viral replication and immune evasion escape, as well as the strategy of targeting LLPS to treat viral infectious diseases.

Phase-separated nucleocapsid protein of SARS-CoV-2 suppresses cGAS-DNA recognition by disrupting cGAS-G3BP1 complex.

Currently, the incidence and fatality rate of SARS-CoV-2 remain continually high worldwide. COVID-19 patients infected with SARS-CoV-2 exhibited decreased type I interferon (IFN-I) signal, along with limited activation of antiviral immune responses as well as enhanced viral infectivity. Dramatic progresses have been made in revealing the multiple strategies employed by SARS-CoV-2 in impairing canonical RNA sensing pathways. However, it remains to be determined about the SARS-CoV-2 antagonism of cGAS-mediated activation of IFN responses during infection. In the current study, we figure out that SARS-CoV-2 infection leads to the accumulation of released mitochondria DNA (mtDNA), which in turn triggers cGAS to activate IFN-I signaling. As countermeasures, SARS-CoV-2 nucleocapsid (N) protein restricts the DNA recognition capacity of cGAS to impair cGAS-induced IFN-I signaling. Mechanically, N protein disrupts the assembly of cGAS with its co-factor G3BP1 by undergoing DNA-induced liquid-liquid phase separation (LLPS), subsequently impairs the double-strand DNA (dsDNA) detection ability of cGAS. Taken together, our findings unravel a novel antagonistic strategy by which SARS-CoV-2 reduces DNA-triggered IFN-I pathway through interfering with cGAS-DNA phase separation.

Modulation of virus-induced neuroinflammation by the autophagy receptor SHISA9 in mice.

Microglia and astrocytes are subgroups of brain glia cells that support and protect neurons within the central nervous system (CNS). At early stages of viral infection in the CNS, they are predominant responding cells and lead to recruitment of peripheral immune cells for viral clearance. Inhibitor of nuclear factor κB kinase subunit epsilon (IKKi) is critical for type I interferon signalling and inflammation, which modulate heterogenic immune responses during CNS infection. Balanced autophagy is vital to maintain brain integrity, yet regulation of autophagy and immune activity within brain glia cells is poorly understood. Here we identify SHISA9 as an autophagy cargo receptor that mediates the autophagy-dependent degradation of IKKi during herpes simplex virus type 1 infection. IKKi is recognized by SHISA9 through unanchored K48-linked poly-ubiquitin chains and bridged to autophagosome membrane components GABARAPL1. Single-cell RNA sequencing analysis shows that SHISA9 has temporal characteristics while modulating both antiviral and inflammatory responses in microglia and astrocytes at different stages during viral infection. We found that Shisa9-/- mice are highly susceptible to herpes simplex virus encephalitis, have pathogenic astrocytes and display more severe neuroinflammation compared with wild-type mice. Taken together, our study unravels a critical role of selective autophagy by orchestrating immune heterogeneity of different CNS resident cells through the SHISA9-IKKi axis.

Metabolic enzyme UAP1 mediates IRF3 pyrophosphorylation to facilitate innate immune response.

Post-translational modifications (PTMs) of proteins are crucial to guarantee the proper biological functions in immune responses. Although protein phosphorylation has been extensively studied, our current knowledge of protein pyrophosphorylation, which occurs based on phosphorylation, is very limited. Protein pyrophosphorylation is originally considered to be a non-enzymatic process, and its function in immune signaling is unknown. Here, we identify a metabolic enzyme, UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1), as a pyrophosphorylase for protein serine pyrophosphorylation, by catalyzing the pyrophosphorylation of interferon regulatory factor 3 (IRF3) at serine (Ser) 386 to promote robust type I interferon (IFN) responses. Uap1 deficiency significantly impairs the activation of both DNA- and RNA-viruse-induced type I IFN pathways, and the Uap1-deficient mice are highly susceptible to lethal viral infection. Our findings demonstrate the function of protein pyrophosphorylation in the regulation of antiviral responses and provide insights into the crosstalk between metabolism and innate immunity.

Palmitoylation prevents sustained inflammation by limiting NLRP3 inflammasome activation through chaperone-mediated autophagy.

As a key component of the inflammasome, NLRP3 is a critical intracellular danger sensor emerging as an important clinical target in inflammatory diseases. However, little is known about the mechanisms that determine the kinetics of NLRP3 inflammasome stability and activity to ensure effective and controllable inflammatory responses. Here, we show that S-palmitoylation acts as a brake to turn NLRP3 inflammasome off. zDHHC12 is identified as the S-acyltransferase for NLRP3 palmitoylation, which promotes its degradation through the chaperone-mediated autophagy pathway. Zdhhc12 deficiency in mice enhances inflammatory symptoms and lethality following alum-induced peritonitis and LPS-induced endotoxic shock. Notably, several disease-associated mutations in NLRP3 are associated with defective palmitoylation, resulting in overt NLRP3 inflammasome activation. Thus, our findings identify zDHHC12 as a repressor of NLRP3 inflammasome activation and uncover a previously unknown regulatory mechanism by which the inflammasome pathway is tightly controlled by the dynamic palmitoylation of NLRP3.

Suppression of ACE2 SUMOylation protects against SARS-CoV-2 infection through TOLLIP-mediated selective autophagy.

In addition to investigating the virology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), discovering the host-virus dependencies are essential to identify and design effective antiviral therapy strategy. Here, we report that the SARS-CoV-2 entry receptor, ACE2, conjugates with small ubiquitin-like modifier 3 (SUMO3) and provide evidence indicating that prevention of ACE2 SUMOylation can block SARS-CoV-2 infection. E3 SUMO ligase PIAS4 prompts the SUMOylation and stabilization of ACE2, whereas deSUMOylation enzyme SENP3 reverses this process. Conjugation of SUMO3 with ACE2 at lysine (K) 187 hampers the K48-linked ubiquitination of ACE2, thus suppressing its subsequent cargo receptor TOLLIP-dependent autophagic degradation. TOLLIP deficiency results in the stabilization of ACE2 and elevated SARS-CoV-2 infection. In conclusion, our findings suggest selective autophagic degradation of ACE2 orchestrated by SUMOylation and ubiquitination as a potential way to combat SARS-CoV-2 infection.

Erratum: Dual Feedforward Loops Modulate Type I Interferon Responses and Induce Selective Gene Expression during TLR4 Activation.

[This corrects the article DOI: 10.1016/j.isci.2020.100881.].

Autophagy Activation Induces p62-Dependent Autophagic Degradation of Dengue Virus Capsid Protein During Infection.

In the past decade, dengue virus infection is one of the most prevalent and rapidly spreading arthropod-borne diseases worldwide with about 400 million infections every year. Although it has been reported that the dengue virus could take advantage of autophagy to promote its propagation, the association between selective autophagy and the dengue virus remains largely unclear. Here, we demonstrated that dengue virus capsid protein, the key viral protein for virus assembly, maturation, and replication, underwent autophagic degradation after autophagy activation. Autophagy cargo receptor p62 delivered ubiquitinated capsid protein to autophagosomes for degradation, which could be enhanced by Torin 1 treatments. Further study revealed that the association between p62 and viral capsid protein was dependent on the ubiquitin-binding domain of p62, and the poly-ubiquitin conjugated at lysine 76 of capsid protein served as a recognition signal for autophagy. Consistently, p62 deficiency in Huh7 cells led to the enhancement of dengue virus replication. Our study revealed that p62 targeted dengue virus capsid protein for autophagic degradation in a ubiquitin-dependent manner, which might uncover the potential roles of p62 in restricting dengue virus replication.

Targeting Selective Autophagy as a Therapeutic Strategy for Viral Infectious Diseases.

Autophagy is an evolutionarily conserved lysosomal degradation system which can recycle multiple cytoplasmic components under both physiological and stressful conditions. Autophagy could be highly selective to deliver different cargoes or substrates, including protein aggregates, pathogenic proteins or superfluous organelles to lysosome using a series of cargo receptor proteins. During viral invasion, cargo receptors selectively target pathogenic components to autolysosome to defense against infection. However, viruses not only evolve different strategies to counteract and escape selective autophagy, but also utilize selective autophagy to restrict antiviral responses to expedite viral replication. Furthermore, several viruses could activate certain forms of selective autophagy, including mitophagy, lipophagy, aggrephagy, and ferritinophagy, for more effective infection and replication. The complicated relationship between selective autophagy and viral infection indicates that selective autophagy may provide potential therapeutic targets for human infectious diseases. In this review, we will summarize the recent progress on the interplay between selective autophagy and host antiviral defense, aiming to arouse the importance of modulating selective autophagy as future therapies toward viral infectious diseases.

Palmitoylation facilitates inflammation through suppressing NOD2 degradation mediated by the selective autophagy receptor SQSTM1.

The intracellular pattern recognition receptor NOD2 senses bacterial peptidoglycan to drive proinflammatory and antimicrobial responses. Dysregulation of NOD2 signaling confers susceptibility to several immunological and inflammatory diseases. Although palmitoylation of NOD2 is required for its membrane recruitment and activation, whether palmitoylation can modulate the stability of NOD2 to orchestrate inflammation remains unclear. Recently, we have revealed that S-palmitoylation restricts SQSTM1-mediated selective macroautophagic/autophagic degradation of NOD2, and identified a gain-of-function R444C variant of NOD2 short isoform (NOD2sR444C) in autoinflammatory disease, which induces excessive inflammation through its enhanced S-palmitoylation level and decreased autophagic degradation.

The TRIM14-USP14-BRCC3 complex epigenetically regulates inflammation through inhibiting OPTN-mediated autophagic degradation of KDM4D.

Macroautophagy/autophagy is a conserved eukaryotic process to mediate the degradation of cell organelles and protein aggregates, which participates in a variety of cellular responses, including immune signal transduction. KDM4D functions as an important histone demethylase to regulate gene transcription by inhibiting histone H3K9 trimethylation. Whether autophagy epigenetically regulates the immune response via modulating the stability and activity of KDM4D remains largely unclear. Recently, we identified TRIM14 (tripartite motif-containing 14) as an epigenetic regulator, which recruits USP14 and BRCC3 to form a regulatory complex, and promotes an inflammation response through inhibiting OPTN-mediated autophagic degradation of KDM4D.

An inducible CRISPR/Cas9 screen identifies DTX2 as a transcriptional regulator of human telomerase.

Most tumor cells reactivate telomerase to ensure unlimited proliferation, whereas the expression of human telomerase reverse transcriptase (hTERT) is tightly regulated and rate-limiting for telomerase activity maintenance. Several general transcription factors (TFs) have been found in regulating hTERT transcription; however, a systematic study is lacking. Here we performed an inducible CRISPR/Cas9 KO screen using an hTERT core promoter-driven reporter. We identified numerous positive regulators including an E3 ligase DTX2. In telomerase-positive cancer cells, DTX2 depletion downregulated hTERT transcription and telomerase activity, contributing to progressive telomere shortening, growth arrest, and increased apoptosis. Utilizing BioID, we characterized multiple TFs as DTX2 proximal proteins, among which NFIC functioned corporately with DTX2 in promoting hTERT transcription. Further analysis demonstrated that DTX2 mediated K63-linked ubiquitination of NFIC, which facilitated NFIC binding to the hTERT promoter and enhanced hTERT expression. These findings highlight a new hTERT regulatory pathway that may be exploited for potential cancer therapeutics.

OTUD7B deubiquitinates SQSTM1/p62 and promotes IRF3 degradation to regulate antiviral immunity.

Deubiquitination plays an important role in the regulation of the crosstalk between macroautophagy/autophagy and innate immune signaling, yet its regulatory mechanisms are not fully understood. Here we identify the deubiquitinase OTUD7B as a negative regulator of antiviral immunity by targeting IRF3 (interferon regulatory factor 3) for selective autophagic degradation. Mechanistically, OTUD7B interacts with IRF3, and activates IRF3-associated cargo receptor SQSTM1/p62 (sequestosome 1) by removing its K63-linked poly-ubiquitin chains at lysine 7 (K7) to enhance SQSTM1 oligomerization. Moreover, viral infection increased the expression of OTUD7B, which forms a negative feedback loop by promoting IRF3 degradation to balance type I interferon (IFN) signaling. Taken together, our study reveals a specific role of OTUD7B in mediating the activation of cargo receptors in a substrate-dependent manner, which could be a potential target against excessive immune responses.Abbreviations: Baf A1: bafilomycin A1; CGAS: cyclic GMP-AMP synthase; DDX58/RIG-I: DExD/H-box helicase 58; DSS: dextran sodium sulfate; DUBs: deubiquitinating enzymes; GFP: green fluorescent protein; IFN: interferon; IKKi: IKBKB/IkappaB kinase inhibitor; IRF3: interferon regulatory factor 3; ISGs: interferon-stimulated genes; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; PAMPs: pathogen-associated molecular patterns; SeV: Sendai virus; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; Ub: ubiquitin; WT: wild-type; VSV: vesicular stomatitis virus.

Palmitoylation restricts SQSTM1/p62-mediated autophagic degradation of NOD2 to modulate inflammation.

The nucleotide-binding oligomerization domain protein 2 (NOD2) senses bacterial peptidoglycan to induce proinflammatory and antimicrobial responses. Dysregulation of NOD2 signaling is involved in multiple inflammatory disorders. Recently, S-palmitoylation, a novel type of post-translational modification, is reported to play a crucial role in membrane association and ligand-induced signaling of NOD2, yet its influence on the stability of NOD2 is unclear. Here we show that inhibition of S-palmitoylation facilitates the SQSTM1/p62-mediated autophagic degradation of NOD2, while S-palmitoylation of NOD2 by ZDHHC5 promotes the stability of NOD2. Furthermore, we identify a gain-of-function R444C variant of NOD2 short isoform (NOD2s-R444C) in autoinflammatory disease, which induces excessive inflammation through its high S-palmitoylation level. Mechanistically, the NOD2s-R444C variant possesses a stronger binding ability to ZDHHC5, which promotes its S-palmitoylation, and restricts its autophagic degradation by reducing its interaction with SQSTM1/p62. Taken together, our study reveals the regulatory role of S-palmitoylation in controlling NOD2 stability through the crosstalk with autophagy, and provides insights into the association between dysfunctional S-palmitoylation and the occurrence of inflammatory diseases.

Selective autophagy controls the stability of TBK1 via NEDD4 to balance host defense.

As a core kinase of antiviral immunity, the activity and stability of TANK-binding kinase 1 (TBK1) is tightly controlled by multiple post-translational modifications. Although it has been demonstrated that TBK1 stability can be regulated by ubiquitin-dependent proteasome pathway, it is unclear whether another important protein degradation pathway, autophagosome pathway, can specifically affect TBK1 degradation by cargo receptors. Here we report that E3 ubiquitin ligase NEDD4 functions as a negative regulator of type I interferon (IFN) signaling by targeting TBK1 for degradation at the late stage of viral infection, to prevent the host from excessive immune response. Mechanically NEDD4 catalyzes the K27-linked poly-ubiquitination of TBK1 at K344, which serves as a recognition signal for cargo receptor NDP52-mediated selective autophagic degradation. Taken together, our study reveals the regulatory role of NEDD4 in balancing TBK1-centered type I IFN activation and provides insights into the crosstalk between selective autophagy and antiviral signaling.

RNA-induced liquid phase separation of SARS-CoV-2 nucleocapsid protein facilitates NF-κB hyper-activation and inflammation.

The ongoing 2019 novel coronavirus disease (COVID-19) caused by SARS-CoV-2 has posed a worldwide pandemic and a major global public health threat. The severity and mortality of COVID-19 are associated with virus-induced dysfunctional inflammatory responses and cytokine storms. However, the interplay between host inflammatory responses and SARS-CoV-2 infection remains largely unknown. Here, we demonstrate that SARS-CoV-2 nucleocapsid (N) protein, the major structural protein of the virion, promotes the virus-triggered activation of NF-κB signaling. After binding to viral RNA, N protein robustly undergoes liquid-liquid phase separation (LLPS), which recruits TAK1 and IKK complex, the key kinases of NF-κB signaling, to enhance NF-κB activation. Moreover, 1,6-hexanediol, the inhibitor of LLPS, can attenuate the phase separation of N protein and restrict its regulatory functions in NF-κB activation. These results suggest that LLPS of N protein provides a platform to induce NF-κB hyper-activation, which could be a potential therapeutic target against COVID-19 severe pneumonia.

High-throughput screening of functional deubiquitinating enzymes in autophagy.

Macroautophagy/autophagy, a eukaryotic homeostatic process that sequesters cytoplasmic constituents for lysosomal degradation, is orchestrated by a number of autophagy-related (ATG) proteins tightly controlled by post-translational modifications. However, the involvement of reversible ubiquitination in the regulation of autophagy remains largely unclear. Here, we performed a single-guide RNA-based screening assay to investigate the functions of deubiquitinating enzymes (DUBs) in regulating autophagy. We identified previously unrecognized roles of several DUBs in modulating autophagy at multiple levels by targeting various ATG proteins. Mechanistically, we demonstrated that STAMBP/AMSH (STAM-binding protein) promotes the stabilization of ULK1 by removing its lysine 48 (K48)-linked ubiquitination, whereas OTUD7B mediates the degradation of PIK3 C3 by enhancing its K48-linked ubiquitination, thus positively or negatively affects autophagy flux, respectively. Together, our study elaborated on the broad involvement of DUBs in regulating autophagy and uncovered the critical roles of the reversible ubiquitination in the modification of ATG proteins.Abbreviations: ATG: autophagy-related; Baf A1: bafilomycin A1; DUB: deubiquitinating enzyme; EBSS: Earle's balanced salt solution; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OTUD7B: OTU domain-containing protein 7B; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; sgRNA: single-guide RNA; SQSTM1/p62: sequestosome 1; STAMBP/AMSH: STAM-binding protein; ULK1: unc-51 like autophagy activating kinase 1; USP: ubiquitin specific peptidase.

Selective autophagy controls the stability of transcription factor IRF3 to balance type I interferon production and immune suppression.

IRF3 (interferon regulatory factor 3) is one of the most critical transcription factors in antiviral innate immune signaling, which is ubiquitously expressed in a variety of cells. Although it has been demonstrated that IRF3 can provoke multiple cellular processes during viral infection, including type I interferon (IFN) production, the mechanisms underlying the precise regulation of IRF3 activity are still not completely understood. Here, we report that selective macroautophagy/autophagy mediated by cargo receptor CALCOCO2/NDP52 promotes the degradation of IRF3 in a virus load-dependent manner. Deubiquitinase PSMD14/POH1 prevents IRF3 from autophagic degradation by cleaving the K27-linked poly-ubiquitin chains at lysine 313 on IRF3 to maintain its basal level and IRF3-mediated type I IFN activation. The autophagic degradation of IRF3 mediated by PSMD14 or CALCOCO2 ensures the precise control of IRF3 activity and fine-tunes the immune response against viral infection. Our study reveals the regulatory role of PSMD14 in balancing IRF3-centered IFN activation with immune suppression and provides insights into the crosstalk between selective autophagy and type I IFN signaling.Abbreviations: ATG5: autophagy related gene 5; Baf A1: bafilomycin A1; BECN1: beclin 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CGAS: cyclic GMP-AMP synthase; DDX58/RIG-I: DExD/H-box helicase 58; DUBs: deubiquitinating enzymes; IFN: interferon; IRF3: interferon regulatory factor 3; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; PAMPs: pathogen-associated molecule patterns; PBMC: peripheral blood mononuclear cell; PSMD14/POH1: proteasome 26S subunit, non-ATPase 14; RIPA: RLR-induced IRF3-mediated pathway of apoptosis; SeV: Sendai virus; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; Ub: ubiquitin; WT: wild type.

Interplay of m6A and H3K27 trimethylation restrains inflammation during bacterial infection.

While N 6-methyladenosine (m6A) is the most prevalent modification of eukaryotic messenger RNA (mRNA) involved in various cellular responses, its role in modulating bacteria-induced inflammatory response remains elusive. Here, we showed that loss of the m6A reader YTH-domain family 2 (YTHDF2) promoted demethylation of histone H3 lysine-27 trimethylation (H3K27me3), which led to enhanced production of proinflammatory cytokines and facilitated the deposition of m6A cotranscriptionally. Mechanistically, the mRNA of lysine demethylase 6B (KDM6B) was m6A-modified and its decay mediated by YTHDF2. YTHDF2 deficiency stabilized KDM6B to promote H3K27me3 demethylation of multiple proinflammatory cytokines and subsequently enhanced their transcription. Furthermore, we identified H3K27me3 as a barrier for m6A modification during transcription. KDM6B recruits the m6A methyltransferase complex to facilitate the methylation of m6A in transcribing mRNA by removing adjacent H3K27me3 barriers. These results revealed cross-talk between m6A and H3K27me3 during bacterial infection, which has broader implications for deciphering epitranscriptomics in immune homeostasis.