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.

MicroRNA-302 Cluster Downregulates Enterovirus 71-Induced Innate Immune Response by Targeting KPNA2.

Enterovirus 71 (EV71) induces significantly elevated levels of cytokines and chemokines, leading to local or systemic inflammation and severe complications. As shown in our previous study, microRNA (miR) 302c regulates influenza A virus-induced IFN expression by targeting NF-κB-inducing kinase. However, little is known about the role of the miR-302 cluster in EV71-mediated proinflammatory responses. In this study, we found that the miR-302 cluster controls EV71-induced cytokine expression. Further studies demonstrated that karyopherin α2 (KPNA2) is a direct target of the miR-302 cluster. Interestingly, we also found that EV71 infection upregulates KPNA2 expression by downregulating miR-302 cluster expression. Upon investigating the mechanisms behind this event, we found that KPNA2 intracellularly associates with JNK1/JNK2 and p38, leading to translocation of those transcription factors from the cytosol into the nucleus. In EV71-infected patients, miR-302 cluster expression was downregulated and KPNA2 expression was upregulated compared with controls, and their expression levels were closely correlated. Taken together, our work establishes a link between the miR-302/ KPNA2 axis and EV71-induced cytokine expression and represents a promising target for future antiviral therapy.

MicroRNA-302a suppresses influenza A virus-stimulated interferon regulatory factor-5 expression and cytokine storm induction.

During influenza A virus (IAV) infection, cytokine storms play a vital and critical role in clinical outcomes. We have previously reported that microRNA (miR)-302c regulates IAV-induced IFN expression by targeting the 3'-UTR of nuclear factor κB (NF-κB)-inducing kinase. In the current study, we found that miR-302a, another member of the miR-302 cluster, controls the IAV-induced cytokine storm. According to results from cell-based and knockout mouse models, IAV induces a cytokine storm via interferon regulatory factor-5 (IRF-5). We also found that IAV infection up-regulates IRF-5 expression and that IRF-5 in turn promotes IAV replication. Furthermore, we observed that IRF-5 is a direct target of miR-302a, which down-regulated IRF-5 expression by binding its 3'-UTR. Moreover, IAV increased IRF-5 expression by down-regulating miR-302a expression. Interestingly, miR-302a inhibited IAV replication. In IAV-infected patients, miR-302a expression was down-regulated, whereas IRF-5 expression was up-regulated. Taken together, our work uncovers and defines a signaling pathway implicated in an IAV-induced cytokine storm.

Gene Expression and Antiviral Activity of Interleukin-35 in Response to Influenza A Virus Infection.

Interleukin-35 (IL-35) is a newly described member of the IL-12 family. It has been reported to inhibit inflammation and autoimmune inflammatory disease and can increase apoptotic sensitivity. Little is known about the role of IL-35 during viral infection. Herein, high levels of IL-35 were found in peripheral blood mononuclear cells and throat swabs from patients with seasonal influenza A virus (IAV) relative to healthy individuals. IAV infection of human lung epithelial and primary cells increased levels of IL-35 mRNA and protein. Further studies demonstrated that IAV-induced IL-35 transcription is regulated by NF-κB. IL-35 expression was significantly suppressed by selective inhibitors of cyclooxygenase-2 (COX-2) and inducible nitric-oxide synthase, indicating their involvement in IL-35 expression. Interestingly, IL-35 production may have suppressed IAV RNA replication and viral protein synthesis via induction of type I and III interferons (IFN), leading to activation of downstream IFN effectors, including double-stranded RNA-dependent protein kinase, 2',5'-oligoadenylate synthetase, and myxovirus resistance protein. IL-35 exhibited extensive antiviral activity against the hepatitis B virus, enterovirus 71, and vesicular stomatitis virus. Our results demonstrate that IL-35 is a novel IAV-inducible cytokine, and its production elicits antiviral activity.

Set7 facilitates hepatitis C virus replication via enzymatic activity-dependent attenuation of the IFN-related pathway.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, usually resulting in persistent infection involving hepatic steatosis, cirrhosis, and hepatocellular carcinoma via escape of the host's immune response. Set7 is a lysine-specific methyltransferase that is involved in gene regulation and virus replication. However, the mechanism underlying the immune evasion between HCV and Set7 is not well understood. In this study, we observed that the expression of Set7 in Huh7.5.1 cells was upregulated by HCV infection, and high levels of Set7 expression were also found in the sera, PBMCs, and liver tissue of HCV patients relative to healthy individuals. Further investigation showed that Set7 enhanced HCV replication in an enzymatic activity-dependent manner. Moreover, our data showed that Set7 decreased the expression of virus-induced IFN and IFN-related effectors, such as dsRNA-activated protein kinase and 2',5'-oligoadenylate synthetase. Further investigation suggested that Set7 suppressed the endogenous IFN expression by reducing the nuclear translocation of IFN regulatory factor 3/7 and the p65 subunit of NF-κB and reduced IFN-induced dsRNA-activated protein kinase and 2',5'-oligoadenylate synthetase via attenuation of the phosphorylation of STAT1 and STAT2. Additionally, IFN receptors, including IFNAR1 and IFNAR2, which are located upstream of the JAK/STAT pathway, were reduced by Set7. Taken together, our results reveal that Set7 facilitates HCV replication through the attenuation of IFN signaling pathways and IFN-related effectors.

HCV NS4B induces apoptosis through the mitochondrial death pathway.

The hepatitis C virus (HCV) NS4B protein is known to induce the formation of a membranous web that is thought to be the site of viral RNA replication. However, the exact functions of NS4B remain poorly characterized. In this study, we found that NS4B induced apoptosis in 293T cells and Huh7 cells, as confirmed by Hoechst staining, DNA fragmentation, and annexin V/PI assays. Furthermore, protein immunoblot analysis demonstrated that NS4B triggered the cleavage of caspase 3, caspase 7, and poly(ADP-ribose) polymerase (PARP). Further studies revealed that NS4B induced the activation of caspase 9, the reduction of mitochondrial membrane potential and the release of cytochrome c from the mitochondria. However, NS4B expression did not trigger XBP1 mRNA splicing and increase the expression of binding immunoglobulin protein (BiP, or GRP78) and C/EBP homologous protein (CHOP), which serves as the indicators of ER stress. Taken together, our results suggest that HCV NS4B induces apoptosis through the mitochondrial death pathway.

Dual effects of interleukin-18: inhibiting hepatitis B virus replication in HepG2.2.15 cells and promoting hepatoma cells metastasis.

Interleukin-18 (IL-18) has been reported to inhibit hepatitis B virus (HBV) replication in the liver of HBV transgenic mice; however, the molecular mechanism of its antiviral effect has not been fully understood. In the present study, it was shown that IL-18 and its receptors (IL-18R) were constitutively expressed in hepatoma cell lines HepG2 and HepG2.2.15 as well as normal liver cell line HL-7702. We demonstrated that IL-18 directly inhibited HBV replication in HepG2.2.15 cells via downregulating the activities of HBV core and X gene promoters. The suppressed HBV replication by IL-18 could be rescued by the administration of BAY11-7082, an inhibitor of transcription factor NF-κB. On the other hand, it was of interest that IL-18 promoted HepG2 cell metastasis and migration dose dependently in both wound-healing assays and Transwell assays. The underlying mechanism could be partially attributable to the increased activities of extracellular matrix metalloproteinase (MMP)-9, MMP-3, and MMP-2 by IL-18, which upregulated the mRNA levels of MMP-3 and MMP-9 in a NF-κB-dependent manner. Furthermore, it was confirmed that expression of IL-18/IL-18R and most MMPs were remarkably upregulated in hepatocellular carcinoma (HCC) liver cancer tissue specimens, suggesting that IL-18/IL-18R-triggered signaling pathway was closely related to HCC metastasis in vivo. Therefore, we revealed the dual effects of IL-18 in human hepatocytes: it not only inhibited HBV replication but also promoted hepatoma cells metastasis and migration. NF-κB played a critical role in both effects. Our work contributed to a deeper understanding of the biological function of IL-18 in human hepatocytes.

Expression, purification and immunogenic characterization of hepatitis C virus recombinant E1E2 protein expressed by Pichia pastoris yeast.

Development of an effective vaccine may be the key in the control of hepatitis C virus (HCV) infection. Recent studies have shown that HCV envelope proteins can induce broadly neutralizing antibodies against conserved domain for HCV binding to the cellular receptors. So HCV envelope proteins are considered as the major HCV vaccine candidate. In this study, we used Pichia pastoris yeast to express truncated HCV E1E2 protein, which consists of E1 residues 187-346 and E2 residues 381-699. The yeast can produce high level of recombinant HCV E1E2 protein. The protein has complex glycosylation and can bind to CD81, the putative HCV receptor. Moreover, the purified protein can efficiently induce anti-E1E2 antibodies in rabbits, which are able to neutralize two kinds of HCV pseudotype particles derived from HCV genotype 1a and 1b, as well as HCV virions derived from HCV genotype 2a. These findings indicate that the recombinant E1E2 glycoprotein is effective in inducing broadly neutralizing antibodies, and is a potent HCV vaccine candidate.

WITHDRAWN: USP14 stabilizes calcineurin and regulates NFAT/AP-1 dependent IL-4 transcription as a novel calcineurin-binding protein.

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Hepatitis C virus NS2/3 protease regulates HCV IRES-dependent translation and NS5B RdRp activity.

Chronic hepatitis C virus (HCV) infection often leads to liver cancer. NS2/3 protease is the first of two virally encoded proteases required for HCV polyprotein processing. In this report, we investigated the function of NS2/3 protease on HCV replication and translation. Cells transfected with plasmids encoding wild-type or mutant NS2/3 and a dual-luciferase reporter construct containing an HCV internal ribosome entry site (IRES) were used to examine the effect of NS2/3 protease on translation of HCV RNA. Cells transfected with plasmids encoding wild-type or mutant NS2/3, pcDNA-NS5B and a reporter plasmid were used to examine the effect of NS2/3 protease on HCV replication. The results showed that both autocleavage processing and the uncleaved form of NS2/3 protease specifically decrease HCV IRES-directed translation, while the uncleaved form of NS2/3 protease decreases HCV NS5B RdRp activity (replication), indicating that autoregulation by NS2/3 protease of HCV replication and translation may play an important role in persistent HCV infection.

Hepatitis C virus (HCV) NS2 protein up-regulates HCV IRES-dependent translation and down-regulates NS5B RdRp activity.

Chronic hepatitis C virus (HCV) infection often leads to liver cancer. The HCV NS2 protein is a hydrophobic transmembrane protein that associates with several cellular proteins in mammalian cells. In this report, we investigated the function of NS2 protein on HCV replication and translation by using a transient cell-based expression system. Cells co-transfected with pcDNA3.1 (-)-NS2 and the dual-luciferase reporter construct containing the HCV IRES were used to detect the effect of NS2 protein on HCV translation. Cells co-transfected with pcDNA3.1(-)-NS2, pcDNA-NS5B and a reporter plasmid were used to detect the effect of NS2 protein on HCV replication. The results showed that HCV NS2 protein up-regulated HCV IRES-dependent translation in a specific and dose-dependent manner in Huh7 cells but not in HeLa and HepG2 cells, and NS2 protein inhibited NS5B RdRp activity in a dose-independent manner in all three cell lines. These findings may suggest a novel mechanism by which HCV modulates its NS5B replication and IRES-dependent translation and facilitates virus persistence.

The nucleocapsid protein of SARS-associated coronavirus inhibits B23 phosphorylation.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is responsible for SARS infection. Nucleocapsid (N) protein of SARS-CoV encapsidates the viral RNA and plays an important role in virus particle assembly and release. In this study, the N protein of SARS-CoV was found to associate with B23, a phosphoprotein in nucleolus, in vitro and in vivo. Mapping studies localized the critical N sequences for this interaction to amino acid residues 175-210, which included a serine/arginine (SR)-rich domain. In vitro phosphorylation assay showed that the N protein inhibited the B23 phosphorylation at Thr199.

Torque teno virus (SANBAN isolate) ORF2 protein suppresses NF-kappaB pathways via interaction with IkappaB kinases.

Since the first discovery of Torque teno virus (TTV) in 1997, many researchers focused on its epidemiology and transcriptional regulation, but the function of TTV-encoded proteins remained unknown. The function of the TTV open reading frame (ORF) in the nuclear factor kappaB (NF-kappaB) pathway has not yet been established. In this study, we found for the first time that the TTV ORF2 protein could suppress NF-kappaB activity in a dose-dependent manner in the canonical NF-kappaB pathway. By Western blot analysis, we proved that the TTV ORF2 protein did not alter the level of NF-kappaB expression but prevented the p50 and p65 subunits from entering the nucleus due to the inhibition of IkappaBalpha protein degradation. Further immunoprecipitation assays showed that the TTV ORF2 protein could physically interact with IKKbeta as well as IKKalpha, but not IKKgamma. Luciferase assays and Western blot experiments showed that the TTV ORF2 protein could also suppress NF-kappaB activity in the noncanonical NF-kappaB pathway and block the activation and translocation of p52. Finally, we found that the TTV ORF2 protein inhibited the transcription of NF-kappaB-mediated downstream genes (interleukin 6 [IL-6], IL-8, and COX-2) through down-regulation of NF-kappaB. Together, these data indicate that the TTV ORF2 protein suppresses the canonical and noncanonical NF-kappaB pathways, suggesting that the TTV ORF2 protein may be involved in regulating the innate and adaptive immunity of organisms, contributing to TTV pathogenesis, and even be related to some diseases.

Up-regulation of IL-6 and TNF-alpha induced by SARS-coronavirus spike protein in murine macrophages via NF-kappaB pathway.

The clinical picture of severe acute respiratory syndrome (SARS) is characterized by an over-exuberant immune response with lung lymphomononuclear cells infilteration and proliferation that may account for tissue damage more than the direct effect of viral replication. To understand how cells response in the early stage of virus-host cell interaction, in this study, a purified recombinant S protein was studied for stimulating murine macrophages (RAW264.7) to produce proinflammatory cytokines (IL-6 and TNF-alpha) and chemokine IL-8. We found that direct induction of IL-6 and TNF-alpha release in the supernatant in a dose-, time-dependent manner and highly spike protein-specific, but no induction of IL-8 was detected. Further experiments showed that IL-6 and TNF-alpha production were dependent on NF-kappaB, which was activated through I-kappaBalpha degradation. These results suggest that SARS-CoV spike protein may play an important role in the pathogenesis of SARS, especially in inflammation and high fever.

Expression, purification and characterization of the Hepatitis B virus entire envelope large protein in Pichia pastoris.

The current HBsAg vaccine has performed a vital role in preventing the transmission of HBV during the past 20 years. However, a number of individuals still show no response or a low response to the vaccine. In the present study, the HBV envelope large protein gene was cloned into the eukaryotic expression vector pPIC9k and was subsequently expressed in the yeast Pichia pastoris. The HBV large protein (L protein) was produced and secreted into the medium, where some of the L protein formed particles. The soluble L protein and particles were purified by column chromatography and sucrose density gradient centrifugation. Western blot analysis demonstrated that the particle was composed of both HBV L and S protein. To compare the antigenicity of the L protein and HBsAg, rabbits were immunized with the soluble L protein and the commercially available HBV vaccine and the increasing level of antibodies was determined by ELISA. The results showed that the anti-HBsAg antibody, from rabbits injected with the L protein at a dose of 2 and 10microg, was detected on day 14, whereas rabbits vaccinated with 10 and 2microg HBsAg did not develop antibodies until day 21 and 28, respectively. The antibody level in groups inoculated with the L protein was approximately 50% higher than in the group injected with HBsAg using the same dose. Furthermore, 2microg L protein induced a significant and rapid anti-HBsAg antibody response than 10microg HBsAg. Therefore, we suggest that the L protein is an ideal candidate for a new generation HB vaccine to protect people from HBV infection.

HCV NS2 protein inhibits cell proliferation and induces cell cycle arrest in the S-phase in mammalian cells through down-regulation of cyclin A expression.

Chronic hepatitis C virus (HCV) infection often leads to liver cancer. NS2 protein is a HCV hydrophobic transmembrane protein that associates with several cellular proteins in mammalian cells. In this report, we investigated the functions of NS2 protein by examining its effects on cell growth and cell cycle progression. Stable NS2-expressing HeLa and Vero cell lines were established by transfection of the cells with pcDNA3.1(-)-NS2 followed by selection of the transfected cells in the presence of G418. We found that the proliferation rates of both NS2-expressing cell lines were inhibited by 40-50% compared with the control cells that were transfected with pcDNA3.1(-) control vector. Cell cycle analysis of these NS2-expressing cell lines shows that the proportion of cells in the S-phase increased significantly compared to that of control cells that do not express NS2 protein, suggesting NS2 protein induces cell cycle arrest in the S-phase. Further studies showed that the induction of cell cycle arrest in the S-phase by NS2 protein is associated with the decrease of cyclin A level. In contrast, the expression of NS2 protein does not affect the levels of cyclin-dependent kinase CDK2, CDK4, cyclin D1, or cyclin E. Our results suggest that HCV NS2 protein inhibits cell growth and induces the cell cycle arrest in the S-phase through down-regulation of cyclin A expression, which may be beneficial to HCV viral replication. Our findings not only provide information in the understanding mechanism of HCV infection, but also provide guidance for the future development of potential therapeutics for the prevention and treatment of the viral infection.

[The purification of HBV full-length PreS protein in Pichia pastoris].

The Pichia pastoris strain GS115-PreS could produce a high expression level of full-length PreS protein that secreted to the supernatant after methanol induction in the fermentation. The Western blot analysis showed a single band with expected molecular mass of 48kD and that the major component of the particles was the full-length PreS protein (PreS1 + PreS2 + S) and small envelope protein (S) of 48 and 28 kD, respectively. Electron microscopy image showed PreS particles with 30 nm in diameter. The supernatants of the fermentation were desalted and concentrated. Purified PreS protein was obtained by DEAE-SFF anion exchange column chromatography and the PreS particles were obtained by ultracentrifugation and sucrose density gradient. The ELISA assay results proved that both full-length PreS protein and particles showed high immunogenicity and specificity. P/N ratio further demonstrated that the immunogenicity of the particles is higher than the full-length PreS protein.

Activation of NF-kappaB by the full-length nucleocapsid protein of the SARS coronavirus.

The severe acute respiratory syndrome coronavirus (SARS-CoV) is the major causative agent for the worldwide outbreak of SARS in 2003. The mechanism by which SARS-CoV causes atypical pneumonia remains unclear. The nuclear factor kappa B (NF-kappaB) is a key transcription factor that activates numerous genes involved in cellular immune response and inflammation. Many studies have shown that NF-kappaB plays an important role in the pathogenesis of lung diseases. In this study, we investigated the possible regulatory interaction between the SARS-CoV nucleocapsid (N) protein and NF-kappaB by luciferase activity assay. Our results showed that the SARS-CoV N protein can significantly activate NF-kappaB only in Vero E6 cells, which are susceptible to SARS-CoV infection, but not in Vero or HeLa cells. This suggests that NF-kappaB activation is cell-specific. Furthermore, NF-kappaB activation in Vero E6 cells expressing the N protein is dose-dependent. Further experiments showed that there is more than one function domain in the N protein responsible for NF-kappaB activation. Our data indicated the possible role of the N protein in the pathogenesis of SARS.

Hepatitis C virus non-structural 5A protein can enhance full-length core protein-induced nuclear factor-kappaB activation.

AIM: To study the effects of hepatitis C virus (HCV) core and non-structural 5A (NS5A) proteins on nuclear factor-kappaB (NF-kappaB) activity for understanding their biological function on chronic hepatitis caused by HCV infection. METHODS: Luciferase assay was used to measure the activity of NF-kappaB in three different cell lines cotransfected with a series of deletion mutants of core protein alone or together with NS5A protein using pNF-kappaB-Luc as a reporter plasmid. Western blot and indirect immunofluorescence assays were used to confirm the expression of proteins and to detect their subcellular localization, respectively. Furthermore, Western blot was also used to detect the expression levels of NF-kappaB/p65, NF-kappaB/p50, and inhibitor kappaB-a (IkappaB-a). RESULTS: The wild-type core protein (C191) and its mutant segments (C173 and C158) could activate NF-kappaB in Huh7 cells only and activation caused by (C191) could be enhanced by NS5A protein. Moreover, the full-length core protein and its different deletion mutants alone or together with NS5A protein did not enhance the expression level of NF-kappaB. The NF-kappaB activity was augmented due to the dissociation of NF-kappaB-IkappaB complex and the degradation of IkappaB-a. CONCLUSION: NF-kappaB is the key transcription factor that can activate many genes that are involved in the cellular immune response and inflammation. Coexpression of the full-length core protein along with NS5A can enhance the NF-kappaB activation, and this activation may play a significant role in chronic liver diseases including hepatocellular carcinoma associated with HCV infection.