Tripartite Motif Protein 25 Research Articles

Hantaan virus inhibits type I interferon response by targeting RLR signaling pathways through TRIM25

Hantaan virus (HTNV) is responsible for hemorrhagic fever with renal syndrome (HFRS), primarily due to its ability to inhibit host innate immune responses, such as type I interferon (IFN-I). In this study, we conducted a transcriptome analysis to identify host factors regulated by HTNV nucleocapsid protein (NP) and glycoprotein. Our findings demonstrate that NP and Gc proteins inhibit host IFN-I production by manipulating the retinoic acid-induced gene I (RIG-I)-like receptor (RLR) pathways. Further analysis reveals that HTNV NP and Gc proteins target upstream molecules of MAVS, such as RIG-I and MDA-5, with Gc exhibiting stronger inhibition of IFN-I responses than NP. Mechanistically, NP and Gc proteins interact with tripartite motif protein 25 (TRIM25) to competitively inhibit its interaction with RIG-I/MDA5, suppressing RLR signaling pathways. Our study unveils a cross-talk between HTNV NP/Gc proteins and host immune response, providing valuable insights into the pathogenic mechanism of HTNV.

Interleukin-22 facilitates the interferon-λ-mediated production of tripartite motif protein 25 to inhibit replication of duck viral hepatitis A virus type 1

The innate immune system provides a defense against invading pathogens by inducing various interferon (IFN)-stimulated genes (ISGs). We recently reported that tripartite motif protein 25 (TRIM25), an important ISG, was highly upregulated in duck embryo hepatocyte cells (DEFs) after infection with duck viral hepatitis A virus type 1 (DHAV-1). However, the mechanism of upregulation of TRIM25 remains unknown. Here we reported that interleukin-22 (IL-22), whose expression was highly facilitated in DEFs and various organs of 1-day-old ducklings after DHAV-1 infection, highly enhanced the IFN-λ-induced production of TRIM25. The treatment with IL-22 neutralizing antibody or the overexpression of IL-22 highly suppressed or facilitated TRIM25 expression, respectively. The phosphorylation of signal transducer and activator of transcription 3 (STAT3) was crucial for the process of IL-22 enhancing IFN-λ-induced TRIM25 production, which was suppressed by WP1066, a novel inhibitor of STAT3 phosphorylation. The overexpression of TRIM25 in DEFs resulted in a high production of IFNs and reduced DHAV-1 replication, whereas the attenuated expression of IFNs and facilitated replication of DHAV-1 were observed in the RNAi group, implying that TRIM25 defended the organism against DHAV-1 propagation by inducing the production of IFNs. In summary, we reported that IL-22 activated the phosphorylation of STAT3 to enhance the IFN-λ-mediated TRIM25 expression and provide a defense against DHAV-1 by inducing IFN production.

Expression of Zinc-Finger Antiviral Protein in hCMEC/D3 Human Cerebral Microvascular Endothelial Cells: Effect of a Toll-Like Receptor 3 Agonist

Introduction: Invasion of viruses into the brain causes viral encephalitis, which can be fatal and causes permanent brain damage. The blood-brain barrier (BBB) protects the brain by excluding harmful substances and microbes. Brain microvascular endothelial cells are important components of the BBB; however, the mechanisms of antiviral reactions in these cells have not been fully elucidated. Zinc-finger antiviral protein (ZAP) is a molecule that restricts the infection of various viruses, and there are 2 major isoforms: ZAPL and ZAPS. Toll-like receptor 3 (TLR3), a pattern-recognition receptor against viral double-stranded RNA, is implicated in antiviral innate immune reactions. The aim of this study was to investigate the expression of ZAP in cultured hCMEC/D3 human brain microvascular endothelial cells treated with an authentic TLR3 agonist polyinosinic-polycytidylic acid (poly IC). Methods: hCMEC/D3 cells were cultured and treated with poly IC. Expression of ZAPL and ZAPS mRNA was investigated using quantitative reverse transcription-polymerase chain reaction, and protein expression of these molecules was examined using western blotting. The role of nuclear factor-κB (NF-κB) was examined using the NF-κB inhibitor, SN50. The roles of interferon (IFN)-β, IFN regulatory factor 3 (IRF3), tripartite motif protein 25 (TRIM25), and retinoic acid-inducible gene-I (RIG-I) in poly IC-induced ZAPS expression were examined using RNA interference. Propagation of Japanese encephalitis virus (JEV) was examined using a focus-forming assay. Results: ZAPS mRNA and protein expression was upregulated by poly IC, whereas the change of ZAPL mRNA and protein levels was minimal. Knockdown of IRF3 or TRIM25 decreased the poly IC-induced upregulation of ZAPS, whereas knockdown of IFN-β or RIG-I did not affect ZAPS upregulation. SN50 did not affect ZAPS expression. Knockdown of ZAP enhanced JEV propagation. Conclusion: ZAPL and ZAPS were expressed in hCMEC/D3 cells, and ZAPS expression was upregulated by poly IC. IRF3 and TRIM25 are involved in poly IC-induced upregulation of ZAPS. ZAP may contribute to antiviral reactions in brain microvascular endothelial cells and protect the brain from invading viruses such as JEV.

TRIM25 and DEAD-Box RNA Helicase DDX3X Cooperate to Regulate RIG-I-Mediated Antiviral Immunity.

The cytoplasmic retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiate interferon (IFN) production and antiviral gene expression in response to RNA virus infection. Consequently, RLR signalling is tightly regulated by both host and viral factors. Tripartite motif protein 25 (TRIM25) is an E3 ligase that ubiquitinates multiple substrates within the RLR signalling cascade, playing both ubiquitination-dependent and -independent roles in RIG-I-mediated IFN induction. However, additional regulatory roles are emerging. Here, we show a novel interaction between TRIM25 and another protein in the RLR pathway that is essential for type I IFN induction, DEAD-box helicase 3X (DDX3X). In vitro assays and knockdown studies reveal that TRIM25 ubiquitinates DDX3X at lysine 55 (K55) and that TRIM25 and DDX3X cooperatively enhance IFNB1 induction following RIG-I activation, but the latter is independent of TRIM25’s catalytic activity. Furthermore, we found that the influenza A virus non-structural protein 1 (NS1) disrupts the TRIM25:DDX3X interaction, abrogating both TRIM25-mediated ubiquitination of DDX3X and cooperative activation of the IFNB1 promoter. Thus, our results reveal a new interplay between two RLR-host proteins that cooperatively enhance IFN-β production. We also uncover a new and further mechanism by which influenza A virus NS1 suppresses host antiviral defence.

Ectopic Expression of TRIM25 Restores RIG-I Expression and IFN Production Reduced by Multiple Enteroviruses 3Cpro.

Enteroviruses (EVs) 3C proteins suppress type I interferon (IFN) responses mediated by retinoid acid-inducible gene I (RIG-I), while an E3 ubiquitin ligase, tripartite motif protein 25 (TRIM25)-mediated RIG-I ubiquitination is essential for RIG-I antiviral activity. Therefore, whether the effect of EVs 3C on RIG-I is associated with TRIM25 expression is worth to be further investigated. Here, we demonstrate that 3C proteins of EV71 and coxsackievirus B3 (CVB3) reduced not only RIG-I expression but also TRIM25 expression through protease cleavage activity, while overexpression of TRIM25 restored RIG-I expression and IFN-β production reduced by 3C proteins. Further investigation confirmed that the two amino acids and functional domains in TRIM25 required for RIG-I ubiquitination and TRIM25 structural conformation were essential for the recovery of RIG-I expression. Moreover, we also observed that TRIM25 could rescue RIG-I expression reduced by 3C proteins of CVA6 and EV-D68 but not CVA16. Our findings provide an insightful interpretation of 3C-mediated host innate immune suppression and support TRIM25 as an attractive target against multiple EVs infection.

Tripartite motif protein 25 is associated with epirubicin resistance in hepatocellular carcinoma cells via regulating PTEN/AKT pathway.

Tripartite motif protein 25 (TRIM25) expression was altered in various human cancers. Herein, we found that the expression of TRIM25 was elevated in hepatocellular carcinoma (HCC) tissues and cell lines. Knockdown of TRIM25 increased the sensitivity of HCC HepG2 cells to epirubicin (EPI), as indicated by reduced cell viability, enhanced cell apoptosis, and downregulated P-glycoprotein (P-gp) and multiple drug-resistance protein 1 (MRP1). Moreover, TRIM25 knockdown strengthened the effects of EPI on phosphatase and tensin homolog (PTEN) and phosphorylated (p)-AKT. However, overexpression of TRIM25 exerted an opposite effect, weakening the sensitivity of Huh7 to EPI, and obviously increasing PTEN and reducing p-AKT. Most important, all the changes induced by TRIM25 overexpression in Huh7 were reversed with additional treatment of LY294002 (an AKT pathway inhibitor). Notably, coimmunoprecipitation experiments confirmed the interaction between TRIM25 and PTEN. Knockdown of TRIM25 resulted in reduced ubiquitination of PTEN protein. Collectively, our data suggested that TRIM25 enhanced EPI resistance via modulating PTEN/AKT pathway, and targeting TRIM25 may enhance the sensitivity of HCC cells toward chemotherapy drugs.

Nucleocapsid protein of porcine reproductive and respiratory syndrome virus antagonizes the antiviral activity of TRIM25 by interfering with TRIM25-mediated RIG-I ubiquitination

Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV), and is characterized by respiratory diseases in piglet and reproductive disorders in sow. Identification of sustainable and effective measures to mitigate PRRSV transmission is a pressing problem. The nucleocapsid (N) protein of PRRSV plays a crucial role in inhibiting host innate immunity during PRRSV infection. In the current study, a new host-restricted factor, tripartite motif protein 25 (TRIM25), was identified as an inhibitor of PRRSV replication. Co-immunoprecipitation assay indicated that the PRRSV N protein interferes with TRIM25–RIG-I interactions by competitively interacting with TRIM25. Furthermore, N protein inhibits the expression of TRIM25 and TRIM25-mediated RIG-I ubiquitination to suppress interferon β production. Furthermore, with increasing TRIM25 expression, the inhibitory effect of N protein on the ubiquitination of RIG-I diminished. These results indicate for the first time that TRIM25 inhibits PRRSV replication and that the N protein antagonizes the antiviral activity by interfering with TRIM25-mediated RIG-I ubiquitination. This not only provides a theoretical basis for the development of drugs to control PRRSV replication, but also better explains the mechanism through which the PRRSV N protein inhibits innate immune responses of the host.

Positive regulatory role of c-Src-mediated TRIM25 tyrosine phosphorylation on RIG-I ubiquitination and RIG-I-mediated antiviral signaling pathway

Retinoic acid-inducible gene I (RIG-I) detects viral RNAs and induces antiviral responses. During viral RNA recognition by RIG-I, tripartite motif protein 25 (TRIM25) plays a critical regulatory role by inducing K63-linked RIG-I polyubiquitination. Previous proteomics analysis revealed several phosphorylation sites on TRIM25, including tyrosine 278 (Y278), yet the roles of these modifications remain elusive. Here, we demonstrated that TRIM25 interacted with c-Src and underwent tyrosine phosphorylation by c-Src kinase upon viral infection and the phosphorylation is required for the complete activation of RIG-I signaling. Analysis using a c-Src inhibitor and TRIM25 mutant, in which tyrosine 278 is substituted by phenylalanine (Y278F), suggested that the phosphorylation positively regulates K63-linked polyubiquitination of RIG-I and subsequent antiviral signaling. The TRIM25 Y278F mutant displayed decreased E3-ubiquitin ligase activity in vitro, suggesting that this phosphorylation event affects the E3-ligase activity of TRIM25. Thus, we provide a molecular mechanism of c-Src-mediated positive regulation of RIG-I signaling.

TRIM25 is associated with cisplatin resistance in non-small-cell lung carcinoma A549 cell line via downregulation of 14-3-3σ

Lung cancer, in particular, non-small cell lung cancer (NSCLC), is the leading cause of cancer-related mortality. Cis-Diamminedichloroplatinum (cisplatin, CDDP) as first-line chemotherapy for NSCLC, but resistance occurs frequently. We previously reported that Tripartite motif protein 25 (TRIM25) was highly expressed in cisplatin-resistant human lung adenocarcinoma A549 cells (A549/CDDP) in comparison with its parental A549 cells. Herein, we take a further step to demonstrate the association of TRIM25 and cisplatin resistance and also the underlying mechanisms. Knockdown of TRIM25 by RNA interference in A549/CDDP cells decreased half maximal inhibitory concentration (IC50) values and promoted apoptosis in response to cisplatin, whereas overexpression of TRIM25 had opposite effects. More importantly, we found that concomitant knockdown of 14-3-3σ and TRIM25 absolutely reversed the decreased MDM2, increased p53, increased cleaved-Capsese3 and decreased IC50 value induced by knockdown of TRIM25 individually, suggesting that TRIM25 mediated cisplatin resistance primarily through downregulation of 14-3-3σ. Our results indicate that TRIM25 is associated with cisplatin resistance and 14-3-3σ-MDM2-p53 signaling pathway is involved in this process, suggesting targeting TRIM25 may be a potential strategy for the reversal of cisplatin resistance.

The Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Inhibits Type I Interferon Production by Interfering with TRIM25-Mediated RIG-I Ubiquitination.

Severe acute respiratory syndrome (SARS) is a respiratory disease, caused by a coronavirus (SARS-CoV), that is characterized by atypical pneumonia. The nucleocapsid protein (N protein) of SARS-CoV plays an important role in inhibition of type I interferon (IFN) production via an unknown mechanism. In this study, the SARS-CoV N protein was found to bind to the SPRY domain of the tripartite motif protein 25 (TRIM25) E3 ubiquitin ligase, thereby interfering with the association between TRIM25 and retinoic acid-inducible gene I (RIG-I) and inhibiting TRIM25-mediated RIG-I ubiquitination and activation. Type I IFN production induced by poly I·C or Sendai virus (SeV) was suppressed by the SARS-CoV N protein. SARS-CoV replication was increased by overexpression of the full-length N protein but not N amino acids 1 to 361, which could not interact with TRIM25. These findings provide an insightful interpretation of the SARS-CoV-mediated host innate immune suppression caused by the N protein.IMPORTANCE The SARS-CoV N protein is essential for the viral life cycle and plays a key role in the virus-host interaction. We demonstrated that the interaction between the C terminus of the N protein and the SPRY domain of TRIM25 inhibited TRIM25-mediated RIG-I ubiquitination, which resulted in the inhibition of IFN production. We also found that the Middle East respiratory syndrome CoV (MERS-CoV) N protein interacted with TRIM25 and inhibited RIG-I signaling. The outcomes of these findings indicate the function of the coronavirus N protein in modulating the host's initial innate immune response.

Disturbed P53-MDM2 Feedback Loop Contributes to Thoracic Aortic Dissection Formation and May be a Result of TRIM25 Overexpression

The development of thoracic aortic dissection (TAD) is attributed to a broad range of degenerative, genetic, structural, oxidative, apoptotic, and acquired disease states. In this study, we examined the role of the disturbed p53-MDM2 (murine double minute 2) feedback loop in the formation of TAD, and one of a potential feedback loop regulator, TRIM25 (tripartite motif protein-25). Surgical specimens of the aorta from TAD patients (n=10) and controls (n=10) were tested for α-smooth muscle actin (α-SMA), p53, MDM2, and TRIM25 by western blot, immunohistochemical staining, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), respectively. When compared with controls, western blot shows that the protein levels of p53, MDM2, and TRIM25 were increased significantly in the aortic media of TAD patients. qRT-PCR further verified that the mRNA expression of MDM2 and TRIM25 was also increased 6- and 4-folds, respectively, in the TAD media of the aortic wall. Immunohistochemistry results showed significantly decreased staining of α-SMA, smooth muscle cells, and more collagen deposition in the media of the aortic wall from patients with TAD. This study provided a new insight into the disturbed p53-MDM2 feedback loop in the pathogenesis of TAD, and this may be because of the TRIM25 overexpression.

Selenocysteine as a Latent Bioorthogonal Electrophilic Probe for Deubiquitylating Enzymes.

Deubiquitylating enzymes (DUBs) remove ubiquitin (Ub) from various cellular proteins and render eukaryotic ubiquitylation a dynamic process. The misregulation of protein ubiquitylation is associated with many human diseases, and there is an urgent need to identify specific DUBs associated with therapeutically relevant targets of Ub. We report the development of two facile selenocysteine-based strategies to generate the DUB probe dehydroalanine (Dha). Optimized oxidative or alkylative elimination of Se yielded Dha at the C-terminus of Ub. The high utility of alkylative elimination, which is compatible with multiple thiols in Ub targets, was demonstrated by generating a probe derived from the Ub ligase tripartite motif protein 25 (TRIM-25). Successful capture of the TRIM-25-associated DUB, ubiquitin-specific protease 15, demonstrated the versatility of our chemical strategy for identifying target-specific DUBs.

Regulation of MDA5-MAVS Antiviral Signaling Axis by TRIM25 through TRAF6-Mediated NF-κB Activation.

Tripartite motif protein 25 (TRIM25), mediates K63-linked polyubiquitination of Retinoic acid inducible gene I (RIG-I) that is crucial for downstream antiviral interferon signaling. Here, we demonstrate that TRIM25 is required for melanoma differentiation-associated gene 5 (MDA5) and MAVS mediated activation of NF-κB and interferon production. TRIM25 is required for the full activation of NF-κB at the downstream of MAVS, while it is not involved in IRF3 nuclear translocation. Mechanical studies showed that TRIM25 is involved in TRAF6-mediated NF-κB activation. These collectively indicate that TRIM25 plays an additional role in RIG-I/MDA5 signaling other than RIG-I ubiquitination via activation of NF-κB.

Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein

RIG-I (retinoic acid inducible gene I)-mediated antiviral signaling serves as the first line of defense against viral infection. Upon detection of viral RNA, RIG-I undergoes TRIM25 (tripartite motif protein 25)-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that TRK-fused gene (TFG) protein, previously identified as a TRIM25-interacting protein, binds TRIM25 upon virus infection and negatively regulates RIG-I-mediated type-I IFN signaling. RIG-I-mediated IFN production and nuclear factor (NF)-κB signaling pathways were upregulated by the suppression of TFG expression. Furthermore, vesicular stomatitis virus (VSV) replication was significantly inhibited by small inhibitory hairpin RNA (shRNA)-mediated knockdown of TFG, supporting the suppressive role of TFG in RIG-I-mediated antiviral signaling. Interestingly, suppression of TFG expression increased not only RIG-I-mediated signaling but also MAVS (mitochondrial antiviral signaling protein)-induced signaling, suggesting that TFG plays a pivotal role in negative regulation of RNA-sensing, RIG-I-like receptor (RLR) family signaling pathways.

Molecular characterization of tripartite motif protein 25 (TRIM25) involved in ERα-mediated transcription in the Korean rose bitterling Rhodeus uyekii

Tripartite motif-containing 25 (TRIM25), also known as estrogen-responsive finger protein (EFP), plays an essential role in cell proliferation and innate immunity. In the present study, we isolated and characterized the TRIM25 cDNA of the Korean rose bitterling Rhodeus uyekii, designated RuTRIM25. It encodes an open reading frame of 669 amino acids containing an N-terminal RBCC motif composed of a RING domain, two B boxes, and a coiled-coil domain and a C-terminal B30.2 (PRY/SPRY) domain. RuTRIM25 shows strong homology (79.7%) to zebrafish TRIM25 and shared 32.4-28.8% homology with TRIM25 from other species, including mammals. RuTRIM25 mRNA was expressed ubiquitously. It was highly expressed in the ovary, spleen, and liver and moderately in the stomach and intestine of normal Korean rose bitterling. The intracellular localization of RuTRIM25 in HEK293T cells was diffusely localized in the cytoplasm and its RING domain deletion mutant (RuTRIM25ΔR) was detected diffusely with some aggregates in the cytoplasm. RuTRIM25, but not RuTRIM25ΔR, is ubiquitinated in vivo. Ectopic expression of RuTRIM25 synergistically activated the estrogen receptor (ER)-mediated luciferase reporter activity in a dose-dependent manner in HEK293T cells. Together, these results suggest that the RuTRIM25 regulates the ER-mediated transcription in fish similarly to its mammalian counterpart.

Negative Role of RIG-I Serine 8 Phosphorylation in the Regulation of Interferon-β Production

RIG-I (retinoic acid-inducible gene I) and TRIM25 (tripartite motif protein 25) have emerged as key regulatory factors to induce interferon (IFN)-mediated innate immune responses to limit viral replication. Upon recognition of viral RNA, TRIM25 E3 ligase binds the first caspase recruitment domain (CARD) of RIG-I and subsequently induces lysine 172 ubiquitination of the second CARD of RIG-I, which is essential for the interaction with downstream MAVS/IPS-1/CARDIF/VISA and, thereby, IFN-β mRNA production. Although ubiquitination has emerged as a major factor involved in RIG-I activation, the potential contribution of other post-translational modifications, such as phosphorylation, to the regulation of RIG-I activity has not been addressed. Here, we report the identification of serine 8 phosphorylation at the first CARD of RIG-I as a negative regulatory mechanism of RIG-I-mediated IFN-β production. Immunoblot analysis with a phosphospecific antibody showed that RIG-I serine 8 phosphorylation steady-state levels were decreased upon stimulation of cells with IFN-β or virus infection. Substitution of serine 8 in the CARD RIG-I functional domain with phosphomimetic aspartate or glutamate results in decreased TRIM25 binding, RIG-I ubiquitination, MAVS binding, and downstream signaling. Finally, sequence comparison reveals that only primate species carry serine 8, whereas other animal species carry an asparagine, indicating that serine 8 phosphorylation may represent a primate-specific regulation of RIG-I activation. Collectively, these data suggest that the phosphorylation of RIG-I serine 8 operates as a negative switch of RIG-I activation by suppressing TRIM25 interaction, further underscoring the importance of RIG-I and TRIM25 connection in type I IFN signal transduction.

Phosphorylation-Mediated Negative Regulation of RIG-I Antiviral Activity

Recognition of invading viruses by the host is elicited by cellular sensors which trigger signaling cascades that lead to type I interferon (IFN) gene expression. Retinoic acid-inducible gene I (RIG-I) has emerged as a key receptor for the detection of viral RNA in the cytosol, inducing IFN-mediated innate immune responses to limit viral replication through its interaction with MAVS (also called IPS-1, CARDIF, or VISA). Upon the recognition of viral RNA, the Lys-172 residue of RIG-I undergoes ubiquitination induced by tripartite motif protein 25 (TRIM25), an essential protein for antiviral signal transduction. Here we demonstrate that phosphorylation represents another regulatory mechanism for RIG-I-mediated antiviral activity. Using protein purification and mass spectrometry analysis, we identified three phosphorylation sites in the amino-terminal caspase recruitment domains (CARDs) of RIG-I. One of these residues, Thr-170, is located in close proximity to Lys-172, and we speculated that its phosphorylation may affect Lys-172 ubiquitination and functional activation of RIG-I. Indeed, a RIG-I mutant carrying a phosphomimetic Glu residue in place of Thr-170 loses TRIM25 binding, Lys-172 ubiquitination, MAVS binding, and downstream signaling ability. This suggests that phosphorylation of RIG-I at Thr-170 inhibits RIG-I-mediated antiviral signal transduction. Immunoblot analysis with a phospho-specific antibody showed that the phosphorylation of the RIG-I Thr-170 residue is present under normal conditions but rapidly declines upon viral infection. Our results indicate that Thr-170 phosphorylation and TRIM25-mediated Lys-172 ubiquitination of RIG-I functionally antagonize each other. While Thr-170 phosphorylation keeps RIG-I latent, Lys-172 ubiquitination enables RIG-I to form a stable complex with MAVS, thereby inducing IFN signal transduction.

Actin and RIG-I/MAVS Signaling Components Translocate to Mitochondria upon Influenza A Virus Infection of Human Primary Macrophages

Influenza A virus is one of the most important causes of respiratory infection. During viral infection, multiple cell signaling cascades are activated, resulting in the production of antiviral cytokines and initiation of programmed cell death of virus-infected cells. In the present study, we have used subcellular proteomics to reveal the host response to influenza A infection at the protein level in human macrophages. Macrophages were infected with influenza A virus, after which the cytosolic and mitochondrial cell fractions were prepared and analyzed by using two-dimensional electrophoresis for protein separation and mass spectrometry for protein identification. In cytosolic proteomes, the level of several heat shock proteins and fragments of cytoskeletal proteins was clearly up-regulated during influenza A virus infection. In mitochondrial proteomes, simultaneously with the expression of viral proteins, the level of intact actin and tubulin was highly up-regulated. This was followed by translocation of the components of antiviral RNA recognition machinery, including RIG-I (retinoic acid-inducible protein I), TRADD (TNFR1-associated death domain protein), TRIM25 (tripartite motif protein 25), and IKKepsilon (inducible IkappaB kinase), onto the mitochondria. Cytochalasin D, a potent inhibitor of actin polymerization, clearly inhibited influenza A virus-induced expression of IFN-beta, IL-29, and TNF-alpha, suggesting that intact actin cytoskeleton structure is crucial for proper activation of antiviral response. At late phases of infection mitochondrial fragmentation of actin was seen, indicating that actin fragments, fractins, are involved in disruption of mitochondrial membranes during apoptosis of virus-infected cells. In conclusion, our results suggest that actin network interacts with mitochondria to regulate both antiviral and cell death signals during influenza A virus infection.

Roles of RIG-I N-terminal tandem CARD and splice variant in TRIM25-mediated antiviral signal transduction

The caspase recruitment domain (CARD) of intracellular adaptors and sensors plays a critical role in the assembly of signaling complexes involved in innate host defense against pathogens and in the regulation of inflammatory responses. The cytosolic receptor retinoic acid-inducible gene-I (RIG-I) recognizes viral RNA in a 5'-triphosphate-dependent manner and initiates an antiviral signaling cascade. Upon viral infection, the N-terminal CARDs of RIG-I undergo the K(63)-linked ubiquitination induced by tripartite motif protein 25 (TRIM25), critical for the interaction of RIG-I with its downstream signaling partner MAVS/VISA/IPS-1/Cardif. Here, we demonstrate the distinct roles of RIG-I first and second CARD in TRIM25-mediated RIG-I ubiquitination: TRIM25 binds the RIG-I first CARD and subsequently ubiquitinates its second CARD. The T(55)I mutation in RIG-I first CARD abolishes TRIM25 interaction, whereas the K(172)R mutation in the second CARD eliminates polyubiquitin attachment. The necessity of the intact tandem CARD for RIG-I function is further evidenced by a RIG-I splice variant (SV) whose expression is robustly up-regulated upon viral infection. The RIG-I SV carries a short deletion (amino acids 36-80) within the first CARD and thereby loses TRIM25 binding, CARD ubiquitination, and downstream signaling ability. Furthermore, because of its robust inhibition of virus-induced RIG-I multimerization and RIG-I-MAVS signaling complex formation, this SV effectively suppresses the RIG-I-mediated IFN-beta production. This study not only elucidates the vital role of the intact tandem CARD for TRIM25-mediated RIG-I activation but also identifies the RIG-I SV as an off-switch regulator of its own signaling pathway.