The tumor suppressor PTEN has a critical role in antiviral innate immunity.

The gene encoding PTEN is one of the most frequently mutated tumor suppressor-encoding genes in human cancer. While PTEN's function in tumor suppression is well established, its relationship to anti-microbial immunity remains unknown. Here we found a pivotal role for PTEN in the induction of type I interferon, the hallmark of antiviral innate immunity, that was independent of the pathway of the kinases PI(3)K and Akt. PTEN controlled the import of IRF3, a master transcription factor responsible for IFN-ß production, into the nucleus. We further identified a PTEN-controlled negative phosphorylation site at Ser97 of IRF3 and found that release from this negative regulation via the phosphatase activity of PTEN was essential for the activation of IRF3 and its import into the nucleus. Our study identifies crosstalk between PTEN and IRF3 in tumor suppression and innate immunity.

The Zinc-Finger Protein ZCCHC3 Binds RNA and Facilitates Viral RNA Sensing and Activation of the RIG-I-like Receptors.

Recognition of viral RNA by the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiates innate antiviral immune response. How the binding of viral RNA to and activation of the RLRs are regulated remains enigmatic. In this study, we identified ZCCHC3 as a positive regulator of the RLRs including RIG-I and MDA5. ZCCHC3 deficiency markedly inhibited RNA virus-triggered induction of downstream antiviral genes, and ZCCHC3-deficient mice were more susceptible to RNA virus infection. ZCCHC3 was associated with RIG-I and MDA5 and functions in two distinct processes for regulation of RIG-I and MDA5 activities. ZCCHC3 bound to dsRNA and enhanced the binding of RIG-I and MDA5 to dsRNA. ZCCHC3 also recruited the E3 ubiquitin ligase TRIM25 to the RIG-I and MDA5 complexes to facilitate its K63-linked polyubiquitination and activation. Thus, ZCCHC3 is a co-receptor for RIG-I and MDA5, which is critical for RLR-mediated innate immune response to RNA virus.

Tankyrases inhibit innate antiviral response by PARylating VISA/MAVS and priming it for RNF146-mediated ubiquitination and degradation.

During viral infection, sensing of viral RNA by retinoic acid-inducible gene-I-like receptors (RLRs) initiates an antiviral innate immune response, which is mediated by the mitochondrial adaptor protein VISA (virus-induced signal adaptor; also known as mitochondrial antiviral signaling protein [MAVS]). VISA is regulated by various posttranslational modifications (PTMs), such as polyubiquitination, phosphorylation, O-linked ß-d-N-acetylglucosaminylation (O-GlcNAcylation), and monomethylation. However, whether other forms of PTMs regulate VISA-mediated innate immune signaling remains elusive. Here, we report that Poly(ADP-ribosyl)ation (PARylation) is a PTM of VISA, which attenuates innate immune response to RNA viruses. Using a biochemical purification approach, we identified tankyrase 1 (TNKS1) as a VISA-associated protein. Viral infection led to the induction of TNKS1 and its homolog TNKS2, which translocated from cytosol to mitochondria and interacted with VISA. TNKS1 and TNKS2 catalyze the PARylation of VISA at Glu137 residue, thereby priming it for K48-linked polyubiquitination by the E3 ligase Ring figure protein 146 (RNF146) and subsequent degradation. Consistently, TNKS1, TNKS2, or RNF146 deficiency increased the RNA virus-triggered induction of downstream effector genes and impaired the replication of the virus. Moreover, TNKS1- or TNKS2-deficient mice produced higher levels of type I interferons (IFNs) and proinflammatory cytokines after virus infection and markedly reduced virus loads in the brains and lungs. Together, our findings uncover an essential role of PARylation of VISA in virus-triggered innate immune signaling, which represents a mechanism to avoid excessive harmful immune response.

The PB1 protein of influenza A virus inhibits the innate immune response by targeting MAVS for NBR1-mediated selective autophagic degradation.

Influenza A virus (IAV) has evolved various strategies to counteract the innate immune response using different viral proteins. However, the mechanism is not fully elucidated. In this study, we identified the PB1 protein of H7N9 virus as a new negative regulator of virus- or poly(I:C)-stimulated IFN induction and specifically interacted with and destabilized MAVS. A subsequent study revealed that PB1 promoted E3 ligase RNF5 to catalyze K27-linked polyubiquitination of MAVS at Lys362 and Lys461. Moreover, we found that PB1 preferentially associated with a selective autophagic receptor neighbor of BRCA1 (NBR1) that recognizes ubiquitinated MAVS and delivers it to autophagosomes for degradation. The degradation cascade mediated by PB1 facilitates H7N9 virus infection by blocking the RIG-I-MAVS-mediated innate signaling pathway. Taken together, these data uncover a negative regulatory mechanism involving the PB1-RNF5-MAVS-NBR1 axis and provide insights into an evasion strategy employed by influenza virus that involves selective autophagy and innate signaling pathways.

ZFYVE1 negatively regulates MDA5- but not RIG-I-mediated innate antiviral response.

The retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I and melanoma differentiation-associated gene 5 (MDA5), sense cytoplasmic viral RNA and initiate innate antiviral responses. How RIG-I and MDA5 are differentially regulated remains enigmatic. In this study, we identified the guanylate-binding protein (GBP) and zinc-finger FYVE domain-containing protein ZFYVE1 as a negative regulator of MDA5- but not RIG-I-mediated innate antiviral responses. ZFYVE1-deficiency promoted MDA5- but not RIG-I-mediated transcription of downstream antiviral genes. Comparing to wild-type mice, Zfyve1-/- mice were significantly protected from lethality induced by encephalomyocarditis virus (EMCV) that is sensed by MDA5, whereas Zfyve1-/- and Zfyve1+/+ mice were comparable to death induced by vesicular stomatitis virus (VSV) that is sensed by RIG-I. Mechanistically, ZFYVE1 interacted with MDA5 but not RIG-I. ZFYVE1 bound to viral RNA and decreased the ligand binding and oligomerization of MDA5. These findings suggest that ZFYVE1 acts as a specific negative regulator of MDA5-mediated innate immune responses by inhibiting its ligand binding and oligomerization.

The Nucleoprotein of H7N9 Influenza Virus Positively Regulates TRAF3-Mediated Innate Signaling and Attenuates Viral Virulence in Mice.

H7N9 influenza A virus (IAV) is an emerged contagious pathogen that may cause severe human infections, even death. Understanding the precise cross talk between virus and host is vital for the development of effective vaccines and therapeutics. In the present study, we identified the nucleoprotein (NP) of H7N9 IAV as a positive regulator of RIG-I like receptor (RLR)-mediated signaling. Based on a loss-of-function strategy, we replaced H1N1 (mouse-adapted PR8 strain) NP with H7N9 NP, by using reverse genetics, and found that the replication and pathogenicity of recombinant PR8-H7N9NP (rPR8-H7N9NP) were significantly attenuated in cells and mice. Biochemical and cellular analyses revealed that H7N9 NP specifically interacts with tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) after viral infection. Subsequently, we identified a PXXQXS motif in the H7N9 NP that may be a determinant for the NP and TRAF3 interaction. Furthermore, H7N9 NP stabilized TRAF3 expression via competitively binding to TRAF3 with cellular inhibitor of apoptosis 2 (cIAP2), leading to the inhibition of the Lys48-linked polyubiquitination and degradation of TRAF3. Taken together, these data uncover a novel mechanism by which the NP of H7N9 IAV positively regulates TRAF3-mediated type I interferon signaling. Our findings provide insights into virus and host survival strategies that involve a specific viral protein that modulates an appropriate immune response in hosts.IMPORTANCE The NS1, PB2, PA-X, and PB1-F2 proteins of influenza A virus (IAV) are known to employ various strategies to counteract and evade host defenses. However, the viral components responsible for the activation of innate immune signaling remain elusive. Here, we demonstrate for the first time that the NP of H7N9 IAV specifically associates with and stabilizes the important adaptor molecule TRAF3, which potentiates RLR-mediated type I interferon induction. Moreover, we reveal that this H7N9 NP protein prevents the interaction between TRAF3 and cIAP2 that mediates Lys48-linked polyubiquitination of TRAF3 for degradation. The current study revealed a novel mechanism by which H7N9 NP upregulates TRAF3-mediated type I interferon production, leading to attenuation of viral replication and pathogenicity in cells and mice. Our finding provides a possible explanation for virus and host commensalism via viral manipulation of the host immune system.

USP19 Inhibits TNF-α- and IL-1ß-Triggered NF-κB Activation by Deubiquitinating TAK1.

The dynamic regulations of ubiquitination and deubiquitination play important roles in TGF-ß-activated kinase 1 (TAK1)-mediated NF-κB activation, which regulates various physiological and pathological events. We identified ubiquitin-specific protease (USP)19 as a negative regulator of TNF-α- and IL-1ß-triggered NF-κB activation by deubiquitinating TAK1. Overexpression of USP19 but not its enzymatic inactive mutant inhibited TNF-α- and IL-1ß-triggered NF-κB activation and transcription of downstream genes, whereas USP19 deficiency had the opposite effects. Usp19-/- mice produced higher levels of inflammatory cytokines and were more susceptible to TNF-α- and IL-1ß-triggered septicemia death compared with their wild-type littermates. Mechanistically, USP19 interacted with TAK1 in a TNF-α- or IL-1ß-dependent manner and specifically deconjugated K63- and K27-linked polyubiquitin chains from TAK1, leading to the impairment of TAK1 activity and the disruption of the TAK1-TAB2/3 complex. Our findings provide new insights to the complicated molecular mechanisms of the attenuation of the inflammatory response.

Phosphoproteomics Analysis Reveals a Potential Role of CHK1 in Regulation of Innate Immunity through IRF3.

Inhibitors of checkpoint kinase 1 (CHK1), a central component of DNA damage and cell cycle checkpoint response, represent a promising new cancer therapy, but the global cellular functions they regulate through phosphorylation are poorly understood. To elucidate the CHK1-regulated phosphorylation network, we performed a global quantitative phosphoproteomics analysis, which revealed 142 phosphosites whose phosphorylation levels were significantly different following treatment with the CHK1 inhibitor SCH 900776. Bioinformatics analysis identified phosphoproteins that function in ATR-CHK1 signaling, DNA replication, and DNA repair. Furthermore, IRF3 phosphorylation at S173 and S175 was significantly reduced following treatment with SCH 900776. Our findings indicate that the CHK1-dependent regulation of IRF3 phosphorylation at S173 and S175 may play a role in the induction of innate immune response after replication stress or DNA damage, which suggests a potential function of CHK1 in the innate immune response. Data are available via ProteomeXchange with identifier PXD015125.

A Naturally Occurring Deletion in the Effector Domain of H5N1 Swine Influenza Virus Nonstructural Protein 1 Regulates Viral Fitness and Host Innate Immunity.

Nonstructural protein 1 (NS1) of influenza A virus regulates innate immune responses via various mechanisms. We previously showed that a naturally occurring deletion (the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus impairs the inhibition of type I interferon (IFN) in chicken fibroblasts and attenuates virulence in chickens. Here we found that the virus bearing this deletion in its NS1 effector domain showed diminished inhibition of IFN-related cytokine expression and attenuated virulence in mice. We further showed that deletion of the EALQR motif disrupted NS1 dimerization, impairing double-stranded RNA (dsRNA) sequestration and competitive binding with RIG-I. In addition, the EALQR-deleted NS1 protein could not bind to TRIM25, unlike full-length NS1, and was less able to block TRIM25 oligomerization and self-ubiquitination, further impairing the inhibition of TRIM25-mediated RIG-I ubiquitination compared to that with full-length NS1. Our data demonstrate that the EALQR deletion prevents NS1 from blocking RIG-I-mediated IFN induction via a novel mechanism to attenuate viral replication and virulence in mammalian cells and animals.IMPORTANCE H5 highly pathogenic avian influenza viruses have infected more than 800 individuals across 16 countries, with an overall case fatality rate of 53%. Among viral proteins, nonstructural protein 1 (NS1) of influenza virus is considered a key determinant for type I interferon (IFN) antagonism, pathogenicity, and host range. However, precisely how NS1 modulates virus-host interaction, facilitating virus survival, is not fully understood. Here we report that a naturally occurring deletion (of the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus disrupted NS1 dimerization, which diminished the blockade of IFN induction via the RIG-I signaling pathway, thereby impairing virus replication and virulence in the host. Our study demonstrates that the EALQR motif of NS1 regulates virus fitness to attain a virus-host compromise state in animals and identifies this critical motif as a potential target for the future development of small molecular drugs and attenuated vaccines.

Glycogen synthase kinase 3ß regulates IRF3 transcription factor-mediated antiviral response via activation of the kinase TBK1.

Viral infection activates transcription factors IRF3 and NF-κB, which collaborate to induce type I interferons (IFNs). Here, we identified glycogen synthase kinase 3ß (GSK3ß) as an important regulator for virus-triggered IRF3 and NF-κB activation, IFN-ß induction, and cellular antiviral response. Overexpression of GSK3ß potentiated virus-induced activation of IRF3 and transcription of the IFNB1 gene, whereas reduced expression or deletion of GSK3ß impaired virus-induced IRF3 and NF-κB activation, transcription of the IFNB1 gene, as well as cellular antiviral response. GSK3ß physically associated with the kinase TBK1 in a viral infection-dependent manner. GSK3ß promoted TBK1 self-association and autophosphorylation at Ser172, which is critical for virus-induced IRF3 activation and IFN-ß induction. The effect of GSK3ß on virus-induced signaling is independent of its kinase activity. Our findings suggest that GSK3ß plays important roles in virus-triggered IRF3 activation by promoting TBK1 activation and provide new insights to the molecular mechanisms of cellular antiviral response.

TRIM8 Negatively Regulates TLR3/4-Mediated Innate Immune Response by Blocking TRIF-TBK1 Interaction.

TLR-mediated signaling pathways play critical roles in host defense against microbials. However, dysregulation of innate immune and inflammatory responses triggered by TLRs would result in harmful damage to the host. Using a Trim8 gene-knockout mouse model, we show that tripartite motif (TRIM) 8 negatively regulates TLR3- and TLR4-mediated innate immune and inflammatory responses. TRIM8 deficiency leads to increased polyinosinic-polycytidylic acid- and LPS-triggered induction of downstream anti-microbial genes including TNF, Il6, Rantes, and Ifnb, evaluated serum cytokine levels, and increased susceptibility of mice to polyinosinic-polycytidylic acid- and LPS-induced inflammatory death as well as Salmonella typhimurium infection-induced loss of body weight and septic shock. TRIM8 interacted with Toll/IL-1 receptor domain-containing adapter-inducing IFN-ß and mediated its K6- and K33-linked polyubiquitination, leading to disruption of the Toll/IL-1 receptor domain-containing adapter-inducing IFN-ß-TANK-binding kinase-1 association. Our findings uncover an additional mechanism on the termination of TLR3/4-mediated inflammatory and innate immune responses.

The ubiquitin ligase RNF5 regulates antiviral responses by mediating degradation of the adaptor protein MITA.

Viral infection activates transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. MITA (also known as STING) has recently been identified as an adaptor that links virus-sensing receptors to IRF3 activation. Here, we showed that the E3 ubiquitin ligase RNF5 interacted with MITA in a viral-infection-dependent manner. Overexpression of RNF5 inhibited virus-triggered IRF3 activation, IFNB1 expression, and cellular antiviral response, whereas knockdown of RNF5 had opposite effects. RNF5 targeted MITA at Lys150 for ubiquitination and degradation after viral infection. Both MITA and RNF5 were located at the mitochondria and endoplasmic reticulum (ER) and viral infection caused their redistribution to the ER and mitochondria, respectively. We further found that virus-induced ubiquitination and degradation of MITA by RNF5 occurred at the mitochondria. These findings suggest that RNF5 negatively regulates virus-triggered signaling by targeting MITA for ubiquitination and degradation at the mitochondria.

Duck Tembusu Virus Nonstructural Protein 1 Antagonizes IFN-ß Signaling Pathways by Targeting VISA.

Duck Tembusu virus (DTMUV) is an emergent infectious pathogen that has caused severe disease in ducks and huge economic losses to the poultry industry in China since 2009. Previously, we showed that DTMUV inhibits IFN-ß induction early in infection; however, the mechanisms of the inhibition of innate immune responses remain poorly understood. In this study, we screened DTMUV-encoded structural and nonstructural proteins using reporter assays and found that DTMUV NS1 markedly suppressed virus-triggered IFN-ß expression by inhibiting retinoic acid-inducible gene I-like receptor signaling. Moreover, we found that DTMUV NS1 specifically interacted with the C-terminal domain of virus-induced signaling adaptor and impaired the association of retinoic acid-inducible gene I or melanoma differentiation-associated gene 5 and virus-induced signaling adaptor, thereby downregulating the retinoic acid-inducible gene I-like receptor-mediated signal transduction and cellular antiviral responses, leading to evasion of the innate immune response. Together, our findings reveal a novel mechanism manipulated by DTMUV to circumvent the host antiviral immune response.

The VP3 structural protein of foot-and-mouth disease virus inhibits the IFN-ß signaling pathway.

Foot-and-mouth disease is a frequently occurring disease of cloven-hoofed animals that is caused by infection with the foot-and-mouth virus (FMDV). FMDV circumvents the type-I IFN response by expressing proteins that antagonize cellular innate immunity, such as leader protease and 3C protease. We identified the FMDV structural protein VP3 as a negative regulator of the virus-triggered IFN-ß signaling pathway. Expression of FMDV VP3 inhibited the Sendai virus-triggered activation of IFN regulatory factor-3 and the expression of retinoic acid-inducible gene-I/melanoma differentiation-associated protein-5. Transient transfection and coimmunoprecipitation confirmed that the structural protein VP3 interacts with virus-induced signaling adapter (VISA), which is dependent on the C-terminal aa 111-220 of VP3. In addition, we found that FMDV VP3 inhibits the expression of VISA by disrupting its mRNA. Taken together, our findings reveal a novel strategy used by the structural VP3 protein of FMDV to evade host innate immunity.-Li, D., Yang, W., Yang, F., Liu, H., Zhu, Z., Lian, K., Lei, C., Li, S., Liu, X., Zheng, H., Shu, H. The VP3 structural protein of foot-and-mouth disease virus inhibits the IFN-ß signaling pathway.

The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation.

Viral infection triggers activation of transcription factors such as NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. Here, we identified MITA as a critical mediator of virus-triggered type I IFN signaling by expression cloning. Overexpression of MITA activated IRF3, whereas knockdown of MITA inhibited virus-triggered activation of IRF3, expression of type I IFNs, and cellular antiviral response. MITA was found to localize to the outer membrane of mitochondria and to be associated with VISA, a mitochondrial protein that acts as an adaptor in virus-triggered signaling. MITA also interacted with IRF3 and recruited the kinase TBK1 to the VISA-associated complex. MITA was phosphorylated by TBK1, which is required for MITA-mediated activation of IRF3. Our results suggest that MITA is a critical mediator of virus-triggered IRF3 activation and IFN expression and further demonstrate the importance of certain mitochondrial proteins in innate antiviral immunity.

WDFY1 mediates TLR3/4 signaling by recruiting TRIF.

Toll-like receptors (TLRs) are pattern recognition receptors that sense a variety of pathogens, initiate innate immune responses, and direct adaptive immunity. All TLRs except TLR3 recruit the adaptor MyD88 to ultimately elicit inflammatory gene expression, whereas TLR3 and internalized TLR4 use TIR-domain-containing adaptor TRIF for the induction of type I interferon and inflammatory cytokines. Here, we identify the WD repeat and FYVE-domain-containing protein WDFY1 as a crucial adaptor protein in the TLR3/4 signaling pathway. Overexpression of WDFY1 potentiates TLR3- and TLR4-mediated activation of NF-κB, interferon regulatory factor 3 (IRF3), and production of type I interferons and inflammatory cytokines. WDFY1 depletion has the opposite effect. WDFY1 interacts with TLR3 and TLR4 and mediates the recruitment of TRIF to these receptors. Our findings suggest a crucial role for WDFY1 in bridging the TLR-TRIF interaction, which is necessary for TLR signaling.

Krüppel-like factor 6 is a co-activator of NF-κB that mediates p65-dependent transcription of selected downstream genes.

The transcription factor NF-κB plays a pivotal role in a broad range of physiological and pathological processes, including development, inflammation, and immunity. How NF-κB integrates activating signals to expression of specific sets of target genes is of great interest. Here, we identified Krüppel-like factor 6 (KLF6) as a co-activator of NF-κB after TNFα and IL-1ß stimulation. Overexpression of KLF6 enhanced TNFα- and IL-1ß-induced activation of NF-κB and transcription of a subset of downstream genes, whereas knockdown of KLF6 had opposite effects. KLF6 interacted with p65 in the nucleus and bound to the promoters of target genes. Upon IL-1ß stimulation, KLF6 was recruited to promoters of a subset of NF-κB target genes in a p65-dependent manner, which was in turn required for the optimal binding of p65 to the target gene promoters. Our findings thus identified KLF6 as a previously unknown but essential co-activator of NF-κB and provided new insight into the molecular regulation of p65-dependent gene expression.

FoxO1 negatively regulates cellular antiviral response by promoting degradation of IRF3.

Viral infection causes activation of the transcription factor IRF3, which is critical for production of type I interferons (IFNs) and innate antiviral immune response. How virus-induced type I IFN signaling is controlled is not fully understood. Here we identified the transcription factor FoxO1 as a negative regulator for virus-triggered IFN-ß induction. Overexpression of FoxO1 inhibited virus-triggered ISRE activation, IFN-ß induction as well as cellular antiviral response, whereas knockdown of FoxO1 had opposite effects. FoxO1 interacted with IRF3 in a viral infection-dependent manner and promoted K48-linked polyubiquitination and degradation of IRF3 in the cytosol. Furthermore, FoxO1-mediated degradation of IRF3 was independent of the known E3 ubiquitin ligases for IRF3, including RBCK1 and RAUL. Our findings thus suggest that FoxO1 negatively regulates cellular antiviral response by promoting IRF3 ubiquitination and degradation, providing a previously unknown mechanism for control of type I IFN induction and cellular antiviral response.

Comparative transcriptome analysis reveals molecular adaptations underlying distinct immunity and inverted resting posture in bats.

Understanding how natural selection shapes unique traits in mammals is a central topic in evolutionary biology. The mammalian order Chiroptera (bats) is attractive for biologists as well as the general public due to their specific traits of extraordinary immunity and inverted resting posture. However, genomic resources for bats that occupy key phylogenetic positions are not sufficient, which hinders comprehensive investigation of the molecular mechanisms underpinning the origin of specific traits in bats. Here, we sequenced the transcriptomes of 5 bats that are phylogenetically divergent and occupy key positions in the phylogenetic tree of bats. In combination with the available genomes of 19 bats and 21 other mammals, we built a database consisting of 10 918 one-to-one ortholog genes and reconstructed phylogenetic relationships of these mammals. We found that genes related to immunity, bone remodeling, and cardiovascular system are targets of natural selection along the ancestral branch of bats. Further analyses revealed that the T cell receptor signaling pathway involved in immune adaptation is specifically enriched in bats. Moreover, molecular adaptations of bone remodeling, cardiovascular system, and balance sensing may help to explain the reverted resting posture in bats. Our study provides valuable transcriptome resources, enabling us to tentatively identify genetic changes associated with bat-specific traits. This work is among the first to advance our understanding of the molecular underpinnings of inverted resting posture in bats, which could provide insight into healthcare applications such as hypertension in humans.

The nucleoprotein of influenza A virus inhibits the innate immune response by inducing mitophagy.

Mitophagy is a form of autophagy that plays a key role in maintaining the homeostasis of functional mitochondria in the cell. Viruses have evolved various strategies to manipulate mitophagy to escape host immune responses and promote virus replication. In this study, the nucleoprotein (NP) of H1N1 virus (PR8 strain) was identified as a regulator of mitophagy. We revealed that NP-mediated mitophagy leads to the degradation of the mitochondria-anchored protein MAVS, thereby blocking MAVS-mediated antiviral signaling and promoting virus replication. The NP-mediated mitophagy is dependent on the interaction of NP with MAVS and the cargo receptor TOLLIP. Moreover, Y313 of NP is a key residue for the MAVS-NP interaction and NP-mediated mitophagy. The NPY313F mutation significantly attenuates the virus-induced mitophagy and the virus replication in vitro and in vivo. Taken together, our findings uncover a novel mechanism by which the NP of influenza virus induces mitophagy to attenuate innate immunity.Abbreviations: ACTB: actin beta; ATG7: autophagy related 7; ATG12: autophagy related 12; CCCP: carbonyl cyanide 3-chlorophenyl hydrazone; co-IP: co-immunoprecipitation; COX4/COXIV: cytochrome c oxidase subunit 4; DAPI: 4',6-diamidino-2-phenylindole, dihydrochloride; EID50: 50% egg infective dose; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HEK: human embryonic kidney; hpi: hours post-infection; IAV: influenza A virus; IFN: interferon; IP: immunoprecipitation; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; Mdivi-1: mitochondrial division inhibitor 1; MLD50: 50% mouse lethal dose; MOI: multiplicity of infection; NBR1: NBR1 autophagy cargo receptor; NP: nucleoprotein; PB1: basic polymerase 1; RFP: red fluorescent protein; RIGI: RNA sensor RIG-I; RIGI-N: RIGI-CARD; SeV: Sendai virus; SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TOLLIP: toll interacting protein; TOMM20: translocase of outer mitochondrial membrane 20; TUBA: tubulin alpha; Vec: empty vector; vRNP: viral ribonucleoprotein.