Crosstalk between Autophagy and RLR Signaling

Autophagy plays a homeostatic role in regulating cellular metabolism by degrading unwanted intracellular materials and acts as a host defense mechanism by eliminating infecting pathogens, such as viruses. Upon viral infection, host cells often activate retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling to induce the transcription of type I interferons, thus establishing the first line of the innate antiviral response. In recent years, numerous studies have shown that virus-mediated autophagy activation may benefit viral replication through different actions on host cellular processes, including the modulation of RLR-mediated innate immunity. Here, an overview of the functional molecules and regulatory mechanism of the RLR antiviral immune response as well as autophagy is presented. Moreover, a summary of the current knowledge on the biological role of autophagy in regulating RLR antiviral signaling is provided. The molecular mechanisms underlying the crosstalk between autophagy and RLR innate immunity are also discussed.


Introduction
Autophagy is a fundamentally catabolic process that degrades unnecessary intracellular materials to support the recycling of nutrients, thus promoting the regeneration of energy sources and balancing metabolic homeostasis. In addition to basal autophagy, a variety of stimuli, including deprivation of nutrients, aggregation and/or unfolding of proteins, damage to organelles, and infection by pathogens, can specifically activate autophagy to resolve cellular stresses. Deregulation of autophagy is involved in the pathogenesis of human diseases, such as cancer, neurodegenerative diseases, infectious diseases, and metabolic disorders. Several viral infections may induce autophagy to reconstitute the membranous compartments for virus replication and to repress the innate antiviral response, which benefits virus replication. In contrast, virus-triggered autophagy may be used to restrict viral growth by eliminating the infecting virus, acting as a host defense mechanism against viral infection. Upon virus infection, pattern recognition receptors (PRRs), such as retinoic acid-inducible I (RIG-I)-like receptors (RLRs), recognize pathogen-associated molecular patterns (PAMPs) and subsequently trigger a downstream signal cascade to induce the production of interferons (IFNs) and cytokines, thus stimulating innate antiviral immunity. However, to achieve successful replication, viruses have evoked strategies to repress the RLR-triggered antiviral response and may also interfere with autophagic degradation. In recent years, numerous studies have provided insights into the molecular interactions between virus-activated autophagy and RLR innate immune signaling. In this review, we first summarize the current understanding of the molecular regulation of RLR signaling and autophagy. Then, we provide an overview of the physiological importance and molecular mechanism by which autophagy regulates the antiviral response of RLRs. . The free CARDs of RIG-I and MDA5 undergo self-oligomerization and interact with the CARDs of MAVS on mitochondria. The formed MAVS aggregates in turn to recruit the ubiquitin E3 ligases (TNF) receptor-associated factor 2 (TRAF2), TRAF5, and TRAF6, which produce polyubiquitin chains. The polyubiquitin chain then induces the recruitment of nuclear factor kappa-light-chain-enhancer of activated B (NF-kB) essential modulator (NEMO), which subsequently activates TANK binding kinase 1 (TBK1) to phosphorylate interferon response factor 3 (IRF3). Phosphorylated IRF3 then translocates into the nucleus and subsequently activates the gene expression of type I interferons (IFNs).
In addition to RIG-I and MDA5, mitochondrial antiviral signaling (MAVS, also known as IFN-beta promoter stimulator-1 [IPS-1], virus-induced signaling adaptor [VISA], and CARD adaptor inducing IFN-β [Cardif]) also contains a CARD at its N-terminal regions ( Figure 1A), which can interact with the CARDs of RIG-I and MDA5 and a transmembrane domain at the C-terminus for localization on the outer membrane of mitochondria (MOM) ( Figure 1B) [35][36][37][38]. The association between the CARDs of RIG-I/MDA5 and MAVS activates downstream signaling. Moreover, oligomerization of MAVS through N-terminal CARDs assembles into protease-and detergent-resistant prion-like structures on the MOM and promotes the recruitment of the ubiquitin E3 ligases tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), TRAF5, and TRAF6 [39,40]. Subsequently, these ubiquitin E3 ligases produce the polyubiquitin chain that recruits the nuclear factor kappa-light-chain-enhancer of activated B (NF-kB) essential modulator (NEMO), which in turn activates TANK binding kinase 1 (TBK1) and inhibitor of nuclear factor kappa B (Ik-B) kinase (IKK) to phosphorylate interferon response factor 3 (IRF3) and Ik-B, respectively ( Figure 1B) [39,40]. The homodimers of phosphorylated IRF3 and NF-kB then translocate into the nucleus and transactivate the gene expression of IFN and proinflammatory cytokines, respectively ( Figure 1B) [39,40]. Then, type I IFNs, such as The free CARDs of RIG-I and MDA5 undergo self-oligomerization and interact with the CARDs of MAVS on mitochondria. The formed MAVS aggregates in turn to recruit the ubiquitin E3 ligases (TNF) receptor-associated factor 2 (TRAF2), TRAF5, and TRAF6, which produce polyubiquitin chains. The polyubiquitin chain then induces the recruitment of nuclear factor kappa-light-chain-enhancer of activated B (NF-kB) essential modulator (NEMO), which subsequently activates TANK binding kinase 1 (TBK1) to phosphorylate interferon response factor 3 (IRF3). Phosphorylated IRF3 then translocates into the nucleus and subsequently activates the gene expression of type I interferons (IFNs).
In the past decade, mounting lines of evidence have demonstrated that the assembly of filamentous and highly ordered structures of RLRs, including RIG-I and MDA5, critically activates RLR antiviral signaling by enhancing their binding affinity to dsRNA and MAVS [110][111][112][113][114][115][116][117][118]. In the presence of an unanchored Lys-63-linked polyubiquitin chain and ATP hydrolysis, the CARDs of RIG-I assemble on RNA and form a tightly bound tetramer, which forms single-competent aggregates with the CARDs of MAVS [110][111][112]118]. In contrast to RIG-I, the head-to-tail assembly of MDA5 forms a filament along the long dsRNA sequence, allowing MDA5 to discriminate between self-and nonself RNA via assembly and disassembly dynamics [113][114][115][116]. It is also noted that ATP hydrolysis is required to disassemble MDA5 filaments rather than to oligomerize MDA5 [113][114][115][116]. In addition to RLRs, viral infection induces the aggregation of MAVS on the MOM and forms prion-like fibers that are resistant to detergent and proteases [39,40]. MAVS aggregates on the MOM and activates RLR antiviral signaling not only by associating with the RIG-I tetramer to form single-competent aggregates [110][111][112]118] but also by recruiting ubiquitin E3 ligases to produce polyubiquitin chains [39,40]. In addition to mitochondria, MAVS localized on peroxisomes and mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) also serves as a platform for innate antiviral immunity [119][120][121]. In particular, viral infection induces the assembly of a RIG-I translocon consisting of RIG-I, TRIM25, and 14-3-3ε on MAMs to activate the RLR antiviral response by promoting RIG-I polyubiquitination and interaction with MAVS [121].

Gene Polymorphism(s) of RLR and Modulation of RLR Antiviral Signaling
Single nucleotide polymorphisms (SNPs) in RLR have been identified and shown to regulate IFN response and affect viral susceptibility. The mutation of serine at residue 183 of RIG-I to isoleucine (Ser183Ile) has been shown to inhibit the polyinosinic acid-polycytidylic acid (poly ([I:C]), 5 -triphosphate-RNA, Sendai virus (SeV), and IAV-induced activation of IFN-β promoter, presumably increasing the formation of RIG-I self association [122,123]. In addition, the mutation of arginine at cysteine (Arg7Cys) also reportedly decreases the activation of the IFN-β promoter in IAV-infected cells [124]. In contrast, a nonsense mutation of glutamic acid at residue 627 (E627) leads to deletion of the C-terminal region and dsRNA binding activity of MDA5, which represses the poly (I:C)-triggered activation of the IFN-β promoter [123]. In addition, an inherited missense mutation in MDA5 (mutation of lysine at residue 365 to glutamic acid, K365E) also reportedly represses the poly (I:C)induced activation of the IFN-β promoter and the IFN-driven ISRE activation of the ISRE promoter [125]. Additionally, K365E mutant MDA5 increases the replication of human rhinovirus (HRV) in patient-derived nasal epithelial cells [125]. Type 1 diabetes (T1D)associated variants in MDA5, including mutations of alanine at residue 946 to threonine (A946T), isoleucine at residue 923 to valine (I923V), and glutamic acid at residue 627 to the stop codon (E627X), have also been demonstrated to decrease the rotavirus (RV)-induced upregulation of IFN-β mRNA and increase the replication of RV in infected cells [126].
These studies collectively indicate that SNPs and germline mutations of RLR may disturb innate antiviral immunity and enhance susceptibility to virus infection.
Since autophagy involves the rearrangement of intracellular membranes, several viruses exploit activated autophagy in infected cells to form multi-vesicular compartments for viral RNA replication, such as poliovirus and HRV [333,334]. Analogously, other viruses that cause a major burden in the human population, including HCV [335][336][337], hepatitis B virus (HBV) [338,339], DENV [340,341], and enteroviruses [342,343], were also shown to utilize autophagy in order to organize membranous structures for virus replication. Notably, SARS-CoV-2 infection has recently been reported to induce the accumulation of autophagosomes, in which viral RNAs replicate, presumably through the repression of autophagosome-lysosome fusion by the viral genome-encoded protein open reading frame 3a (ORF3a) [344][345][346]. Similarly, CVB3 infection has been shown to block autophagosome fusion through the viral protease-mediated proteolysis of SNARE complex, ultimately promoting accumulated autophagosome for the replication of the virus [347]. Therefore, viral-induced autophagy may play a distinct role in positively promoting virus growth by generating membranous vesicles for the replication of viral RNA. In addition, viral-induced autophagy functions in the nonlytic and intercellular spread of nonenveloped viruses, such as poliovirus, through an unconventional secretion pathway [348]. Moreover, autophagy reportedly participates in the viral envelopment of HBV [349], the egress of HCV [350], the maturation of infectious particles of DENV [351], and the prevention of cell death induced by DENV and CHIKV [352,353]. Together, these studies indicate that viral-induced autophagy may affect many aspects of the virus life cycle to benefit viral growth.

Regulation of RLR Signaling by Autophagy
In recent years, autophagy has emerged as playing a functional role(s) in negatively regulating RLR-mediated antiviral signaling. Several viruses activate autophagy and thus target the degradation of functional molecules and organelles involved in the regulation of the RLR IFN response. In this section, we therefore summarize the current knowledge on how autophagy represses RLR innate immunity according to the molecular targets of RLR signaling degraded by autophagy. The molecular mechanism and physiological significance of autophagy in RLR antiviral immunity is also discussed.

The Interaction between the ATG12-ATG5 Conjugate and CARDs of RIG-I and MDA5
The integration of autophagy into the RLR antiviral immune response was first uncovered by findings showing the enhancement of the vesicular stomatitis virus (VSV)-induced RLR-mediated IFN response in the infected ATG5-deficient mouse embryonic fibroblasts (MEFs) ( Table 1) [354]. The increased RLR antiviral IFN immunity in VSV-infected cells lacking autophagy was characterized by increases in IFN-β mRNA levels and IRF3 phosphorylation, which were accompanied by a decrease in the viral infectivity of infected cells. Apart from VSV-infected cells, gene knockout of ATG5 also leads to hyperactivation of the dsRNA (poly[I:C])-induced type IFN I response, as shown by the increased mRNA levels of IFN-4α, IFN-β, interleukin-6 (IL-6), and C-X-C motif chemokine 10 (CXCL-10, also named IP-10). Reciprocally, overexpression of wild-type (WT) ATG5, rather than ATG5 K103R (Lys130 mutated to arginine [Arg]), which is unable to conjugate with ATG12 in cells, repressed the dsRNA-induced increase in promoter activities of IFN-4α, NF-kB, and IFN-β. In addition, the ATG12-ATG5 conjugate was shown to physically interact with RLRs in human embryonic kidney (HEK293) cells, including RIG-I and MAVS, through CARD binding. These studies collectively suggest that the ATG12-ATG5 conjugate, an important regulator of autophagosome maturation, may specifically bind to the CARDs of RIG-I and MAVS, leading to repression of RLR signaling and inhibition of the antiviral response (Table 1) (Figure 4) [354]. in SeV-infected cells and relieved the repressive effect of NEDD4 on SeV-induced IFN-β, ISG56/IFIT1, and ISG54/IFIT2 mRNA levels. Together, these studies indicate a repressive role of the NEDD4/NDP52 axis in the regulation of RLR antiviral immunity by targeting TBK1 for degradation (Table 1) (Figure 4) [375].

Removal of Mitochondria by Autophagy
Mitochondria and the associated ER membrane represent platforms for the assembly of RLR-associated molecules and signaling transduction of RLR antiviral immunity [119][120][121]. In recent years, it has been interesting and remains questionable whether alteration of mitochondrial dynamics and mitochondrial turnover regulate RLR antiviral signaling. Tal et al. firstly showed that interference with autophagy led to an increase in type I IFN response, which was associated with the accumulation of mitochondria [355]. In this study, the authors reported that gene silencing of ATG5 in MEF increased the dsRNA-induced production and secretion of IFN-α and IL-6, thus enhancing VSV infection (Table 1) [355]. In addition, ATG5-knockdown cells were shown to contain accumulated mitochondria, as demonstrated by the enhanced fluorescence intensity of MitoTracker (MitoTracker Green and MitoTracker Red)-labeled mitochondria in a flow cytometry assay and the upregulated mitochondrial DNA expression in a Southern blotting analysis. Along with these results, the expression of MAVS was indicated to be increased in ATG5 knockout MEFs, as shown by Western blotting and immunofluorescence (IFA) staining coupled flow cytometry assays. Additionally, overexpression of MAVS increased IFN-α mRNA levels in ATG5-deficient cells. Moreover, MitoSOX fluorescence probe labeling and flow cytometry experimental results showed that interruption of the autophagic process in MEF cells by ATG5 knockout led to an increase in the level of mitochondrial reactive oxygen species (ROS). In addition, treatment of ATG5-deficient MEFs with the antioxidant N-acetyl-L-cysteine (NAC) not only decreased the ROS level in mitochondria but also alleviated dsRNA-induced upregulation of IFN-α expression. In contrast, induction of mitochondrial ROS accumulation by an inhibitor of the mitochondrial electron transfer complex 1, rotenone, in autophagy-competent cells increased the dsRNA-activated IFN-α mRNA levels, which was further potentiated in rotenone-treated ATG5-deficient cells.
In conclusion, these findings imply that autophagy plays a homeostatic role in the regulation of RLR antiviral immunity by eliminating mitochondria containing ROS, which may trigger overexpression of MAVS and hyperactivation of RLR signaling (Table 1

Repression of Flaviviral PAMP-Triggered RLR Innate Immunity by Autophagy
In addition to VSV and dsRNA-induced IFN antiviral responses, viral-activated autophagy was also demonstrated to repress the HCV PAMP-induced IFN antiviral response (Table 1) [356,357]. The RIG-I N-terminal fragment (RIG-I N) has been previously shown to sufficiently activate the type I IFN response [22] and gene knockdown of ATG5 increased the RIG-I N-induced activation of IFN-β and ISRE promoters in human hepatoma Huh7 cells. Additionally, the HCV (JFH1 strain, genotype 2a) PAMP (3 -UTR and PU/UC within HCV RNA genome)-triggered IFN antiviral response was potentiated in cells with ATG5 gene silencing, as shown by the enhancement of IFN-β promoter activity and an increase in IFN-β mRNA level. The upregulated transactivation of IFN-β led to the upregulated expression of IFN-induced protein with tetratricopeptide repeats 1 (IFIT1, also named ISG56) and triggered paracrine antiviral immunity against HCV replication. In addition to HCV PAMPs, ATG5 knockdown also enhanced the DENV PAMP (3 -UTR)-induced activation of the IFN-β promoter and upregulated the expression of ISG56/IFIT1. Moreover, nutrient-starvation-induced autophagy triggered by treatment with Earle's balanced salt solution (EBSS), Hank's balanced salt solution (HBSS), or the mTOR inhibitor rapamycin was shown to repress HCV PAMP-activated IFN-β promoter activity. In a similar fashion, activation of autophagy through the unfolded protein response (UPR) by dithiothreitol (DTT) and tunicamycin treatment also inhibited the activation of the IFN-β promoter in HCV PAMP-treated cells. Furthermore, interference with autophagosome-lysosome fusion by the chemicals chloroquine (CQ) and bafilomycin (BAF-A1) and gene silencing of Rab7 and LAMP2 significantly inhibited IFN-β promoter activation. Overall, these results provide the first line of evidence showing that HCV activates autophagy to repress the RLR-mediated IFN response and suggest that pharmacological modulation of autophagy may alter innate antiviral immunity (Table 1) [356,357]. Soon after this study, another study showed that gene knockdown of Beclin-1 in HCV (H77 strain, genotype 1a)-infected immortalized human hepatocytes (IHHs) inhibited viral-activated autophagy and virus infectivity, which coincidently increased the mRNA levels of IFN-α, IFN-β, 2 -5 -oligoadenylate synthetase 1 (OAS1), and IFN-α-inducible protein 27 (IFI27) [358]. Additionally, inhibition of autophagy by silencing ATG7 gene expression analogously upregulated the mRNA expression of IFN-α, OAS1, and IFI27 and simultaneously reduced the infectious titer of HCV. Moreover, interference with HCV-induced autophagy promoted the apoptosis of infected IHHs. Again, these studies indicate that HCV may use autophagy to repress the RLR innate immune response (Table 1) [358]; however, whether and how RLR signaling molecules are regulated by autophagy in HCV-infected cells remain largely unclear.

Autophagic Degradation of TRAF6 by p62/SQSTM1
HCV (JFH1) infection was shown to induce autophagy through activating unfolded protein response (UPR) in an in vitro cell culture model [356,376]. Gene silencing of the molecules involved in autophagy and UPR inhibits the replication of HCV viral RNA [356,376], suggesting that HCV-activated autophagy benefits viral growth. In addition, HCV (JFH1) infection leads to mitochondrial fission and promotes the PINK1/Parkindependent mitophagy [377,378], thus attenuating cell apoptosis in the infected cells and promoting the establishment of viral persistence. These studies collectively indicate that HCV infection induces general autophagy and triggers selective autophagy to promote mitochondrial turnover.
Chan et al. first discovered that HCV (JFH1) infection led to the degradation of TRAF6, a ubiquitin E3 ligase necessary for producing polyubiquitin chains that recruit NEMO and activate RLR antiviral signaling (Table 1) [359]. Treatment with an autolysosome inhibitor, BAF-A1, restored the expression of TRAF6 in HCV-infected cells. Further studies revealed that HCV infection led to the colocalization of TRAF6 within viral-induced autophagic vacuoles and its interaction with p62/SQSTM1. Overexpression of TRAF6 inhibited the replication of HCV viral RNA, whereas depletion of TRAF6 gene expression increased the levels of HCV viral RNA and the viral infectivity of infected cells. Furthermore, TRAF6 knockdown was shown to reduce NF-kB promoter activity and decrease the mRNA and protein levels of IL-6 and tumor necrosis factor-α (TNF-α) in HCV-infected cells. These findings suggest that autophagy may negatively regulate the RLR-mediated type I IFN response by promoting the degradation of TRAF6 (Table 1) (Figure 4) [359].

Autophagic Degradation of RIG-I
RIG-I is a degradative substrate of autophagy induced by RNA viruses, including VSV and H1N1 IAV (Table 1) [360]. Du et al. reported that leucine-rich repeat containing protein 25 (LRRC25) significantly repressed RIG-I N-induced activation of the ISRE promoter in HEK293 cells (Table 1) [360]. The challenge of human leukemia monocytes, THP-1 cells with VSV and poly (I:C) resulted in the upregulation of LRRC25. Overexpression of LRRC25 repressed the poly (I:C)-and SeV-induced activation of the IFN-β and ISRE promoters, accompanied by enhanced VSV replication in HEK293 cells. In contrast, gene knockout of LRRC25 in THP-1 cells further increased VSV-triggered IRF3 phosphorylation and IFN-β production and simultaneously decreased the replication of VSV. Analogously, gene silencing of LRRC25 in human peripheral blood mononuclear cells (PBMCs) also led to the hyperactivation of H1N1 IAV-induced IRF3 phosphorylation and upregulation of IFN-β, IFIT1, and IFIT2 mRNA expression. In addition, VSV infection in THP-1 and PBMC cells and treatment with poly (I:C) in HEK293 cells promoted the interaction between LRRC25 and the CARDs of RIG-I and MDA5. Moreover, overexpression of LRRC25 in HEK293 cells promoted the degradation of RIG-I, which was abrogated by treatment with CQ and NH 4 Cl, inhibitors of autolysosome maturation and 3-MA, and gene knockout of ATG5 and Beclin-1. Moreover, the authors demonstrated that LRRC25 can mediate the interaction between RIG-I and p62/SQSTM1 in poly (I:C)-treated and VSV-infected cells by simultaneously binding to these two proteins. Gene knockout of p62/SQSTM1 reversed the LRRC25-induced degradation of RIG-I. It was noted that protein ubiquitination of RIG-I was not required for p62/SQSTM1-mediated targeting. Furthermore, gene knockout of ISG15 remarkably blocked the interaction between LRRC25 and RIG-I and reduced LRRC25-triggered RIG-I degradation, implying that ISG15 serves as a critical signal for the degradation of RIG-I by LRRC25. These studies collectively indicate that LRRC25 inhibits type I IFN response by promoting the degradation of RIG-I via ISG15 and p62/SQSTM1 (Table 1) [360]. In addition to LRRC25, the mitochondria-associated ubiquitin E3 ligase, membrane-associated ring-CH-type finger 5 (MARCH5), was also shown to promote the Lys-48-linked ubiquitination of RIG-I at the Lys193 and Lys203 residues within the CARDs of RIG-I, leading to the degradation of RIG-I and inhibition of RLR antiviral immunity (Table 1 protein also inhibited IFN antiviral response through inducing mitophagy [363], arguing again that mitophagy plays a negative role in regulating RLR IFN response (Table 1) (Figure 4).

Autophagic Degradation of RIG-I and MDA5 by CCDC50
Apart from the known cargo receptors for selective autophagy, coiled-coil domaincontaining protein 50 (CCDC50) was recently reported to function as a novel cargo receptor of selective autophagy for targeting RIG-I and MDA5 for degradation (Table 1) [364]. Gene silencing of CCDC50 in mouse BMDMs and BMDCs amplified the SeV-induced increase in IFN-β mRNA levels and production of IFN-β and increased the phosphorylation of TBK1 in SeV-infected cells. SeV infection led to increases in the mRNA and protein levels of CCDC50. In a similar fashion, gene knockout of CCDC50 in mice potentiated the SeVand VSV-induced type 1 IFN response, restricted the viral replication of VSV, and protected VSV-infected mice from death. Overexpression of CCDC50 in HEK293 cells diminished the SeV-triggered activation of the IFN-β, ISRE, and NF-kB promoters, whereas gene knockout of CCDC50 increased the IFN-β mRNA and protein levels and repressed the production of infectious titers in VSV-infected cells. In addition, overexpression of CCDC50 led to the degradation of RIG-I and MDA5, while CCDC50 knockout prolonged the protein halflives of RIG-I and MDA5. Moreover, SeV infection enhanced the interaction of CCDC50 with RIG-I and MDA5, and ectopic expression of CCDC50 in SeV-infected cells promoted the degradation of these two proteins, which was attenuated by 3-MA, CQ, and NH 4 Cl treatment. Additionally, SeV infection resulted in the interaction of CCDC50 with LC3B and p62/SQSTM1 and enhanced the colocalization between CCDC50 and autophagic vacuoles containing GFP-LC3B and p62/SQSTM1 puncta. Moreover, CCDC50 specifically bound to Lys-63-linked ubiquitinated RIG-I and MDA5 and interacted with LC3B, thereby targeting RIG-I and MDA5 for autophagy for degradation. These findings collectively suggest that CCDC50 may decrease the antiviral IFN response of RLR by targeting RIG-I and MDA5 for autophagic degradation (Table 1) (Figure 4) [364].

Autophagic Degradation of MAVS by NDP52/Calcoco2
In addition to RIG-I and MDA5, MAVS have been extensively shown to be degraded by autophagy in repressing RLR antiviral response. Jin et al. first reported that selective autophagy may negatively regulate RLR-triggered IFN antiviral immunity through Tetherin (Table 1) [365]. In this study, the authors demonstrated that ectopic expression of Tetherin promoted the viral replication of VSV and SeV in infected A549 cells (human adenocarcinoma alveolar basal epithelial cells), accompanied by repression of IFN-β activation. Overexpression of Tetherin also reduced the RIG-I N-triggered activation of IFN-β and ISRE promoters in large T antigen-immortalized HEK293 (HEK293T) cells, whereas silencing of Tetherin in HEK293T and A549 cells potentiated the poly (I:C) and 5 -triphosphate-RNAinduced activation of IFN-β and ISRE promoters and elevated the mRNA levels of IFN-β, ISG56/IFIT1, and ISG54 (also named IFIT2). Similarly, gene knockdown of Tetherin led to elevated IFN-β and ISG56/IFIT1 mRNA levels, enhanced phosphorylation of TBK1 and IRF3 in SeV-and H1N1 IAV-infected cells, and coincidentally attenuated the replication of these two viruses. Tetherin abrogated RIG-I N, MDA5 N (the N-terminal fragment of MDA5 has been previously shown to sufficiently activate the type I IFN response [22]), and MAVS-triggered inductions on IFN-β and ISRE promoters. Further studies showed the specific interaction between Tetherin and MAVS, which was enhanced by H1N1 IAV infection. Additionally, poly (I:C) treatment induced the colocalization of Tetherin with MAVS in mitochondria. Ectopic expression of Tetherin resulted in MAVS degradation, which was reversed by treating cells with 3-methyladenine (3-MA), an inhibitor of the class III (PI(3)K) complex, autolysosome inhibitors, CQ, BAF-A1, and ammonia chloride (NH 4 Cl), and gene silencing of ATG5 and Beclin-1. In addition, Tetherin promoted the physical interaction between MAVS and NDP52/Calcoco2. Downregulation of NDP52/Calcoco2 gene expression alleviated the Tetherin-induced degradation of MAVS, attenuated the Tetherinmediated repression of SeV-triggered IFN-β, ISG56/IFIT1, and ISG54/IFIT2 mRNA levels, and repressed the Tetherin-triggered enhancement of virus replication in SeV-infected cells. Moreover, Tetherin promoted the Lys-27-linked ubiquitination of MAVS at the Lys7 residue through the ubiquitin E3 ligase membrane-associated ring finger (C3HC4) 8 (MARCH8). Interference with the ubiquitination of MAVS by gene knockout of MARCH8 and a mutation of the ubiquitination site of MAVS (K7R) protected MAVS from degradation by SeV infection and Tetherin, and amplified SeV-triggered RLR antiviral IFN immunity, including elevated phosphorylation of TBK1 and IRF3 and upregulated mRNA levels of IFN-β, ISG56/IFIT1, and ISG54/IFIT2. Collectively, these studies conclude that Tetherin functions as a negative regulator of the RLR downstream IFN response through MARCH8-mediated ubiquitination of MAVS and autophagic degradation of MAVS via interaction with NDP52/Calcoco2 (Table 1) (Figure 4) [365].
In addition to SeV and VSV, NDP52/Calcoco2 also targets MAVS for degradation to inhibit RLR antiviral signaling in CVB3-infected cells [330]. Mukherjee et al. first reported that CVB3 may attenuate the type I IFN response through 3Cpro protease-mediated cleavage of MAVS and Toll receptor domain-containing adaptor inducing interferon-beta (TRIF) [379]. In addition to MAVS, CVB3 3Cpro targets MDA5 for proteolysis and leads to inhibition of the activation of IFN antiviral immunity [380]. These studies suggest that CVB3 represses the type I IFN immune response through the viral protease-mediated proteolysis of MAVS, TRIF, and MDA5. Interestingly, CVB3 3Cpro was reported to cleave SNAP29 and PLEKHM1, two molecules that critically function in autophagosome-lysosome fusion, thus inhibiting autophagic flux in the infected cells and inducing the accumulation of autophagosome for virus replication [347]. Mohamud et al. showed that p62/SQSTM1 and NDP52/Calcoco2 differentially regulated virus proliferation in CVB3-infected HeLa cells (Table 1) [330]. Gene silencing of NDP52/Calcoco2 dramatically reduced CVB3 replication in the infected cells, whereas p62/SQSTM1 knockdown in CVB3-infected cells elevated viral replication. In addition, overexpression of p62/SQSTM1 reduced CVB3 replication in the infected cells, while ectopic expression of NDP52/Calcoco2 enhanced it in CVB3infected cells. In addition, both NDP52/Calcoco2 and p62/SQSTM1 were demonstrated to interact with the CVB3 VP1 protein. Further analysis revealed that CVB3 infection induced Lys-48-and Lys-63-linked protein ubiquitination of VP1. In addition, gene knockdown of NDP52/Calcoco2, rather than p62/SQSTM1, in CVB3-infected cells led to an increase in MAVS protein levels and the phosphorylation of TBK1. CVB3 infection induced the cleavage of NDP52/Calcoco2 after Q139 treatment by viral protease 3C, and the free C-terminal fragment of NDP52/Calcoco2 was able to promote the degradation of MAVS and the viral replication of CVB3 and repress the activation of poly(I:C)-induced IFN-β production in a similar fashion as the full-length form. These findings suggest that CVB3 infection may mediate the degradation of MAVS through NDP52/Calcoco2, thereby attenuating antiviral signaling downstream of RLR (Table 1 (Table 1) [366]. Overexpression of WT RNF34 but not the E3 ligase-knockout mutant (H342A) of RNF34 significantly reduced the VSV-triggered activation of the IFN-β and NF-kB promoters. In addition, RNF34 overexpression repressed RIG-I N, MAVS, and poly (I:C)-triggered IFN-β activation. Reciprocally, knockdown of RNF34 gene expression elevated the phosphorylation of TBK1 and IRF3 and increased the expression of ISG54/IFIT2 and ISG56/IFIT1 in VSV-infected cells. RNF34 was shown to directly bind to MAVS, as indicated by yeast two-hybrid and glutathione S-transferase (GST) pull-down assays. VSV infection led to colocalization and interaction of RNF34 with MAVS. Additionally, RNF34 promoted Lys-27-and Lys-63-linked ubiquitination of MAVS at Lys311. Moreover, VSV infection led to the degradation of MAVS, which was reversed by NH 4 Cl treatment and gene silencing of RNF34. Furthermore, VSV infection also induced the interaction of MAVS with WT NDP52/Calcoco2, but not with the UBA mutant (D439R/C443K) of NDP52/Calcoco2. In addition, ectopic expression of WT NDP52/Calcoco2, but not the UBA mutant of NDP52/Calcoco2, repressed MAVS-induced activation of the IFN-β promoter. In contrast, gene knockdown of NDP52/Calcoco2 inhibited the VSV-and RNF34induced degradation of MAVS. Furthermore, gene knockdown of RNF34 attenuated the VSV-induced degradation of the mitochondrial proteins TOMM20 and HSP60 and coincidently enhanced IRF3 phosphorylation. Conversely, ectopic expression of RNF34 facilitated the degradation of TOMM20 and HSP60, accompanied by a dramatic decrease in MAVS protein levels. Moreover, the accumulation of deformed mitochondria was found in the RNF34-silenced VSV-infected cells. Additionally, RNF34 was shown to induce mitophagy, as indicated by the increased acidic signal of an mt-Keima reporter, which is generally used for analyzing mitophagic processes [381,382]. These studies collectively suggest that RNF34 promotes the degradation of MAVS through NDP52/Calcoco2 and mitophagy (Table 1 (Table 1) [368]. Recently, Wang et al. reported that the IAV (H1N1 and H5N1 strains) PB1-F2 protein specifically activated autophagy through the formation of autolysosomes (Table 1) [367], as demonstrated by an increased level of LC3-II and enhanced RFP + /GFPfluorescence intensity of an mCherry-GFP-LC3 autophagy reporter, which is typically used to interpret autophagic flux [383]. Further analysis revealed the colocalization of IAV PB1-F2-induced autophagic vacuoles with mitochondria. The IAV PB1-F2 proteins also induced the degradation of mitochondrially encoded cytochrome C oxidase II (MTCO20) and translocase of the outer mitochondrial membrane complex subunit 20 (TOMM20). Moreover, the IAV PB1-F2 protein also increased the RFP + /GFP + signal of the pmRFP-GFP-Mito mitophagy reporter. These results collectively suggest that the IAV PB1-F2 protein can activate mitophagy [367].
In addition, the authors demonstrated that the IAV PB1-F2 protein interacted with the Tu translation elongation factor, mitochondrial (TUFM) [367]. TUFM is a mitochondrial protein which has been shown to bind to NLR family member X1 (NLRX1) and the ATG12-ATG5-ATG16 trimeric complex (Table 1) [369]. TUFM was also demonstrated to activate autophagy and inhibit RLR downstream type I IFN response (Table 1) [369]. Downregulation of endogenous TUFM expression by small interference RNA (siRNA) gene knockdown and CRISPR/Cas9-sgRNA gene knockout inhibited IAV PB1-F2-induced mitophagy, as indicated by the restored protein levels of MTCO2 and TOMM20, and disturbed colocalization of autophagic vacuoles with mitochondria [367]. Moreover, overexpression of IAV PB1-F2 repressed MAVS-induced activation of the IFN-β promoter, whereas knockdown of TUFM reversed this inhibitory effect. The IAV PB1-F2-induced repression of the MAVS-activated IFN-β promoter was reversed by gene silencing of ATG5. Additionally, ectopic expression of IAV PB1-F2 resulted in the degradation of MAVS, which was restored by TUFM knockdown. Gene knockout of TUFM significantly increased the production of IFN in IAV-infected cells, accompanied by the repression of infected cell viral infectivity. Conversely, overexpression of TUFM benefited the viral growth of IAV-infected cells. Finally, the authors demonstrated that IAV PB1-F2 contained an LIR for binding to LC3B and served as a mitophagy receptor. Together, these results indicate that IAV infection may activate PB1-F2/TUFM axis-induced mitophagy to promote the degradation of MAVS, thus repressing RLR IFN immunity (Table 1) (Figure 4).

Autophagic Degradation of MAVS by NBR1 and RNF5
Analogously, ectopic expression of the H7N9 PB1 protein was also shown to inhibit SeV infection and poly(I:C)-induced activation of the IFN-β promoter, ISRE, and NF-κB promoters in HEK293 cells (Table 1) [370]. Accordingly, the overexpression of PB1 potentiated the SeV-and poly(I:C)-triggered upregulation of IFN-β, IFN-stimulated gene 15 (ISG15), ISG56/IFIT1, regulated upon activation normal T-cell expressed and secreted factor (RANTES), and oligoadenylate synthetase-like protein (OASL) mRNA levels. PB1 also diminished the phosphorylation of IRF3, TBK1, and Ik-Bα and reduced RIG-I N, MDA5 N, and MAVS-triggered activation of the IFN-β promoter, suggesting that PB1 significantly inhibited RLR antiviral signaling. Coimmunoprecipitation (co-IP), in vitro GST pull-down, and IFA assays revealed that PB1 directly interacted with MAVS. In addition, PB1 promoted the degradation of MAVS, which was restored by treatment with CQ and BAF-A1, two autolysosome inhibitors, and gene knockout of ATG7. Reciprocally, activation of autophagy by EBSS further enhanced PB1-triggered MAVS degradation. In addition, PB1 promoted the autophagic degradation of MAVS through the induction of RNF5-mediated Lys27-linked ubiquitination of MAVS and the physical interaction with NBR1. Moreover, overexpression of NBR1 reduced RIG-I N-induced IFN-β promoter activation and enhanced the infectivity of H7N9 IAV-infected cells. In contrast, gene knockout of NBR1 increased the SeV-triggered activation of the IFN-β promoter and decreased the H7N9 IAV infectious titer of infected cells. Finally, ectopic expression of RNF5 increased the virus titer of H7N9 IAV-infected cells, whereas RNF5 knockout repressed the viral infectivity of H7N9 IAV-infected cells. These findings indicate that IAV PB1 induces K27-linked ubiquitination of MAVS by RNF5 and promotes the autophagic degradation of MAVS through NBR1, thereby repressing the antiviral response of RLR (Table 1) (Figure 4) [371]. Gene knockout of HFE in H7N9 IAV-infected mice inhibited viral replication and protected the infected mice from H7N9 IAV-induced death. Notably, depletion of MAVS in H7N9 IAV-infected HFE knockout (HFE −/− ) mice restored the infectious titer of infected cells. Further studies demonstrated that HFE gene knockout further potentiated the H7N9 IAV-induced mRNA and protein levels of IFN-β in infected mice, as well as the phosphorylation of TBK1 and STAT1 in H7N9 IAV-infected mouse bone marrow-derived macrophages (BMDMs), suggesting that H7N9 IAV infection induced HFE expression to repress the antiviral IFN response in RLR. In a similar fashion, depletion of HFE gene expression amplified the poly (I:C)-stimulated IFN-β mRNA and MAVS protein levels and VSV-induced TBK1, IRF3, and STAT1 phosphorylation. Ectopic expression of HFE led to a dramatic degradation of mitochondria-and peroxisome-associated MAVS in HEK293 cells. In contrast, HFE gene knockout resulted in the stabilization of MAVS in H7N9 IAV-infected mouse BMDMs, mouse bone marrow-derived dendritic cells (BMDCs), and MEFs. Moreover, HFE was shown to interact with and promote autophagic degradation of MAVS, which was reversed by treatment with CQ and gene silencing of ATG5 and ATG7. Furthermore, p62/SQSTM1 targeted MAVS for degradation by interacting with HFE, and gene knockout of p62/SQSTM1 diminished the HFE-induced degradation of MAVS and inhibited HFE-repressed MAVS-triggered activation of IFN-β and ISRE promoters. Overall, this study shows that HFE promotes autophagic degradation of MAVS by interacting with p62/SQSTM1 in IAV-infected cells, thus repressing downstream RLR antiviral immunity (Table 1) (Figure 4) [371].

Degradation of RIG-I and MAVS by CSFV-Activated Autophagy
Classical swine fever virus (CSFV), an enveloped and positive-stranded RNA virus, was shown to activate autophagy in infected swine kidney cells, PK-15 cells, and 3D4/2 porcine macrophage cells, as indicated by the upregulated level of LC3B-II and the increased number of GFP-LC3-labeled punctate (Table 1) [372]. Interference with the autophagic process by gene silencing of Beclin-1 or LC3B triggered cell apoptosis in CSFV-infected cells and increased the mRNA levels of IFN-α, IFN-β, and ISGs, TNF superfamily member 10 (TNFSF10), and tumor necrosis factor receptor superfamily member 6 (TNFRSF6, also known as Fas/CD95). In addition, deficiency of autophagy by gene knockdown of Beclin-1 or LC3B in CSFV-infected cells resulted in accumulation of ROS, upregulation of mitochondrial DNA, and increased protein levels of RIG-I and MAVS, which were reversed by NAC, an inhibitor of ROS release. In contrast, the induction of ROS by rotenone in CSFV-infected cells increased the level of RIG-I. Moreover, treatment with NAC reduced cell apoptosis and increased viral replication in CSFV-infected autophagy-deficient cells. In contrast, rotenone repressed the production of CSFV in autophagy-repressed cells. Thereafter, Xie et al. showed that CSFV activated autophagy through repression of mTOR and the calcium/calmodulin-dependent protein kinase 2 (CAMKK2/CaMKKβ)-protein kinase AMP-activated catalytic subunit alpha (PRKAA/AMPK) axis [373]. Moreover, the authors demonstrated that CSFV infection repressed the production of type I IFN through the interaction between Beclin-1 and MAVS. These findings indicate that CSFV may activate autophagy to remove ROS, thus preventing the infected cell from undergoing apoptosis and repressing the activation of the RLR antiviral immune response (Table 1) (Figure 4).

Degradation of IRF3 Protein Stability by TRIM21 and NDP52/Calcoco2
Wu et al. reported that SeV infection of A549 cells, THP-1 cells, and PBMCs led to the degradation of IRF3, which was restored by 3-MA treatment and gene knockout of ATG5 and Beclin-1 (Table 1) [374]. Additionally, SeV infection promoted the binding of IRF3 to NDP52/Calcoco2 and enhanced their colocalization. Gene knockout of NDP52/Calcoco2 resulted in the stabilization and dimerization of IRF3 in SeV-infected cells. In addition, SeV infection triggered the Lys-27-linked ubiquitination of IRF3 at K313 by TRIM21 ubiquitin E3 ligase. The ubiquitination of IRF3 by TRIM21 was shown to serve as a signal for NDP52/Calcoco2-mediated autophagic degradation in cells upon SeV infection. In contrast, the DUB enzyme proteasome 26S subunit, non-ATPase 14 (PSMD14), reversed the degradation of IRF3. Gene silencing and gene knockout of PSMD14 destabilized IRF3 in SeV-infected cells. Further analysis revealed that gene silencing of PSMD14 led to an increase in IRF3 protein ubiquitination in SeV-infected cells, and PSMD14 directly removed the conjugation of the polyubiquitin chain from K313 of IRF3, suggesting that PSMD14 is required for maintaining the stability of IRF3. Moreover, the reduction in VSV-induced ISRE promoter activation in PSMD14-knockout HEK293 cells, the decreased TBK1 and IRF3 phosphorylation by gene knockdown of PSMD14 in VSV-infected cells, the repression of VSV-triggered IFN-β production in PSMD14-silenced A549 cells, and the reduction in VSV-activated secretion of IFN-β in PSMD14-knockdown A549 cells together implied that PSMD14 expression is required for maintaining the basal level of IRF3 to induce the RLR IFN response. Gene knockout of NDP52/Calcoco2 and interference with the protein ubiquitination of IRF3 by a mutation on K313 (K313) diminished the PSMD14-enhanced SeV-infection-triggered IFN response. Hence, these findings suggest that autophagy regulates IRF3 stability and the antiviral IFN response of RLR (Table 1) (Figure 4) [374].

Autophagic Degradation of TBK1 through NEDD4 and NDP52/Calcoco2
In addition to IRF3, TBK1 was shown to be regulated by selective autophagy. Xie et al. recently reported that a ubiquitin E3 ligase, neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4), was capable of attenuating the type I antiviral IFN response (Table 1) [375]. Ectopic expression of NEDD4 repressed SeV-, VSV-, and poly (I:C)-triggered activation of IFN-β and ISRE promoters in HEK293T cells. In particular, NEDD4 overexpression in SeV-infected cells diminished the upregulation of IFN-β, ISG56/IFIT1, and ISG54/IFIT2 mRNA levels and simultaneously enhanced viral replication in the infected cells. Additionally, ectopic expression of NEDD4 repressed the RIG-I N, MDA5, MAVS, and TBK1-induced activation of the IFN-β and ISRE promoters. In addition, NEDD4 was shown to bind to TBK1 physically, and SeV infection promoted the interaction and colocalization between these two proteins. Moreover, NEDD4 induced the Lys-27-linked ubiquitination of TBK1 at the K344 residue and promoted the recognition of TBK1 by NDP52/Calccoco2. Loss of NEDD4-triggered ubiquitination of TBK1 and gene knockout of NDP52/Calccoco2 resulted in resistance of TBK1 to degradation by NEDD4 in SeV-infected cells and relieved the repressive effect of NEDD4 on SeV-induced IFN-β, ISG56/IFIT1, and ISG54/IFIT2 mRNA levels. Together, these studies indicate a repressive role of the NEDD4/NDP52 axis in the regulation of RLR antiviral immunity by targeting TBK1 for degradation (Table 1) (Figure 4) [375].

Conclusions and Perspectives
In the past decade, multiple lines of evidence have implied that autophagy plays functional roles in the regulation of RLR antiviral signaling. Both general and selective autophagy may negatively control the activation of RLR innate immunity by targeting RLR signaling-related molecules for degradation, including RIG-I, MDA5, MAVS, IRF3, and TBK1. Additionally, several fundamental cellular events are involved in the autophagic degradation of these RLR signaling components: (1) the ubiquitin E3 ligases mediate the conjugations of different types of polyubiquitin chain linkages, such as Lys-48, Lys-63, and Lys-27-linked ubiquitination, to the degradative substrates; (2) the targeting of ubiquitinated molecules to degradation within the autophagic process by specific cargo receptors; and (3) the attenuation of degradation by DUB-mediated removal of protein ubiquitination. Although the molecular mechanisms underlying the regulation of RLR innate immunity by autophagy have been proposed, several fundamental questions remain. For instance, whether and how oragnellophagy-mediated turnover of damaged organelles, such as mitochondria and peroxisomes, mainly and/or sufficiently represses the antiviral signaling of RLR is still unclear. In addition, it remains questionable whether these identified molecular mechanisms control RLR innate immunity against virus infection by autophagy in a physiologically relevant context, and these mechanisms should be tested in viral infection experimental models using small animals with competent immune systems. Moreover, the spatial and temporal autophagic regulation of the RLR-mediated type I IFN response remains unresolved. Furthermore, numerous SNPs have been extensively identified in the genes encoding autophagic regulators, and most of these have been shown to be relevant to the development and pathogenesis of human diseases, such as cancer and Crohn's disease [128,384]; however, the underlying molecular mechanism remains unclear. Most importantly, whether the SNPs and germline mutations of autophagy-related molecules involved in the repression of RLR antiviral signaling regulate the viral susceptibility and pathogenicity of infecting viruses remains largely unknown. Therefore, further investigations are urgently needed to comprehensively understand the physiological importance of the crosstalk between autophagy and RLR signaling in the balance of host cell-virus interactions and the pathogenesis of human diseases.