Inducible LGALS3BP/90K activates antiviral innate immune responses by targeting TRAF6 and TRAF3 complex.

The galectin 3 binding protein (LGALS3BP, also known as 90K) is a ubiquitous multifunctional secreted glycoprotein originally identified in cancer progression. It remains unclear how 90K functions in innate immunity during viral infections. In this study, we found that viral infections resulted in elevated levels of 90K. Further studies demonstrated that 90K expression suppressed virus replication by inducing IFN and pro-inflammatory cytokine production. Upon investigating the mechanisms behind this event, we found that 90K functions as a scaffold/adaptor protein to interact with TRAF6, TRAF3, TAK1 and TBK1. Furthermore, 90K enhanced TRAF6 and TRAF3 ubiquitination and served as a specific ubiquitination substrate of TRAF6, leading to transcription factor NF-κB, IRF3 and IRF7 translocation from the cytoplasm to the nucleus. Conclusions: 90K is a virus-induced protein capable of binding with the TRAF6 and TRAF3 complex, leading to IFN and pro-inflammatory production.

NAC1 Potentiates Cellular Antiviral Signaling by Bridging MAVS and TBK1.

Intracellular viral RNAs are recognized by the RIG-I-like receptors (RLRs), which signal through the mitochondrial antiviral signaling protein MAVS. MAVS recruits and activates TBK1 kinase, which further phosphorylates and activates the transcription factor IRF3, leading to the induction of type I IFN and downstream antiviral genes. We identified human nucleus accumbens-associated 1 (NAC1), a member of the BTB/POZ family, as a bridge for MAVS and TBK1 that positively regulates the RLR-mediated induction of type I IFN. Overexpression or knockdown of NAC1 could, respectively, enhance or impair Sendai virus-triggered activation of TBK1 and IRF3, as well as induction of IFN-ß. NAC1 also significantly boosted host antiviral responses against multiple RNA viruses. NAC1 was able to interact with MAVS and TBK1 upon viral infection. The BTB/POZ domain (aa 1-133) of NAC1 interacted with MAVS, and the remainder of NAC1 bound to TBK1. Furthermore, NAC1 could promote the recruitment of TBK1 to MAVS. In contrast, knockdown of NAC1 attenuated the interaction between TBK1 and MAVS. Collectively, our study characterizes NAC1 as an important component of RLR-mediated innate immune responses and uncovers a previously unrecognized function of the BTB/POZ family proteins.

Epigenetic Modification Is Regulated by the Interaction of Influenza A Virus Nonstructural Protein 1 with the De Novo DNA Methyltransferase DNMT3B and Subsequent Transport to the Cytoplasm for K48-Linked Polyubiquitination.

The influenza virus nonstructural protein 1 (NS1) is a nonstructural protein that plays a major role in antagonizing host interferon responses during infection. However, a clear role for the NS1 protein in epigenetic modification has not been established. In this study, NS1 was found to regulate the expression of some key regulators of JAK-STAT signaling by inhibiting the DNA methylation of their promoters. Furthermore, DNA methyltransferase 3B (DNMT3B) is responsible for this process. Upon investigating the mechanisms underlying this event, NS1 was found to interact with DNMT3B but not DNMT3A, leading to the dissociation of DNMT3B from the promoters of the corresponding genes. In addition, the interaction between NS1 and DNMT3B changed the localization of DNMT3B from the nucleus to the cytosol, resulting in K48-linked ubiquitination and degradation of DNMT3B in the cytosol. We conclude that NS1 interacts with DNMT3B and changes its localization to mediate K48-linked polyubiquitination, subsequently contributing to the modulation of the expression of JAK-STAT signaling suppressors.IMPORTANCE The nonstructural protein 1 (NS1) of the influenza A virus (IAV) is a multifunctional protein that counters cellular antiviral activities and is a virulence factor. However, the involvement of NS1 in DNA methylation during IAV infection has not been established. Here, we reveal that the NS1 protein binds the cellular DNMT3B DNA methyltransferase, thereby inhibiting the methylation of the promoters of genes encoding suppressors of JAK-STAT signaling. As a result, these suppressor genes are induced, and JAK-STAT signaling is inhibited. Furthermore, we demonstrate that the NS1 protein transports DNMT3B to the cytoplasm for ubiquitination and degradation. Thus, we identify the NS1 protein as a potential trigger of the epigenetic deregulation of JAK-STAT signaling suppressors and illustrate a novel mechanism underlying the regulation of host immunity during IAV infection.

Inducible microRNA-590-5p inhibits host antiviral response by targeting the soluble interleukin-6 (IL6) receptor.

MicroRNA (miR)-590-5p has been identified as an important regulator of some signaling pathways such as cell proliferation and tumorigenesis. However, little is known about its role during viral infection. Here, we report that miR-590-5p was significantly induced by various viruses and effectively potentiated virus replication in different viral infection systems. Furthermore, miR-590-5p substantially attenuated the virus-induced expression of type I and type III interferons (IFNs) and inflammatory cytokines, resulting in impaired downstream antiviral signaling. Interleukin-6 receptor (IL6R) was identified as a target of miR-590-5p. Interestingly, the role of miR-590-5p in virus-triggered signaling was abolished in IL6R knockout cells, and this could be rescued by restoring the expression of the soluble IL6R (sIL6R) but not the membrane-bound IL6R (mIL6R), suggesting that sIL6R is indispensable for miR-590-5p in modulating the host antiviral response. Furthermore, miR-590-5p down-regulated endogenous sIL6R and mIL6R expression through a translational repression mechanism. These findings thus uncover a previously uncharacterized role and the underlying mechanism of miR-590-5p in the innate immune response to viral infection.

Fibronectin Facilitates Enterovirus 71 Infection by Mediating Viral Entry.

Fibronectin (FN) is a high-molecular-weight extracellular matrix protein that contains the RGDS motif, which is required to bind to integrins. Synthetic RGDS peptides have been reported to compete with FN to bind to the cell surface and inhibit the function of FN. Here, we identified that synthetic RGDS peptides significantly inhibit human enterovirus 71 (EV71) infection in cell cultures. In addition, mice treated with RGDS peptides and infected with EV71 had a significantly higher survival rate and a lower viral load than the control group. Because RGDS peptides affect the function of FN, we questioned whether FN may play a role in virus infection. Our study indicates that overexpression of FN enhanced EV71 infection. In contrast, knockout of FN significantly reduced viral yield and decreased the viral binding to host cells. Furthermore, EV71 entry, rather than intracellular viral replication, was blocked by FN inhibitor pretreatment. Next, we found that FN could interact with the EV71 capsid protein VP1, and further truncated-mutation assays indicated that the D2 domain of FN could interact with the N-terminal fragment of VP1. Taken together, our results demonstrate that the host factor FN binds to EV71 particles and facilitates EV71 entry, providing a potential therapy target for EV71 infection.IMPORTANCE Hand, foot, and mouth disease outbreaks have occurred frequently in recent years, sometimes causing severe neurological complications and even death in infants and young children worldwide. Unfortunately, no effective antiviral drugs are available for human enterovirus 71 (EV71), one of the viruses that cause hand, foot, and mouth disease. The infection process and the host factors involved remain unknown, although several receptors have been identified. In this study, we found that the host factor fibronectin (FN) facilitated EV71 replication by interacting with EV71 particles and further mediated their entry. The RGDS peptide, an FN inhibitor, significantly inhibited EV71 replication in both RD cells and mice. In conclusion, our research identified a new host factor involved in EV71 infection, providing a new potential antiviral target for EV71 treatment.

Inducible TAP1 Negatively Regulates the Antiviral Innate Immune Response by Targeting the TAK1 Complex.

The innate immune response is critical for host defense and must be tightly controlled, but the molecular mechanisms responsible for its negative regulation are not yet completely understood. In this study, we report that transporter 1, ATP-binding cassette, subfamily B (TAP1), a virus-inducible endoplasmic reticulum-associated protein, negatively regulated the virus-triggered immune response. In this study, we observed upregulated expression of TAP1 following virus infection in human lung epithelial cells (A549), THP-1 monocytes, HeLa cells, and Vero cells. The overexpression of TAP1 enhanced virus replication by inhibiting the virus-triggered activation of NF-κB signaling and the production of IFNs, IFN-stimulated genes, and proinflammatory cytokines. TAP1 depletion had the opposite effect. In response to virus infection, TAP1 interacted with the TGF-ß-activated kinase (TAK)1 complex and impaired the phosphorylation of TAK1, subsequently suppressing the phosphorylation of the IκB kinase complex and NF-κB inhibitor α (IκBα) as well as NF-κB nuclear translocation. Our findings collectively suggest that TAP1 plays a novel role in the negative regulation of virus-triggered NF-κB signaling and the innate immune response by targeting the TAK1 complex.

Soluble IL-6 Receptor and IL-27 Subunit p28 Protein Complex Mediate the Antiviral Response through the Type III IFN Pathway.

Previously, we demonstrated that the soluble IL-6R (sIL-6R) plays an important role in the host antiviral response through induction of type I IFN and sIL-6R-mediated antiviral action via the IL-27 subunit p28; however, the mechanism that underlies sIL-6R and p28 antiviral action and whether type III IFN is involved remain unknown. In this study, we constructed a sIL-6R and p28 fusion protein (sIL-6R/p28 FP) and demonstrated that the fusion protein has stronger antiviral activity than sIL-6R alone. Consequently, knockout of sIL-6R inhibited virus-triggered IFN-λ1 expression. In addition, sIL-6R/p28 FP associated with mitochondrial antiviral signaling protein and TNFR-associated factor 6, the retinoic acid-inducible gene I adapter complex, and the antiviral activity mediated by sIL-6R/p28 FP was dependent on mitochondrial antiviral signaling protein. Furthermore, significantly reduced binding of p50/p65 and IFN regulatory factor 3 to the IFN-λ1 promoter was observed in sIL-6R knockout cells compared with the control cells. Interestingly, a novel heterodimer of c-Fos and activating transcription factor 1 was identified as a crucial transcriptional activator of IFN-λ1 The sIL-6R/p28 FP upregulated IFN-λ1 expression by increasing the binding abilities of c-Fos and activating transcription factor 1 to the IFN-λ1 promoter via the p38 MAPK signaling pathway. In conclusion, these results demonstrate the important role of sIL-6R/p28 FP in mediating virus-induced type III IFN production.

Inducible Major Vault Protein Plays a Pivotal Role in Double-Stranded RNA- or Virus-Induced Proinflammatory Response.

Pathogen invasion triggers robust antiviral cytokine production via different transcription factor signaling pathways. We have previously demonstrated that major vault protein (MVP) induces type I IFN production during viral infection; however, little is known about the role of MVP in proinflammatory responses. In this study, we found in vitro that expression of MVP, IL-6, and IL-8 was inducible upon dsRNA stimulation or viral infection. Moreover, MVP was essential for the induction of IL-6 and IL-8, as impaired expression of IL-6 and IL-8 in MVP-deficient human PBMCs, human lung epithelial cells (A549), and THP-1 monocytes, as well as in murine splenocytes, peritoneal macrophages, and PBMCs from MVP-knockout (MVP(-/-)) mice, was observed. Upon investigation of the underlying mechanisms, we demonstrated that MVP acted in synergy with AP-1 (c-Fos) and CCAAT/enhancer binding protein (C/EBP)ß-liver-enriched transcriptional activating protein to activate the IL6 and IL8 promoters. Introduction of mutations into the AP-1 and C/EBPß binding sites on the IL6 and IL8 promoters resulted in the loss of synergistic activation with MVP. Furthermore, we found that MVP interacted with both c-Fos and C/EBPß. The interactions promoted nuclear translocation and recruitment of these transcription factors to IL6 and IL8 promoter regions. In the MVP(-/-) mouse model, significantly decreased expression of early antiviral cytokines resulted in higher viral titer in the lung, higher mortality, and heavier lung damage after infection with lethal influenza A virus. Taken together, our findings help to delineate a novel role of MVP in host proinflammatory response.

Inducible CYP4F12 enhances Hepatitis C virus infection via association with viral nonstructural protein 5B.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) functions as an RNA-dependent RNA polymerase in the HCV replication complex derived from the endoplasmic reticulum in hepatic cells. In this study, NS5B was used as bait in a yeast two-hybrid assay to screen a human liver cDNA library. We confirmed that CYP4F12, a member of the cytochrome P450 superfamily, interacted with NS5B. Furthermore, overexpression of CYP4F12 facilitated HCV replication. In contrast, knockdown of CYP4F12 by specific shRNA decreased HCV replication and viral protein expression. Moreover, our results demonstrated that HCV infection increased the binding of the transcription factor SREBP1 to the CYP4F12 promoter and activated the promoter activity, which indicated that HCV infection increased the expression of CYP4F12 through the SREBP1 pathway. Our results showed that HCV infection induced expression of CYP4F12 protein, which bound to the HCV replication complex to facilitate viral replication.

Human hepatitis B virus surface and e antigens inhibit major vault protein signaling in interferon induction pathways.

BACKGROUND & AIMS: We previously demonstrated that major vault protein (MVP) is a novel virus-induced host factor and its expression upregulates type-I interferon production, leading to cellular antiviral response. However, it remains unclear whether the antiviral function of MVP is impaired during hepatitis B virus (HBV) infection and what mechanisms are involved. Therefore, the aim of this study was to assess whether HBV can alter MVP expression despite the lack of type-I IFN induction and shed light on the underlying mechanisms HBV utilizes to evade host innate immune response. METHODS: The ability of HBV surface and e antigens to inhibit MVP signaling in interferon induction pathways was evaluated by co-immunoprecipitation, immunofluorescence, quantitative RT-PCR, Western blot and reporter assays. RESULTS: In our current study, we found high levels of MVP in peripheral blood mononuclear cells, sera, and liver tissue from HBV-infected patients relative to healthy individuals. We determined that MVP intracellularly associates with MyD88, an adapter protein involved in virus-triggered induction of type-I IFN. Protein truncation analysis revealed that the middle domain of MVP (amino acid residues 310-620) was essential for MyD88 binding. Conversely, HBV inhibited MVP-induced type-I IFN production by suppressing MVP/MyD88 interaction. HBV antigens, both HBsAg and HBeAg, suppressed this interaction by competitively binding to the essential MyD88 binding region of MVP and limiting downstream IFN signaling. CONCLUSIONS: MVP is a virus-induced protein capable of binding with MyD88 leading to type-I IFN production. HBV may evade an immune response by disrupting this interaction and limiting type-I IFN antiviral activity.

An unexpected role for the ketogenic diet in triggering tumor metastasis by modulating BACH1-mediated transcription.

We have found that the ketogenic (Keto) diet is able to, unexpectedly, promote the metastatic potential of cancer cells in complementary mouse models. Notably, the Keto diet-induced tumor metastasis is dependent on BTB domain and CNC homolog 1 (BACH1) and its up-regulation of pro-metastatic targets, including cell migration-inducing hyaluronidase 1, in response to the Keto diet. By contrast, upon genetic knockout or pharmacological inhibition of endogenous BACH1, the Keto diet-mediated activation of those targets is largely diminished, and the effects on tumor metastasis are completely abolished. Mechanistically, upon administration of the Keto diet, the levels of activating transcription factor 4 (ATF4) are markedly induced. Through direct interaction with BACH1, ATF4 is recruited to those pro-metastatic target promoters and enhances BACH1-mediated transcriptional activation. Together, these data implicate a distinct transcription regulatory program of BACH1 for tumor metastasis induced by the Keto diet. Our study also raises a potential health risk of the Keto diet in human patients with cancer.

Deciphering the acetylation code of p53 in transcription regulation and tumor suppression.

Although it is well-established that p53-mediated tumor suppression mainly acts through its ability in transcriptional regulation, the molecular mechanisms of this regulation are not completely understood. Among a number of regulatory modes, acetylation of p53 attracts great interests. p53 was one of the first non-histone proteins found to be functionally regulated by acetylation and deacetylation, and subsequent work has established that reversible acetylation is a general mechanism for regulation of non-histone proteins. Unlike other types of posttranslational modifications occurred during stress responses, the role of p53 acetylation has been recently validated in vivo by using the knock-in mice with both acetylation-defective and acetylation-mimicking p53 mutants. Here, we review the role of acetylation in p53-mediated activities, with a focus on which specific acetylation sites are critical for p53-dependent transcription regulation during tumor suppression and how acetylation of p53 recruits specific "readers" to execute its promoter-specific regulation of different targets. We also discuss the role of p53 acetylation in differentially regulating its classic activities in cell cycle arrest, senescence and apoptosis as well as newly identified unconventional functions such as cell metabolism and ferroptosis.

Methylcrotonoyl-CoA carboxylase 1 potentiates RLR-induced NF-κB signaling by targeting MAVS complex.

RNA virus infections are detected by the RIG-I family of receptors, which signal through the adaptor molecule mitochondrial antiviral signaling (MAVS). MAVS then recruits the adaptor's tumor necrosis factor receptor-associated factor (TRAF) 3 and TRAF6, which in turn activate IRF3 and NF-κB, respectively, to induce interferons (IFNs) and inflammatory responses. Here we show that the biotin-containing enzyme methylcrotonoyl-CoA carboxylase 1 (MCCC1) enhances virus-induced, MAVS-mediated IFN and inflammatory cytokine expression through the NF-κB signaling pathway. MCCC1 knockdown strongly inhibits induction of IFNs and inflammatory cytokines. Furthermore, MCCC1 shows extensive antiviral activity toward RNA viruses, including influenza A virus, human enterovirus 71, and vesicular stomatitis virus. Here, we have elucidated the mechanism underlying MCCC1-mediated inhibition of viral replication. MCCC1 interacts with MAVS and components of the MAVS signalosome and contributes to enhanced production of type I IFNs and pro-inflammatory cytokines by promoting phosphorylation of the IκB kinase (IKK) complex and NF-κB inhibitor-α (IκBα), as well as NF-κB nuclear translocation. This process leads to activation of IFNs and cytokine expression and subsequent activation of IFN-stimulated genes, including double-stranded RNA-dependent protein kinase PKR and myxovirus resistance protein 1. These findings demonstrate that MCCC1 plays an essential role in virus-triggered, MAVS-mediated activation of NF-κB signaling.