RIGI基因敲除HEK293细胞

Accurate control of innate immune responses is required to eliminate invading pathogens and simultaneously avoid autoinflammation and autoimmune diseases. Here, we demonstrate that arginine monomethylation precisely regulates the mitochondrial antiviral-signaling protein (MAVS)-mediated antiviral response. Protein arginine methyltransferase 7 (PRMT7) forms aggregates to catalyze MAVS monomethylation at arginine residue 52 (R52), attenuating its binding to TRIM31 and RIG-I, which leads to the suppression of MAVS aggregation and subsequent activation. Upon virus infection, aggregated PRMT7 is disabled in a timely manner due to automethylation at arginine residue 32 (R32), and SMURF1 is recruited to PRMT7 by MAVS to induce proteasomal degradation of PRMT7, resulting in the relief of PRMT7 suppression of MAVS activation. Therefore, we not only reveal that arginine monomethylation by PRMT7 negatively regulates MAVS-mediated antiviral signaling in vitro and in vivo but also uncover a mechanism by which PRMT7 is tightly controlled to ensure the timely activation of antiviral defense.

TAX1BP1 is a selective macroautophagy/autophagy receptor that inhibits NFKB and RIGI-like receptor (RLR) signaling to prevent excessive inflammation and maintain homeostasis. Selective autophagy receptors such as SQSTM1/p62 and OPTN are phosphorylated by the kinase TBK1 to stimulate their selective autophagy function. However, it is unknown if TAX1BP1 is regulated by TBK1 or other kinases under basal conditions or during RNA virus infection. Here, we found that TBK1 and IKBKE/IKKi function redundantly to phosphorylate TAX1BP1 and regulate its autophagic turnover through canonical macroautophagy. TAX1BP1 phosphorylation promotes its localization to lysosomes, resulting in its degradation. Additionally, we found that during vesicular stomatitis virus infection, TAX1BP1 is targeted to lysosomes in an ATG8-family protein-independent manner. Furthermore, TAX1BP1 plays a critical role in the clearance of MAVS aggregates, and phosphorylation of TAX1BP1 controls its MAVS aggrephagy function. Together, our data support a model whereby TBK1 and IKBKE license TAX1BP1-selective autophagy function to inhibit MAVS and RLR signaling. ATG: autophagy related; BafA1: bafilomycin A1; CALCOCO2: calcium binding and coiled-coil domain 2; GFP: green fluorescent protein; IFA: indirect immunofluorescence assay; IFN: interferon; IκB: inhibitor of nuclear factor kappa B; IKK: IκB kinase; IRF: interferon regulatory factor; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MEF: mouse embryonic fibroblast; MOI: multiplicity of infection; IKBKG/NEMO: inhibitor of nuclear factor kappa B kinase regulatory subunit gamma; NFKB: nuclear factor kappa B; OPTN: optineurin; Poly(I:C): polyinosinic-polycytidylic acid; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RIGI: RNA sensor RIG-I; RLR: RIGI-like receptor; SDD-AGE: semi-denaturing detergent-agarose gel electrophoresis; SeV: Sendai virus; SLR: SQSTM1-like receptor; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TNF: tumor necrosis factor; TRAF: TNF receptor associated factor; VSV: vesicular stomatitis virus; ZnF: zinc finger.

Sec8, an exocyst complex subunit, is pivotal in facilitating the docking of exocytic vesicles to fusion sites on the plasma membrane. However, its involvement in the antiviral innate immune response and virus replication remains unclear. In this study, Sec8 is identified as a novel positive regulator of RIG-I, enhancing the IFN-I signaling response against RNA viruses both in vivo and in vitro. Additionally, Sec8 stabilizes RIG-I by inhibiting its ubiquitination and subsequent proteasome-mediated degradation. Mechanistically, STUB1 degrades RIG-I via K48-linked ubiquitination at Lys190, while Sec8 suppresses STUB1 mRNA by reducing the expression of p53 and competes with STUB1 for binding to RIG-I's CARD domain, thereby preventing STUB1-mediated RIG-I degradation. Importantly, Sec8-deficient mice were more susceptible to RNA virus infection compared to wild-type mice. These findings elucidate a mechanism that Sec8 positively regulates RIG-I in the antiviral innate immune response, offering insights for developing novel therapeutic strategies and targeted antiviral medications.

Linear ubiquitination and deubiquitination represent critical regulatory mechanisms in inflammation and cell death, yet their roles in RIG-I-like receptor (RLR)-dependent signaling remain unclear. Here, we identified OTU deubiquitinase with linear linkage specificity (OTULIN) as a negative regulator of RIG-I-dependent type I IFN (IFN-I) signaling. Overexpression of OTULIN markedly attenuated NF-κB and IFNβ reporter activation triggered by RNA viruses or synthetic analogs, suppressed downstream gene expression, impaired IFN-I production, and diminished phosphorylation of IκBα, TBK1, and IRF3, thereby facilitating viral replication. Conversely, knockout of in HeLa and iBMDM cells enhanced these antiviral signaling events and restricted viral replication. Critically, reintroducing into -KO cells via lentiviral transduction reversed this enhanced phenotype, restoring the suppression of IFN-I signaling. Mechanistically, RNA virus infection induced linear ubiquitination of TRAF6 at K104, K142, and K371. LUBAC promoted antiviral innate immune signaling by enhancing the linear ubiquitination of TRAF6, which was antagonized by OTULIN. Notably, the linear ubiquitination of TRAF6 facilitated its K63-linked ubiquitination and strengthened its association with MAVS, amplifying the antiviral response. Furthermore, mice exhibited enhanced antiviral immunity and more efficient viral clearance than wild-type littermates. Collectively, these findings unveil a regulatory role of OTULIN in attenuating RIG-I-dependent IFN-I signaling through removal of linear ubiquitination from TRAF6, highlighting the essential equilibrium between linear ubiquitination and deubiquitination in antiviral innate immunity and immune homeostasis.

RIG-I and STING are critical for mediating the RIG-I and cGAS-STING signaling pathways that guard against viral infection. Here, we report that ubiquitin-specific peptidase 39 (USP39) positively regulates the RIG-I and cGAS-STING pathways to induce antiviral innate immunity in vitro and in vivo. The USP39 deficiency impaired the antiviral immune response of macrophages, leading to low type I IFNs expression, and high RNA and (e.g., VSV, H1N1 PR8) DNA virus (e.g., HSV-1) replication. Moreover, USP39-deficient mice were more sensitive to VSV or HSV-1 infection than control mice. Conversely, USP39 overexpression promoted the antiviral immune response. Mechanistically, we found that USP39 regulates RIG-I protein expression by promoting pre-RIG-I mRNA splicing and maturation. In addition, we also revealed that USP39 interacts with and stabilizes STING protein by deubiquitinating K48-linked polyubiquitin of STING at K288. These data show that USP39 positively regulates RNA and DNA-virus-induced RIG-I and cGAS-STING signaling, respectively, by promoting post-transcriptional control of RIG-I and stabilization of STING. These data provide new insights and potential therapeutic targets to control viral infections.

RIG-I-like receptors (RLR), RIG-I and MDA5, are cytoplasmic viral RNA sensors that recognize viral double-stranded RNAs and trigger signals to induce antiviral responses, including type I interferon production. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) caused the coronavirus disease 2019 pandemic. However, the RLR role in innate immune response to SARS-CoV-2 has not been fully elucidated. Here, we studied the roles of RLR in cytokine expression responding to SARS-CoV-2 and found that not only MDA5 but also RIG-I are involved in innate immune responses in some types of human cells. Transfection of total RNAs extracted from SARS-CoV-2-infected cells into epithelial cells induced IFN-β, IP-10, and Ccl5 mRNA expression. The cytokine expression was reduced by knockout of either RIG-I or MDA5, suggesting that both proteins are required for appropriate innate immune response to SARS-CoV-2. Two viral genomic RNA regions strongly induced type I IFN expression, and a 200-base fragment of viral RNA preferentially induced type I IFN in a RIG-I-dependent manner. In contrast, SARS-CoV-2 infectious particles hardly induced cytokine expression, suggesting viral escape from the host response. Viral 9b protein inhibited RIG-I and MAVS interaction, and viral 7a protein destabilized the TBK1 protein, leading to attenuated IRF-3 phosphorylation required for type I IFN expression. Our data elucidated the mechanism underlying RLR-mediated response to SARS-CoV-2 infection and viral escape from the host innate immune response.

Retinoic acid-inducible gene I (RIG-I) senses viral RNA and instigates an innate immune signaling cascade to induce type I interferon expression. Currently, the regulatory mechanisms controlling RIG-I activation remain to be fully elucidated. Here we show that the FAK family kinase-interacting protein of 200 kDa (FIP200) facilitates RIG-I activation. FIP200 deficiency impaired RIG-I signaling and increased host susceptibility to RNA virus infection. In vivo studies further demonstrated FIP200 knockout mice were more susceptible to RNA virus infection due to the reduced innate immune response. Mechanistic studies revealed that FIP200 competed with the helicase domain of RIG-I for interaction with the two tandem caspase activation and recruitment domains (2CARD), thereby facilitating the release of 2CARD from the suppression status. Furthermore, FIP200 formed a dimer and facilitated 2CARD oligomerization, thereby promoting RIG-I activation. Taken together, our study defines FIP200 as an innate immune signaling molecule that positively regulates RIG-I activation.

Non-structural protein 2 (NSP2) of PRRSV is highly variable and plays crucial roles in the virus's life cycle. To elucidate the function of NSP2 during PRRSV infection, we identified SH3KBP1 as an NSP2-interacting host protein using mass spectrometry. Exogenous SH3KBP1 expression significantly inhibited PRRSV replication by enhancing IFN-I and related ISGs production. Conversely, SH3KBP1 knockdown promoted viral replication by downregulating IFN-I and ISGs levels. In vivo experiments revealed that Sh3kbp1-/- mice were more susceptible to VSV infection, exhibiting reduced serum IFN-β levels. Further investigation showed that SH3KBP1 enhances RIG-I signal transduction by increasing K63-linked polyubiquitination through interaction with the E3 ubiquitin ligase TRIM25. We also found that PRRSV infection and NSP2 overexpression induce the autophagic degradation of SH3KBP1, counteracting the host's innate immune response. A critical interaction site was identified within the third polyproline-arginine motif in NSP2 (453PVPAPR458). Recombinant PRRSV lacking this motif displayed reduced virulence and decreased SH3KBP1 degradation. This study advances our understanding of how PRRSV interferes with the host immune response and offers valuable insights for developing novel attenuated vaccines against PRRSV.

Mitochondria play a crucial role as a hub for innate immune signal transduction, with mitochondrial antiviral signaling protein (MAVS) being a key regulator in the activation of interferon-β (IFN-β) production. It is essential for MAVS to initiate innate antiviral responses against RNA viruses, contributing to the host's defense mechanisms. In this study, we identified the mitochondrial protein Paraneoplastic Ma Family 4 (PNMA4/MOAP1) as a MAVS-interacting protein by using proximity-based labeling technology in THP-1 and discovered that it could enhance retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) signaling pathway. The absence of PNMA4 significantly reduced RNA virus-induced activation of antiviral pathways and made Pnma4 mice more susceptible to infection. On the one hand, PNMA4 enhanced the interaction between RIG-I and MAVS after RNA virus infection. On the other hand, it facilitated RIG-I lysine 63 (K63)-linked polyubiquitination at lysine 657 by TRIM25, thus positively regulating antiviral responses.

Type Ι interferon (IFN) production mediated by retinoic acid-inducible gene 1 (RIG-I) and mitochondrial antiviral signaling protein (MAVS) is essential for antiviral innate immune responses. Here, we report the identification of a novel co-sensor for cytosolic nucleic acids: DEAD/H-box helicase 11 (DDX11), a member of the DExD/H (Asp-Glu-x-Asp/His)-box helicase family. Knockdown or knockout of DDX11 attenuated the ability of cells to increase IFN-β, IFN-stimulated gene 56, and C-X-C motif chemokine ligand 10 in response to SeV and poly (I:C) by blocking the activation of TANK-binding kinase 1 and IFN regulatory factor 3. Nucleic acid sensing by DDX11 was independent of the stimulator of IFN genes but was dependent on RIG-I and MAVS. DDX11 regulated RIG-I-MAVS-mediated IFN signaling by specifically interacting with nucleic acid, RIG-I, and MAVS to enhance RIG-I-double-strand RNA and RIG-I-MAVS binding affinity. Overall, our results identified a critical role for DDX11 in the innate immune response and provided molecular insights into the mechanisms by which DDX11 recognized cytosolic nucleic acid and interacted with RIG-Ι and MAVS for potent IFN signaling and antiviral immunity. IMPORTANCE:Innate immunity is the first and most rapid host defense against virus infection. Recognition of viral RNA by the retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs) initiates innate antiviral immune responses. How the binding of viral RNA to and activation of the RLRs are regulated remains enigmatic. In this study, we identified DEAD/H-box helicase 11 (DDX11) as a positive regulator of the RIG-I-mitochondrial antiviral signaling protein (MAVS)-mediated signaling pathways. Mechanistically, we demonstrated that DDX11 bound to viral RNA, interacted with RIG-I, and promoted their binding to viral RNA. DDX11 also promoted the interaction between RIG-I and MAVS and activation of RIG-I-MAVS signaling. Overall, our results elucidate the role of DDX11 in RIG-I-MAVS-dependent signaling pathways and may shed light on innate immune gene regulation.

U26 is one of the roseolovirus unique genes with unknown function. Human herpesvirus 6B (HHV-6B) pU26 is predicted to be an 8-transmembrane protein containing a mitochondrion location signal. Here, we analyzed U26 function during HHV-6B infection and find that (i) HHV-6B U26 is expressed at a very early stage during HHV-6B infection, and knockdown of it results in a significant decrease of HHV-6B progeny virus production; (ii) U26 inhibits the activation of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)/mitochondrial antiviral signaling protein (MAVS) signaling pathway, an important anti-HHV-6B infection innate immune response, by targeting MAVS protein for degradation; and (iii) a portion of U26 locates to the mitochondria, which could affect the mitochondrial membrane potential and finally leads to MAVS degradation. These findings indicate that HHV-6B U26 is a novel antagonistic viral factor against host innate antiviral immunity. HHV-6B (human herpesvirus 6B) is well known to evade host antiviral responses and establish a lifelong latent infection. How HHV-6B evades RNA recognition is still poorly understood. Our results indicate that HHV-6 U26 plays a vital role in RLR/MAVS signaling pathway activity. Knockout of endogenous MAVS could facilitate HHV-6B replication. The findings in this study could provide new insights into host-virus interactions and help develop a new therapy against HHV-6B infection.

Retinoic acid-inducible gene I-like receptors (RLRs) are important cytosolic pattern recognition receptors (PRRs) that sense viral RNA before mounting a response leading to the activation of type I IFNs. Several viral infections induce epithelial-mesenchymal transition (EMT), even as its significance remains unclear. Here, we show that EMT or an EMT-like process is a general response to viral infections. Our studies identify a previously unknown mechanism of regulation of an important EMT-transcription factor (EMT-TF) Snail during RNA viral infections and describe its possible implication. RNA viral infections, poly(I·C) transfection, and ectopic expression of RLR components induced Snail levels, indicating that RLR pathway could regulate its expression. Detailed examination using mitochondrial antiviral signaling protein knockout (MAVS-KO) cells established that MAVS is essential in this regulation. We identified two interferon-stimulated response elements (ISREs) in the promoter region and demonstrated that they are important in its transcriptional activation by phosphorylated IRF3. Increasing the levels of Snail activated RLR pathway and dramatically limited replication of the RNA viruses dengue virus, Japanese encephalitis virus (JEV), and vesicular stomatitis virus, pointing to their antiviral functions. Knockdown of Snail resulted in a considerable increase in the JEV titer, validating its antiviral functions. Finally, transforming growth factor β-mediated activation was dependent on Snail levels, confirming its important role in type I IFN activation. Thus, EMT-TF Snail is transcriptionally coregulated with type I IFN by RLRs and, in turn, promotes the RLR pathway, further strengthening the antiviral state in the cell. Our work identified an interesting mechanism of regulation of Snail that demonstrates potential coregulation of multiple innate antiviral pathways triggered by RLRs. Identification of antiviral functions of Snail also provides an opportunity to expand the sphere of RLR signaling. RLRs sense viral genomic RNA or the double-stranded RNA intermediates and trigger the activation of type I IFNs. Snail transcription factor, commonly associated with epithelial-mesenchymal transition (EMT), has been reported to facilitate EMT in several viral infections. Many of these reports are based on oncoviruses, leading to the speculation that EMT induced during infection is an important factor in the oncogenesis triggered by these infections. However, our studies reveal that EMT or EMT-like processes during viral infections have important functions in antiviral response. We have characterized a new mechanism of transcriptional regulation of Snail by IRF3 through interferon-stimulated response elements in their promoters, and this finding could have importance in nonviral contexts as well. We also identify that EMT-TF Snail promotes antiviral status of the infected cells through the RLR pathway. This study characterizes a new regulatory mechanism of activation of Snail and establishes its unidentified function in antiviral response.

Retinoic acid-inducible gene I (RIG-I) plays a critical role in the recognition of intracytoplasmic viral RNA. Upon binding to the RNA of invading viruses, the activated RIG-I translocates to mitochondria, where it recruits adapter protein MAVS, causing a series of signaling cascades. In this study, we demonstrated that Hsp70 binding protein 1 (HSPBP1) promotes RIG-I-mediated signal transduction. The overexpression of HSPBP1 can increase the stability of RIG-I protein by inhibiting its K48-linked ubiquitination, and promote the activation of IRF3 and the production of IFN-β induced by Sendai virus. Knockdown and knockout of HSPBP1 leads to down-regulation of virus-induced RIG-I expression, inhibits IRF3 activation, and reduces the production of IFNB1. These results indicate that HSPBP1 positively regulates the antiviral signal pathway induced by inhibiting the K48-linked ubiquitination of RIG-I.

We knocked out the retinoic acid-inducible gene I (- in HEK293 cells CRISPR/Cas9 to reveal the effects of knockout on the key factors in the type I interferon signaling pathway. Three single guide RNAs (sgRNAs) targeting were designed, and the recombination vectors were constructed on the basis of the pX459 vector and used to transfect HEK293 cells, which were screened by puromycin subsequently. Furthermore, a mimic of virus, poly I: C, was used to transfect the cells screened out. knockout was checked by sequencing, real-time quantitative PCR, Western blotting, and immunofluorescence assay. Meanwhile, the expression levels of key factors of type I interferon signaling pathway such as melanoma differentiation-associated gene 5 (MDA5), interferonβ1 (IFNβ1), and nuclear factor-kappa B p65 [NF-κB(p65)], as well as cell viability, were determined. The results showed that two HEK293 cell lines (S1 and S3) with knockout were obtained, which exhibited lower mRNA and protein levels of RIG-I than the wild type HEK293 cells ( < 0.05). The mRNA levels of and in S1 and S3 cells and the protein level of NF-κB(p65) in S3 cells were lower than those in the wild type ( < 0.05). More extranuclear NF-κB(p65) protein was detected in S1 cells than in the wild type after transfection with poly I: C. Plus, the wild-type and S1 cells transfected with poly I: C for 48 h showcased reduced viability ( < 0.05), while S3 cells did not display the reduction in cell viability. In summary, the present study obtained two HEK293 cell lines with knockout CRISPR/Cas9, which provided a stable cell model for exploring the mechanism of type I interferon signaling pathway.