Exosomes mediate Coxsackievirus B3 transmission and expand the viral tropism.

Specific virus-receptor interactions are important determinants in viral host range, tropism and pathogenesis, influencing the location and initiation of primary infection as well as viral spread to other target organs/tissues in the postviremic phase. Coxsackieviruses of Group B (CVB) and its six serotypes (CVB1-6) specifically interact with two receptor proteins, coxsackievirus-adenovirus receptor (CAR) and decay-accelerating factor (DAF), and cause various lesions in most permissive tissues. However, our previous data and other studies revealed that virus receptor-negative cells or tissues can be infected with CVB type 3 (CVB3), which can also effectively replicate. To study this interesting finding, we explored the possibility that exosomes are involved in CVB3 tropism and that exosomes functionally enhance CVB3 transmission. We found that exosomes carried and delivered CVB3 virions, resulting in efficient infection in receptor-negative host cells. We also found that delivery of CVB3 virions attached to exosomes depended on the virus receptor CAR. Importantly, exosomes carrying CVB3 virions exhibited greater infection efficiency than free virions because they accessed various entry routes, overcoming restrictions to viral tropism. In vivo experiments demonstrated that inhibition of exosome coupling with virions attenuated CVB3-induced immunological system dysfunction and reduced mortality. Our study describes a new mechanism in which exosomes contribute to viral tropism, spread, and pathogenesis.

Genome-Wide CRISPR/Cas9 Screening Identifies That Mitochondrial Solute Carrier SLC25A23 Attenuates Type I IFN Antiviral Immunity via Interfering with MAVS Aggregation.

Activation of the mitochondrial antiviral signaling (MAVS) adaptor, also known as IPS-1, VISA, or Cardif, is crucial for antiviral immunity in retinoic acid-inducible gene I (RIG-I)-like receptor signaling. Upon interacting with RIG-I, MAVS undergoes K63-linked polyubiquitination by the E3 ligase Trim31, and subsequently aggregates to activate downstream signaling effectors. However, the molecular mechanisms that modulate MAVS activation are not yet fully understood. In this study, the mitochondrial solute carrier SLC25A23 was found to attenuate type I IFN antiviral immunity using genome-wide CRISPR/Cas9 screening. SLC25A23 interacts with Trim31, interfering with its binding of Trim31 to MAVS. Indeed, SLC25A23 downregulation was found to increase K63-linked polyubiquitination and subsequent aggregation of MAVS, which promoted type I IFN production upon RNA virus infection. Consistently, mice with SLC25A23 knockdown were more resistant to RNA virus infection in vivo. These findings establish SLC25A23 as a novel regulator of MAVS posttranslational modifications and of type I antiviral immunity.

Structural damage in the C. elegans epidermis causes release of STA-2 and induction of an innate immune response.

The epidermis constantly encounters invasions that disrupt its architecture, yet whether the epidermal immune system utilizes damaged structures as danger signals to activate self-defense is unclear. Here, we used a C. elegans epidermis model in which skin-penetrating infection or injury activates immune defense and antimicrobial peptide (AMP) production. By systemically disrupting each architectural component, we found that only disturbance of the apical hemidesmosomes triggered an immune response and robust AMP expression. The epidermis recognized structural damage through hemidesmosomes associated with a STAT-like protein, whose disruption led to detachment of STA-2 molecules from hemidesmosomes and transcription of AMPs. This machinery enabled the epidermis to bypass certain signaling amplification and directly trigger AMP production when subjected to extensive architectural damage. Together, our findings uncover an evolutionarily conserved mechanism for the epithelial barriers to detect danger and activate immune defense.

Cardiomyocyte-derived HMGB1 takes a protective role in CVB3-induced viral myocarditis via inhibiting cardiac apoptosis.

Coxsackievirus B3 (CVB3)-induced viral myocarditis (VMC) is characterized by immune cell infiltration and myocardial damage. High mobility group box 1 (HMGB1) is a highly conserved nuclear DNA-binding protein that participates in DNA replication, transcriptional regulation, repair response and inflammatory response in different disease models. To investigate the exact function of HMGB1 in CVB3-induced VMC, we crossed Hmgb1-floxed (Hmgb1f/f ) mice with mice carrying a suitable Cre recombinase transgenic strain to achieve conditional inactivation of the Hmgb1 gene in a cardiomyocyte-specific manner and to establish myocarditis. In this study, we found that cardiomyocyte-specific Hmgb1-deficient (Hmgb1f/f TgCre/+ ) mice exhibited exacerbated myocardial injury. Hmgb1-deficient cardiomyocytes may promote early apoptosis via the p53-mediated Bax mitochondrial pathway, as evidenced by the higher localization of p53 protein in the cytosol of Hmgb1-deficient cardiomyocytes upon CVB3 infection. Moreover, cardiomyocyte Hmgb1-deficient mice are more susceptible to cardiac dysfunction after infection. This study provides new insights into HMGB1 in VMC pathogenesis and a strategy for appropriate blocking of HMGB1 in the clinical treatment of VMC.

Spider Silk Protein Forms Amyloid-Like Nanofibrils through a Non-Nucleation-Dependent Polymerization Mechanism.

Amyloid fibrils-nanoscale fibrillar aggregates with high levels of order-are pathogenic in some today incurable human diseases; however, there are also many physiologically functioning amyloids in nature. The process of amyloid formation is typically nucleation-elongation-dependent, as exemplified by the pathogenic amyloid-ß peptide (Aß) that is associated with Alzheimer's disease. Spider silk, one of the toughest biomaterials, shares characteristics with amyloid. In this study, it is shown that forming amyloid-like nanofibrils is an inherent property preserved by various spider silk proteins (spidroins). Both spidroins and Aß capped by spidroin N- and C-terminal domains, can assemble into macroscopic spider silk-like fibers that consist of straight nanofibrils parallel to the fiber axis as observed in native spider silk. While Aß forms amyloid nanofibrils through a nucleation-dependent pathway and exhibits strong cytotoxicity and seeding effects, spidroins spontaneously and rapidly form amyloid-like nanofibrils via a non-nucleation-dependent polymerization pathway that involves lateral packing of fibrils. Spidroin nanofibrils share amyloid-like properties but lack strong cytotoxicity and the ability to self-seed or cross-seed human amyloidogenic peptides. These results suggest that spidroins´ unique primary structures have evolved to allow functional properties of amyloid, and at the same time direct their fibrillization pathways to avoid formation of cytotoxic intermediates.

Harnessing ADAR-Mediated Site-Specific RNA Editing in Immune-Related Disease: Prediction and Therapeutic Implications.

ADAR (Adenosine Deaminases Acting on RNA) proteins are a group of enzymes that play a vital role in RNA editing by converting adenosine to inosine in RNAs. This process is a frequent post-transcriptional event observed in metazoan transcripts. Recent studies indicate widespread dysregulation of ADAR-mediated RNA editing across many immune-related diseases, such as human cancer. We comprehensively review ADARs' function as pattern recognizers and their capability to contribute to mediating immune-related pathways. We also highlight the potential role of site-specific RNA editing in maintaining homeostasis and its relationship to various diseases, such as human cancers. More importantly, we summarize the latest cutting-edge computational approaches and data resources for predicting and analyzing RNA editing sites. Lastly, we cover the recent advancement in site-directed ADAR editing tool development. This review presents an up-to-date overview of ADAR-mediated RNA editing, how site-specific RNA editing could potentially impact disease pathology, and how they could be harnessed for therapeutic applications.

USP39 Serves as a Deubiquitinase to Stabilize STAT1 and Sustains Type I IFN-Induced Antiviral Immunity.

Deubiquitinating enzymes (DUBs) are cysteine proteases that reverse the ubiquitination by removing ubiquitins from the target protein. The human genome encodes ∼100 potential DUBs, which can be classified into six families, influencing multiple cellular processes, such as antiviral responses, inflammatory responses, apoptosis, etc. To systematically explore the role of DUBs involved in antiviral immunity, we performed an RNA interference-based screening that contains 97 human DUBs. We identified that ubiquitin-specific protease (USP) 39 expression modulates the antiviral activity, which is, to our knowledge, a previously unknown function of this enzyme. Small interfering RNA knockdown of USP39 significantly enhanced viral replication, whereas overexpression of USP39 had an opposite effect. Mechanistically, USP39 does not affect the production of type I IFN but significantly promotes JAK/STAT downstream of type I signaling by enhancing IFN-stimulated response elements promoter activity and expression of IFN-stimulated genes. Interestingly, USP39, previously considered not to have the deubiquitinase activity, in this study is proved to interact with STAT1 and sustain its protein level by deubiqutination. Furthermore, we found that through novel mechanism USP39 can significantly decrease K6-linked but not K48-linked ubiquitination of STAT1 for degradation. Taken together, these findings uncover that USP39 is, to our knowledge, a new deubiquitinase that positively regulates IFN-induced antiviral efficacy.

Nuclear Sensor Interferon-Inducible Protein 16 Inhibits the Function of Hepatitis B Virus Covalently Closed Circular DNA by Integrating Innate Immune Activation and Epigenetic Suppression.

BACKGROUND AND AIMS: Nuclear-located covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is a determining factor for HBV persistence and the key obstacle for a cure of chronic hepatitis B. However, it remains unclear whether and how the host immune system senses HBV cccDNA and its biological consequences. APPROACH AND RESULTS: Here, we demonstrated that interferon-inducible protein 16 (IFI16) could serve as a unique innate sensor to recognize and bind to HBV cccDNA in hepatic nuclei, leading to the inhibition of cccDNA transcription and HBV replication. Mechanistically, our data showed that IFI16 promoted the epigenetic suppression of HBV cccDNA by targeting an interferon-stimulated response element (ISRE) present in cccDNA. It is of interest that this ISRE was also revealed to play an important role in IFI16-activated type I interferon responses. Furthermore, our data revealed that HBV could down-regulate the expression level of IFI16 in hepatocytes, and there was a negative correlation between IFI16 and HBV transcripts in liver biopsies, suggesting the possible role of IFI16 in suppressing cccDNA function under physiological conditions. CONCLUSIONS: The nuclear sensor IFI16 suppresses cccDNA function by integrating innate immune activation and epigenetic regulation by targeting the ISRE of cccDNA, and IFI16 may present as a therapeutic target against HBV infection.

Consistency of spatial dynamics of HIV-1 and HCV among HIV-1/HCV coinfected drug users in China.

BACKGROUND: As the transmission routes of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) are similar, previous studies based on separate research on HIV-1 and HCV assumed a similar transmission pattern. However, few studies have focused on the possible correlation of the spatial dynamics of HIV-1 and HCV among HIV-1/HCV coinfected patients. METHODS: A total of 310 HIV-1/HCV coinfected drug users were recruited in Yingjiang and Kaiyuan prefectures, Yunnan Province, China. HIV-1 env, p17, pol and HCV C/E2, NS5B fragments were amplified and sequenced from serum samples. The genetic characteristics and spatial dynamics of HIV-1 and HCV were explored by phylogenetic, bootscanning, and phylogeographic analyses. RESULTS: Among HIV-1/HCV coinfected drug users, eight HCV subtypes (1a, 1b, 3a, 3b, 6a, 6n, 6v, and 6u) and two HIV-1 subtypes (subtype B and subtype C), three HIV-1 circulating recombinant forms (CRF01_AE, CRF07_BC and CRF08_BC), and four unique recombinant forms (URF_BC, URF_01B, URF_01C and URF_01BC) were identified. HCV subtype 3b was the most predominant subtype in both Yingjiang and Kaiyuan prefectures. The dominant circulating HIV-1 subtypes for drug users among the two areas were CRF08_BC and URF_BC. Maximum clade credibility trees revealed that both HIV-1 and HCV were transmitted from Yingjiang to Kaiyuan. CONCLUSIONS: The spatial dynamics of HIV-1 and HCV among HIV-1/HCV coinfected drug users seem to have high consistency, providing theoretical evidence for the prevention of HIV-1 and HCV simultaneously.

Non-volatile acylphloroglucinol components from Eucalyptus robusta inhibit Zika virus by impairing RdRp activity of NS5.

Eucalyptus is a large genus of the Myrtaceae family with high value in various fields of industry. Recently, attention has been focused on the functional properties of Eucalyptus extracts. These extracts have been traditionally used to combat various infectious diseases, and volatile oils are usually considered to play a major role. But the positive effects of non-volatile acylphloroglucinols, a class of specialized metabolites with relatively high content in Eucalyptus, should not be neglected. Herein, non-volatile acylphloroglucinols from leaves of Eucalyptus robusta were evaluated for their abilities to inhibit Zika virus (ZIKV) which is associated with severe neurological damage and complications. The results showed eucalyprobusone G, a new symmetrical acylphloroglucinol dimer, possessed the significant ability to inhibit ZIKV without inducing cytotoxicity. The EC50 values of eucalyprobusone G against the African lineage (MR766) and Asian lineage (SZ-WIV01) of ZIKV were 0.43 ± 0.08 and 10.10 ± 3.84 µM which were 110 times and 5.8 times better than those of the reference compound ribavirin, respectively. Further action mode research showed that eucalyprobusone G impairs the viral binding and RdRp activity of NS5. The results broaden the functional properties of Eucalyptus robusta and indicate acylphloroglucinol dimers could be developed as anti-ZIKV agents.

One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies.

BACKGROUND: Spider silk is a proteinaceous fiber with remarkable mechanical properties spun from spider silk proteins (spidroins). Engineering spidroins have been successfully produced in a variety of heterologous hosts and the most widely used expression system is Escherichia coli (E. coli). So far, recombinantly expressed spidroins often form insoluble inclusion bodies (IBs), which will often be dissolved under extremely harsh conditions in a traditional manner, e.g. either 8 mol/L urea or 6 mol/L guanidine hydrochloride, highly risking to poor recovery of bioactive proteins as well as unexpected precipitations during dialysis process. RESULTS: Here, we present a mild solubilization strategy-one-step heating method to solubilize spidroins from IBs, with combining spidroins' high thermal stability with low concentration of urea. A 430-aa recombinant protein (designated as NM) derived from the minor ampullate spidroin of Araneus ventricosus was expressed in E. coli, and the recombinant proteins were mainly present in insoluble fraction as IBs. The isolated IBs were solubilized parallelly by both traditional urea-denatured method and one-step heating method, respectively. The solubilization efficiency of NM IBs in Tris-HCl pH 8.0 containing 4 mol/L urea by one-step heating method was already comparable to that of 7 mol/L urea with using traditional urea-denatured method. The effects of buffer, pH and temperature conditions on NM IBs solubilization of one-step heating method were evaluated, respectively, based on which the recommended conditions are: heating temperature 70-90 °C for 20 min, pH 7.0-10, urea concentration 2-4 mol/L in normal biological buffers. The recombinant NM generated via the one-step heating method held the potential functions with self-assembling into sphere nanoparticles with smooth morphology. CONCLUSIONS: The one-step heating method introduced here efficiently solubilizes IBs under relatively mild conditions compared to the traditional ones, which might be important for the downstream applications; however, this protocol should be pursued carefully in terms of urea-induced modification sensitive applications. Further, this method can be applied under broad buffer, pH and temperature conditions, conferring the potential to apply to other thermal stable proteins.

POM121 inhibits the macrophage inflammatory response by impacting NF-κB P65 nuclear accumulation.

The nuclear pore membrane protein 121 (POM121) was originally thought to be a constitutive protein of the nuclear pore complex (NPC). In addition to being involved in NPC assembly, abnormal POM121 expression has been found to be associated with many diseases. In this study, we explored, in detail, the effect of POM121 on the macrophage inflammatory response and found that its expression was significantly lower in LPS-stimulated macrophages, substantially amplifying pro-inflammatory cytokine (TNF-α and IL-6) production, suggesting that POM121 exerts a potent inhibitory effect on macrophage inflammation. Consistent with this notion, greater susceptibility to LPS-induced acute lung injury (ALI) as well as more severe tissue inflammation were found in POM121fl/fl Lyzm-Cre+ mice compared to those in control mice, as evidenced by the more severe lung injury and inflammation, increased TNF-α and IL-6 production and more abundant proteins in bronchoalveolar lavage fluid (BALF). This inflammation-modulating effect of POM121 relied on its ability to repress the NF-κB signal pathway via inhibition of phosphorylated P65 (phos-P65) nuclear accumulation. In the present study, we reported that in addition to acting as a constitutive NPC component, POM121 also modulated LPS-induced macrophage inflammation via repressing nuclear P65 translocation. Our study may pave the way for regulating LPS-induced massive macrophage inflammation and providing evidence for the functional diversity of nucleoporins (Nups).

Selective deletion of endothelial cell calpain in mice reduces diabetic cardiomyopathy by improving angiogenesis.

AIMS/HYPOTHESIS: The role of non-cardiomyocytes in diabetic cardiomyopathy has not been fully addressed. This study investigated whether endothelial cell calpain plays a role in myocardial endothelial injury and microvascular rarefaction in diabetes, thereby contributing to diabetic cardiomyopathy. METHODS: Endothelial cell-specific Capns1-knockout (KO) mice were generated. Conditions mimicking prediabetes and type 1 and type 2 diabetes were induced in these KO mice and their wild-type littermates. Myocardial function and coronary flow reserve were assessed by echocardiography. Histological analyses were performed to determine capillary density, cardiomyocyte size and fibrosis in the heart. Isolated aortas were assayed for neovascularisation. Cultured cardiac microvascular endothelial cells were stimulated with high palmitate. Angiogenesis and apoptosis were analysed. RESULTS: Endothelial cell-specific deletion of Capns1 disrupted calpain 1 and calpain 2 in endothelial cells, reduced cardiac fibrosis and hypertrophy, and alleviated myocardial dysfunction in mouse models of diabetes without significantly affecting systemic metabolic variables. These protective effects of calpain disruption in endothelial cells were associated with an increase in myocardial capillary density (wild-type vs Capns1-KO 3646.14 ± 423.51 vs 4708.7 ± 417.93 capillary number/high-power field in prediabetes, 2999.36 ± 854.77 vs 4579.22 ± 672.56 capillary number/high-power field in type 2 diabetes and 2364.87 ± 249.57 vs 3014.63 ± 215.46 capillary number/high-power field in type 1 diabetes) and coronary flow reserve. Ex vivo analysis of neovascularisation revealed more endothelial cell sprouts from aortic rings of prediabetic and diabetic Capns1-KO mice compared with their wild-type littermates. In cultured cardiac microvascular endothelial cells, inhibition of calpain improved angiogenesis and prevented apoptosis under metabolic stress. Mechanistically, deletion of Capns1 elevated the protein levels of ß-catenin in endothelial cells of Capns1-KO mice and constitutive activity of calpain 2 suppressed ß-catenin protein expression in cultured endothelial cells. Upregulation of ß-catenin promoted angiogenesis and inhibited apoptosis whereas knockdown of ß-catenin offset the protective effects of calpain inhibition in endothelial cells under metabolic stress. CONCLUSIONS/INTERPRETATION: These results delineate a primary role of calpain in inducing cardiac endothelial cell injury and impairing neovascularisation via suppression of ß-catenin, thereby promoting diabetic cardiomyopathy, and indicate that calpain is a promising therapeutic target to prevent diabetic cardiac complications.

Scaffold attachment factor B suppresses HIV-1 infection of CD4+ T cells by preventing binding of RNA polymerase II to HIV-1's long terminal repeat.

The 5' end of the HIV, type 1 (HIV-1) long terminal repeat (LTR) promoter plays an essential role in driving viral transcription and productive infection. Multiple host and viral factors regulate LTR activity and modulate HIV-1 latency. Manipulation of the HIV-1 LTR provides a potential therapeutic strategy for combating HIV-1 persistence. In this study, we identified an RNA/DNA-binding protein, scaffold attachment factor B (SAFB1), as a host cell factor that represses HIV-1 transcription. We found that SAFB1 bound to the HIV-1 5' LTR and significantly repressed 5' LTR-driven viral transcription and HIV-1 infection of CD4+ T cells. Mechanistically, SAFB1-mediated repression of HIV-1 transcription and infection was independent of its RNA- and DNA-binding capacities. Instead, by binding to phosphorylated RNA polymerase II, SAFB1 blocked its recruitment to the HIV-1 LTR. Of note, SAFB1-mediated repression of HIV-1 transcription from proviral DNA maintained HIV-1 latency in CD4+ T cells. In summary, our findings reveal that SAFB1 binds to the HIV-1 LTR and physically interacts with phosphorylated RNA polymerase II, repressing HIV-1 transcription initiation and elongation. Our findings improve our understanding of host modulation of HIV-1 transcription and latency and provide a new host cell target for improved anti-HIV-1 therapies.

Spontaneous C-cleavage of a truncated intein as fusion tag to produce tag-free VP1 inclusion body nanoparticle vaccine against CVB3-induced viral myocarditis by the oral route.

BACKGROUND: Oral vaccine is highly desired for infectious disease which is caused by pathogens infection through the mucosal surface. The design of suitable vaccine delivery system is ongoing for the antigen protection from the harsh gastric environment and target to the Peyer's patches to induce sufficient mucosal immune responses. Among various potential delivery systems, bacterial inclusion bodies have been widely used as delivery systems in the field of nanobiomedicine. However, a large number of heterologous complex proteins could be difficult to propagate in E. coli and fusion partners are often used to enhance target protein expression. As a safety concern the fusion protein need to be removed from the target protein to get tag-free protein, especially for the production of protein antigen in vaccinology. Until now, there is no report on how to remove fusion tag from inclusion body particles in vitro and in vivo. Coxsackievirus B3 (CVB3) is a leading causative agent of viral myocarditis and orally protein vaccine is high desired for CVB3-induced myocarditis. In this context, we explored a tag-free VP1 inclusion body nanoparticles production protocol though a truncated Ssp DnaX mini-intein spontaneous C-cleavage in vivo and also exploited the VP1 inclusion bodies as an oral protein nanoparticle vaccine to protect mice against CVB3-induced myocarditis. RESULTS: We successfully produced the tag-free VP1 inclusion body nanoparticle antigen of CVB3 and orally administrated to mice. The results showed that the tag-free VP1 inclusion body nanoparticles as an effective antigen delivery system targeting to the Peyer's patches had the capacity to induce mucosal immunity as well as to efficiently protect mice from CVB3 induce myocarditis without any adjuvant. Then, we proposed the use of VP1 inclusion body nanoparticles as good candidate for oral vaccine to against CVB3-induced myocarditis. CONCLUSIONS: Our tag-free inclusion body nanoparticles production procedure is easy and low cost and may have universal applicability to produce a variety of tag-free inclusion body nanoparticles for oral vaccine.

Semaphorin7A aggravates coxsackievirusB3-induced viral myocarditis by increasing α1ß1-integrin macrophages and subsequent enhanced inflammatory response.

Semaphorin7A (Sema7A) has been reported to play various roles in nerve axon growth, tumor suppression, and tissue remodeling, as well as regulation of intestinal inflammation diseases. Viral myocarditis (VMC) characterized by viral-myocardial-cell necrosis and inflammatory cell infiltration is a common clinical disease of the cardiovascular system. However, the role of Sema7A in coxsackievirus B3 (CVB3)-induced VMC has not been reported. In this study, we generated an acute VMC mouse model by CVB3 infection, and manipulated Sema7A expression by in vivo polyethyleneimine-mediated Sema7A down-regulation. Our results indicated that Sema7A was up-regulated in cardiomyocytes during VMC, and that Sema7A down-regulation following short hairpin RNA interference or mAb neutralization effectively protected mice from VMC. Additionally, reduced inflammatory responses were observed along with Sema7A down-regulation. Furthermore, adoptive transfer of α1ß1-integrin macrophages exacerbated CVB3-induced myocarditis, suggesting the significance of α1ß1-integrin macrophages in response to VMC. We observed that co-culture of neonatal myocardiocytes with macrophages increased the percentage of α1ß1-integrin macrophages, while Sema7A neutralization reduced α1ß1-integrin macrophages in heart tissue of VMC mice. These results demonstrated that Sema7A, as an inflammation regulator in CVB3-induced VMC, might interact with α1ß1-integrin in macrophages to enhance the inflammatory response and aggravate disease severity. Our findings provided insight into the potential role of Sema7A as a therapeutic treatment for VMC.

ODC1 inhibits the inflammatory response and ROS-induced apoptosis in macrophages.

Macrophage activation plays a critical role in the innate immune response. Ornithine decarboxylase (ODC1) metabolizes l-ornithine to polyamines and is the rate-limiting enzyme involved in the metabolism of polyamines, which are reportedly involved in cell differentiation, proliferation, and migration. However, the function of ODC1 in immune cells and especially in macrophages, as well as its underlying molecular mechanism, remains unclear. This study revealed the potential ODC1 function and mechanism associated with the lipopolysaccharide (LPS)-, Bacillus Calmette-Guerin (BCG)-, or carbon tetrachloride (CCl4)-induced inflammatory response in macrophages. We found significant upregulation of ODC1 in macrophages following LPS simulation and ODC1-specific suppression of proinflammatory cytokine secretion from macrophages upon stimulation with LPS, BCG and CCl4, respectively, suggesting a role as a common control element of the inflammatory response. Western blotting for nuclear factor-κB and mitogen-activated protein kinases revealed significant inhibition of phosphorylation of multiple transcription factors following ODC1 overexpression in macrophages. Moreover, ODC1 inhibited reactive oxygen species-induced and caspase-dependent apoptosis highlighted by decreased caspase-3 and -9 expression following ODC1 upregulation. These findings indicated that ODC1 was involved in attenuating the inflammatory response upon stimulation of macrophages, making it a potential therapeutic target for inflammatory diseases.

Reactive Oxygen Species-Mediated c-Jun NH2-Terminal Kinase Activation Contributes to Hepatitis B Virus X Protein-Induced Autophagy via Regulation of the Beclin-1/Bcl-2 Interaction.

Autophagy is closely associated with the regulation of hepatitis B virus (HBV) replication. HBV X protein (HBx), a multifunctional regulator in HBV-associated biological processes, has been demonstrated to be crucial for autophagy induction by HBV. However, the molecular mechanisms of autophagy induction by HBx, especially the signaling pathways involved, remain elusive. In the present investigation, we demonstrated that HBx induced autophagosome formation independently of the class I phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling pathway. In contrast, the class III PI3K(VPS34)/beclin-1 pathway was revealed to be critical for HBx-induced autophagosome formation. Further study showed that HBx did not affect the level of VPS34 and beclin-1 expression but inhibited beclin-1/Bcl-2 association, and c-Jun NH2-terminal kinase (JNK) signaling was found to be important for this process. Moreover, it was found that HBx treatment led to the generation of reactive oxygen species (ROS), and inhibition of ROS activity abrogated both JNK activation and autophagosome formation. Of importance, ROS-JNK signaling was also revealed to play an important role in HBV-induced autophagosome formation and subsequent HBV replication. These data may provide deeper insight into the mechanisms of autophagy induction by HBx and help in the design of new therapeutic strategies against HBV infection.IMPORTANCE HBx plays a key role in diverse HBV-associated biological processes, including autophagy induction. However, the molecular mechanisms of autophagy induction by HBx, especially the signaling pathways involved, remain elusive. In the present investigation, we found that HBx induced autophagy independently of the class I PI3K/AKT/mTOR signaling pathway, while the class III PI3K(VPS34)/beclin-1 pathway was revealed to be crucial for this process. Further data showed that ROS-JNK activation by HBx resulted in the release of beclin-1 from its association with Bcl-2 to form a complex with VPS34, thus enhancing autophagosome formation. Of importance, ROS-JNK signaling was also demonstrated to be critical for HBV replication via regulation of autophagy induction. These data help to elucidate the molecular mechanisms of autophagy induction by HBx/HBV and might be useful for designing novel therapeutic approaches to HBV infection.

Deubiquitinase USP2a Sustains Interferons Antiviral Activity by Restricting Ubiquitination of Activated STAT1 in the Nucleus.

STAT1 is a critical transcription factor for regulating host antiviral defenses. STAT1 activation is largely dependent on phosphorylation at tyrosine 701 site of STAT1 (pY701-STAT1). Understanding how pY701-STAT1 is regulated by intracellular signaling remains a major challenge. Here we find that pY701-STAT1 is the major form of ubiquitinated-STAT1 induced by interferons (IFNs). While total STAT1 remains relatively stable during the early stages of IFNs signaling, pY701-STAT1 can be rapidly downregulated by the ubiquitin-proteasome system. Moreover, ubiquitinated pY701-STAT1 is located predominantly in the nucleus, and inhibiting nuclear import of pY701-STAT1 significantly blocks ubiquitination and downregulation of pY701-STAT1. Furthermore, we reveal that the deubiquitinase USP2a translocates into the nucleus and binds to pY701-STAT1, and inhibits K48-linked ubiquitination and degradation of pY701-STAT1. Importantly, USP2a sustains IFNs-induced pY701-STAT1 levels, and enhances all three classes of IFNs- mediated signaling and antiviral activity. To our knowledge, this is the first identified deubiquitinase that targets activated pY701-STAT1. These findings uncover a positive mechanism by which IFNs execute efficient antiviral signaling and function, and may provide potential targets for improving IFNs-based antiviral therapy.

IL-33 enhances macrophage M2 polarization and protects mice from CVB3-induced viral myocarditis.

Viral myocarditis is the inflammation caused by myocardial virus infection, and the coxsackievirus group B3 virus (CVB3) is the most common pathogen. An efficient therapeutic agent against viral myocarditis is currently unavailable. IL-33, a new member of the IL-1 cytokine superfamily, exhibits potential immunotherapeutic effect against inflammatory and autoimmune diseases. However, the functional role of IL-33 in viral myocarditis has not been investigated. To examine the therapeutic role of IL-33 in viral myocarditis, an IL-33 overexpression plasmid (pDisplay-IL-33) and IL-33 knockdown plasmid (pLL3.7-IL-33) were packaged with polyethylenimine and delivered intravenously at the orbital area of BALB/c male mice after CVB3 infection. Then, myocarditis severity was assessed 7days after infection. Results showed that IL-33 up-regulation significantly alleviated the severity of viral myocarditis with an increased cardiac contractive function and survival rate. Mechanistic studies demonstrated that IL-33 can stimulate ST2L+F4/80+ macrophages and ST2L+CD4+T cells in cardiac tissue to express IL-4, which is a potent inducer for macrophage M2 polarization. Mice with adoptive transfer of M2 macrophages exhibited less cardiac inflammation and attenuated myocarditis, suggesting the protective role of M2 macrophage in viral myocarditis. Additionally, IL-4 neutralization abolished the IL-33-mediated cardiac functional improvement in myocarditis mice. Collectively, our findings provide a novel therapeutic role for IL-33 in CVB3-induced myocarditis.