Virus-induced Protein Research Articles

Generation of viperin-knockout zebrafish by CRISPR/Cas9-mediated genome engineering and the effect of this mutation under VHSV infection

Viperin is an important virus-induced protein in animals that negatively participates in RNA viral replication and transcription. The reactive machinery of viperin suggests that it produces a regulatory molecule ddhCTP, which may affect immune regulation. In this study, we investigated the expression pattern of viperin in larval and adult stages of zebrafish by whole-mount in situ hybridization and reverse transcription-quantitative PCR (RT-qPCR). To elucidate the function of viperin, we generated a zebrafish knockout model using the CRISPR/Cas9 method and evaluated the mutation's effects under viral hemorrhagic septicemia virus (VHSV) infections. In zebrafish larvae, viperin was expressed in the brain region, eye, and pharynx, which was confirmed by cryosectioning. In adult zebrafish, blood cells showed the highest levels of viperin expression. In 5 dpf fish challenged with VHSV, the expression of the viral NP protein was significantly enhanced in viperin−/− compared to wild-type fish. In vitro VHSV propagation analysis indicated comparatively higher levels of virus propagation in viperin−/− fish. Mortality analysis confirmed higher mortality rates, and interferon gene expression analysis showed a strong upregulation of interferon (ifn)φ1 and 3 gene in viperin−/− fish infected with VHSV. This study describes the successful generation of a viperin-knockout model and the role of viperin during VHSV infections.

BAG6 negatively regulates the RLR signaling pathway by targeting VISA/MAVS.

The virus-induced signaling adaptor protein VISA (also known as MAVS, ISP-1, Cardif) is a critical adaptor protein in the innate immune response to RNA virus infection. Upon viral infection, VISA self-aggregates to form a sizeable prion-like complex and recruits downstream signal components for signal transduction. Here, we discover that BAG6 (BCL2-associated athanogene 6, formerly BAT3 or Scythe) is an essential negative regulator in the RIG-I-like receptor signaling pathway. BAG6 inhibits the aggregation of VISA by promoting the K48-linked ubiquitination and specifically attenuates the recruitment of TRAF2 by VISA to inhibit RLR signaling. The aggregation of VISA and the interaction of VISA and TRAF2 are enhanced in BAG6-deficient cell lines after viral infection, resulting in the enhanced transcription level of downstream antiviral genes. Our research shows that BAG6 is a critical regulating factor in RIG-I/VISA-mediated innate immune response by targeting VISA.

Lack of Type I Interferon Signaling Ameliorates Respiratory Syncytial Virus-Induced Lung Inflammation and Restores Antioxidant Defenses.

Respiratory syncytial virus (RSV) infection in mouse and human lung is associated with pathogenic inflammation and oxidative injury. RSV impairs antioxidant responses by increasing the degradation of transcription factor NF-E2-related factor 2 (NRF2), which controls the expression of several antioxidant enzymes (AOEs). In addition to its protective effects, type I IFNs have been increasingly recognized as important mediators of host pathogenic responses during acute respiratory viral infections. We used a mouse model of RSV infection to investigate the effect of lack of type I interferon (IFN) receptor on viral-mediated clinical disease, airway inflammation, NRF2 expression, and antioxidant defenses. In the absence of type I IFN signaling, RSV-infected mice showed significantly less body weight loss and airway obstruction, as well as a significant reduction in cytokine and chemokine secretion and airway inflammation. Lack of type I IFN receptor was associated with greatly reduced virus-induced promyelocytic leukemia lung protein expression, which we showed to be necessary for virus-induced NRF2 degradation in a cell model of infection, resulting in restoration of NRF2 levels, AOE expression, and airway antioxidant capacity. Our data support the concept that modulation of type I IFN production and/or signaling could represent an important therapeutic strategy to ameliorate severity of RSV-induced lung disease.

Dantrolene hinders dengue virus-induced upregulation and translocation of calmodulin to cardiac cell nuclei

Dengue virus (DENV) infection elevates intracellular Ca2+ concentration ([Ca2+]i), but it is unknown whether Ca2+ and calmodulin (CaM) are involved in DENV infection. We conducted immunofluorescence and western blot experiments and measured [Ca2+]i examining the effects of DENV infection and drugs that alter Ca2+/CaM functions on CaM translocation, DENV2 infection, protein expression, virus-inducible STAT2 protein abundance, and CREB phosphorylation in H9c2 cells. DENV infection increased CaM expression, its nuclear translocation and NS3 and E viral proteins expression and colocalization in a manner that could be blocked by the ryanodine receptor antagonist dantrolene. DENV infection also increased CREB phosphorylation, an effect inhibited by either dantrolene or the CaM inhibitor W7. Dantrolene substantially hindered infection as assessed by focus assays in Vero cells. These results suggest that Ca2+ and CaM play an important role in DENV infection of cardiac cells and that dantrolene may protect against severe DENV cardiac morbidity.

Anti-dsDNA specific antibody secreting cells are increased in both frequency and abundance in Chronic Rhinosinusitis with Nasal Polyps

Abstract Background We have previously found that anti-dsDNA IgG autoantibodies are increased in Chronic Rhinosinusitis with Nasal Polyps (CRSwNP) patients. Recent studies find that B cells with the Epstein-Barr virus-induced protein 2(EBI2), is critical to the extrafollicular response. We have described elevated numbers of EBI2+ B cells in NP and they were more frequently antibody secreting cells (ASCs). In this study we evaluate whether dsDNA-specific ASCs are increased in NP and further compared the EBI2+ and EBI2− compartments. Methods To investigate this problem, flow cytometry and ELISpot assays were utilized to compare the frequency of total IgG and dsDNA specific IgG ASCs and cells were sorted into EBI2+ and EBI2− B cells from human NP and tonsils. Results Flow cytometry results (n=10 tonsil, 4 NP) found that although tonsils had significantly more B cells than NP, a significantly smaller proportion were CD27+CD38+ plasmablasts (PB). Further ELISpot analysis found NP B cells were more frequently IgG ASCs than tonsil (0.06% and 0.43% in tonsil (n=9) and NP (n=5) respectively, p<0.001). Anti-dsDNA specific ASCs were two-fold more in NP representing 0.95% of IgG ASCs compared with 0.4% in tonsil ASCs (p<0.05). In analysis of ASCs sorted from tonsils (n=3) and NP (n=2), we found anti-dsDNA IgG ASCs were more frequent among EBI2+ compared to EBI2− cells with 10- and 14-fold increases in dsDNA reactivity (p<0.01). Conclusions Although B-cells are significantly more abundant in tonsils, NP derived B-cells more frequently secrete total- and dsDNA-specific IgG. These differences appear to be driven by extrafollicular EBI2+ B-cells suggesting that EBI2+ B cells represent the major pool of autoreactive B-cells observed in CRSwNP and tonsils.

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.

Ac34 protein of AcMNPV promoted progeny virus production and induced the apoptosis in host Sf9 cells.

To analyze the function of Ac34 in Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and elucidate the JNK apoptotic signaling pathway activation in host Spodoptera frugiperda 9 (Sf9) cells induced by the recombinant virus AcMNPV-Ac34-EGFP. AcMNPV is an important species of baculoviruses. First, viral propagation assay indicated that overexpression of Ac34 protein promoted replication of AcMNPV. Quantitative RT-PCR analysis showed that Ac34 increased the transcriptional level of late genes 38k and vp39, which suggested that Ac34 promoted the production of progeny virus by upregulating transcription of late genes. Second, AcMNPV-Ac34-EGFP inhibited the proliferation of Sf9 cells. Moreover, Sf9 cells infected with AcMNPV-Ac34-EGFP resulted in abundant expression of SfP53 and its accumulation in the nucleus. c-Jun N-terminal kinase (JNK) activation requires MKK4 and MKK7 mediated phosphorylation at Thr183 and Tyr185. We found increased levels of p-JNK1/2 in Sf9 cells infected by AcMNPV-Ac34-EGFP, with concomitant induction of Sf9 cell death. Furthermore, treatment of infected Sf9 cells with SP600125 (an inhibitor of JNK pathway) downregulated p-JNK1/2 and influenced the expression of virus-induced apoptosis protein SfP53, as well as Cytochrome C and Bax. AcMNPV-Ac34-EGFP virus upregulated the progeny virus production and triggered apoptosis via activation of the JNK pathway in Sf9 cells. In this work, we unveiled an effective virus replication factor-Ac34 and more importantly, developed a recombinant virus that can be used as an improved version of biopesticide.

Defining the Role of Stress Granules in Innate Immune Suppression by the Herpes Simplex Virus 1 Endoribonuclease VHS.

In response to virus-induced shutoff host protein synthesis, dynamic aggregates containing mRNA, RNA-binding proteins and translation factors termed stress granules (SGs) often accumulate within the cytoplasm. SGs typically form following phosphorylation and inactivation of the eukaryotic translation initiation factor 2α (eIF2α), a substrate of the double-stranded RNA (dsRNA)-activated kinase protein kinase R (PKR). The detection of innate immune sensors and effectors like PKR at SGs suggests a role in pathogen nucleic acid sensing. However, the functional importance of SGs in host innate responses is unclear and has primarily been examined in response to infection with select RNA viruses. During infection with the DNA virus herpes simplex virus 1 (HSV-1), the virus-encoded virion host shutoff (VHS) endoribonuclease is required to restrict interferon production, PKR activation, and SG formation, although the relationship between these activities remains incompletely understood. Here, we show that in cells infected with a VHS-deficient HSV-1 (ΔVHS) dsRNA accumulated and localized to SGs. Surprisingly, formation of dsRNA and its concentration at SGs was not required for beta interferon mRNA induction, indicating that suppression of type I interferon induction by VHS does not stem from its control of dsRNA accumulation. Instead, STING signaling downstream of cGMP-AMP synthase (cGAS)-dependent DNA sensing is required for beta interferon induction. In contrast, significantly less PKR activation is observed when SG assembly is disrupted by ISRIB, an inhibitor of phosphorylated eIF2α-mediated translation repression, or depleting SG scaffolding proteins G3BP1 or TIA1. This demonstrates that PKR activation is intimately linked to SG formation and that SGs form important hubs to potentiate PKR activation during infection.IMPORTANCE Formation of cytoplasmic stress granules that are enriched for innate immune sensors and effectors is suppressed during many viral infections. It is unclear, however, to what extent this is a side effect of viral efforts to maintain protein synthesis or intentional disruption of a hub for innate immune sensing. In this study, we utilize a herpes simplex virus 1 mutant lacking the RNA nuclease VHS which upon infection induces SGs, PKR activation, and beta interferon to address this question. We show that dsRNA is localized to SGs and that SGs can function to promote PKR activation in the context of a DNA virus infection, but we find no evidence to support their importance for interferon induction during HSV-1 infection.

Human Papillomavirus 16 E7 Stabilizes APOBEC3A Protein by Inhibiting Cullin 2-Dependent Protein Degradation.

ABSTRACTAPOBEC3 (A3) mutation signatures have been observed in a variety of human cancer genomes, including those of cervical and head and neck cancers caused by human papillomavirus (HPV) infection. However, the driving forces that promote off-target A3 activity remain mostly unclear. Here, we report a mechanism for the dramatic increase of A3A protein levels in HPV-positive keratinocytes. We show that expression of the viral protein E7 from high-risk HPVs, but not E7 from low-risk HPVs, significantly prolongs the cellular half-life of A3A protein in human keratinocytes and HPV-positive cancer cell lines. We have mapped several residues within the cullin 2 (CUL2) binding motif of HPV16 E7 as being important for mediating A3A protein stabilization. Furthermore, we provide direct evidence that both A3A and HPV16 E7 interact with CUL2, suggesting that the E7-CUL2 complex formed during HPV infection may regulate A3A protein levels in the cell. Using an in vitro cytidine deaminase assay, we show that E7-stabilized A3A remains catalytically active. Taken together, our findings suggest that the HPV oncoprotein E7 dysregulates endogenous A3A protein levels and thus provides novel mechanistic insight into cellular triggers of A3 mutations in HPV-positive cancers.IMPORTANCE Human papillomavirus (HPV) is causally associated with over 5% of all human malignancies. Several recent studies have shown that a subset of cancers, including HPV-positive head and neck and cervical cancers, have distinct mutational signatures potentially caused by members of the APOBEC3 cytidine deaminase family. However, the mechanism that induces APOBEC3 activity in cancer cells is poorly understood. Here, we report that the HPV oncoprotein E7 stabilizes the APOBEC3A (A3A) protein in human keratinocytes by inhibiting ubiquitin-dependent protein degradation in a cullin-dependent manner. Interestingly, the HPV E7-stabilized A3A protein maintains its deaminase activity. These findings provide a new insight into cancer mutagenesis enhanced by virus-induced A3A protein stabilization.

Hepatitis C virus-induced prion protein expression facilitates hepatitis C virus replication.

Hepatitis C virus (HCV) infects approximately 180 million people worldwide. Significant progress has been made since the establishment of in vitro HCV infection models in cells. However, the replication of HCV is complex and not completely understood. Here, we found that the expression of host prion protein (PrP) was induced in an HCV replication cell model. We then showed that increased PrP expression facilitated HCV genomic replication. Finally, we demonstrated that the KKRPK motif on the N-terminus of PrP bound nucleic acids and facilitated HCV genomic replication. Our results provided important insights into how viruses may harness cellular protein to achieve propagation.

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-β-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.

Inducible Rubicon facilitates viral replication by antagonizing interferon production.

The RUN domain Beclin-1-interacting cysteine-rich-containing (Rubicon) protein is involved in the maturation step of autophagy and the endocytic pathway as a Beclin-1-binding partner, but little is known regarding the role of Rubicon during viral infection. Here, we performed functional studies of the identified target in interferon (IFN) signaling pathways associated with Rubicon to elucidate the mechanisms of viral resistance to IFN. The Rubicon protein levels were elevated in peripheral blood mononuclear cells, sera and liver tissues from patients with hepatitis B virus (HBV) infection relative to those in healthy individuals. Assays of the overexpression and knockdown of Rubicon showed that Rubicon significantly promoted HBV replication. In addition, Rubicon knockdown resulted in the inhibition of enterovirus 71, influenza A virus and vesicular stomatitis virus. The expression o0f Rubicon led to the suppression of virus-induced type-I interferon (IFN-α and IFN-β) and type-III interferon (IFN-λ1). Translocation of activated IRF3 and IRF7 from the cytoplasm to the nucleus was involved in this process, and the NF-κB essential modulator (NEMO), a key factor in the IFN pathway, was the target with which Rubicon interacted. Our results reveal a previously unrecognized function of Rubicon as a virus-induced protein that binds to NEMO, leading to the inhibition of type-I interferon production. Rubicon thus functions as an important negative regulator of the innate immune response, enhances viral replication and may play a role in viral immune evasion.

Ribosome Profiling Reveals Translational Upregulation of Cellular Oxidative Phosphorylation mRNAs during Vaccinia Virus-Induced Host Shutoff.

Vaccinia virus infection causes a host shutoff that is marked by global inhibition of host protein synthesis. Though the host shutoff may facilitate reallocation of cellular resources for viral replication and evasion of host antiviral immune responses, it poses a challenge for continuous synthesis of cellular proteins that are important for viral replication. It is, however, unclear whether and how certain cellular proteins may be selectively synthesized during the vaccinia virus-induced host shutoff. Using simultaneous RNA sequencing and ribosome profiling, two techniques quantifying genome-wide levels of mRNA and active protein translation, respectively, we analyzed the responses of host cells to vaccinia virus infection at both the transcriptional and translational levels. The analyses showed that cellular mRNA depletion played a dominant role in the shutoff of host protein synthesis. Though the cellular mRNAs were significantly reduced, the relative translation efficiency of a subset of cellular mRNAs increased, particularly those involved in oxidative phosphorylation that are responsible for cellular energy production. Further experiments demonstrated that the protein levels and activities of oxidative phosphorylation increased during vaccinia virus infection, while inhibition of the cellular oxidative phosphorylation function significantly suppressed vaccinia virus replication. Moreover, the short 5' untranslated region of the oxidative phosphorylation mRNAs contributed to the translational upregulation. These results provide evidence of a mechanism that couples translational control and energy metabolism, two processes that all viruses depend on host cells to provide, to support vaccinia virus replication during a host shutoff.IMPORTANCE Many viral infections cause global host protein synthesis shutoff. While host protein synthesis shutoff benefits the virus by relocating cellular resources to viral replication, it also poses a challenge to the maintenance of cellular functions necessary for viral replication if continuous protein synthesis is required. Here we measured the host mRNA translation rate during a vaccinia virus-induced host shutoff by analyzing total and actively translating mRNAs in a genome-wide manner. This study revealed that oxidative phosphorylation mRNAs were translationally upregulated during vaccinia virus-induced host protein synthesis shutoff. Oxidative phosphorylation is the major cellular energy-producing pathway, and we further showed that maintenance of its function is important for vaccinia virus replication. This study highlights the fact that vaccinia virus infection can enhance cellular energy production through translational upregulation in the context of an overall host protein synthesis shutoff to meet energy expenditure.

Chronic airway inflammation provides a unique environment for B cell activation and antibody production.

B cells play many roles in health and disease. However, little is known about the mechanisms that drive B cell responses in the airways, especially in humans. Chronic rhinosinusitis (CRS) is an inflammatory disease of the upper airways that affects 10% of Europeans and Americans. A subset of CRS patients develop nasal polyps (NPs), which are characterized by type 2 inflammation, eosinophils and group 2 innate lymphoid cells (ILC2s). We have reported that NP contain elevated levels of B cells and antibodies, making NP an ideal system for studying B cells in the airways. We sought to determine the mechanisms that drive B cell activation and antibody production during chronic airway inflammation. We analysed B cells from NP or tonsil, or after ILC2 coculture, by flow cytometry. Antibody production from tissue was measured using Luminex assays and the frequency of antibody-secreting cells by ELISpot. Formation of B cell clusters was assessed using immunohistochemistry. Expression of genes associated with B cell activation and class switch recombination was measured by qRT-PCR. NP contained significantly elevated frequencies of plasmablasts, especially those that expressed the extrafollicular marker Epstein-Barr virus-induced protein 2 (EBI2), but significantly fewer germinal centre (GC) B cells compared with tonsil. Antibody production and the frequency of antibody-secreting cells were significantly elevated in NP, and there was evidence for local class switch recombination in NP. Finally, ILC2s directly induced EBI2 expression on B cells in vitro. Our data suggest there is a unique B cell activation environment within NP that is distinct from classic GC-mediated mechanisms. We show for the first time that ILC2s directly induce EBI2 expression on B cells, indicating that ILC2s may play an important role in B cell responses. B cell-targeted therapies may provide new treatment options for CRSwNP.

Ezrin and HNRNP expression correlate with increased virus release rate and early onset of virus-induced apoptosis of MDCK suspension cells.

With the aim to adapt high-yield adherent cell lines to suspension growth, Madin Darby canine kidney (MDCK) suspension cells were developed recently that achieved comparable influenza virus yields despite an early induction of apoptosis compared to the parental adherent cell line. For both cell lines, a comprehensive study under comparable infection conditions is performed comprising information on: time course of viral infection, antiviral state of cells, virus-induced apoptosis, and virus-induced cellular protein expression for early and late infection with influenza A/PuertoRico/8/34 H1N1. The proteomic analysis is performed with 2D differential gel electrophoreses followed by mass spectrometry. Based on flow cytometric data and on the differential expression of various stress and apoptosis-related proteins, the earlier onset of virus-induced apoptosis is confirmed for suspension cells. Surprisingly, the data indicated an increased virus release rate for suspension cells. These observations correlate with an increased expression of the apical marker protein ezrin, known to play a role in influenza-induced cytoskeletal rearrangement, and the differential expression of heterogeneous nuclear ribonucleoproteins, known to bind actively influenza viral proteins and play a central role in regulating gene expression. Based on these findings, additional studies towards the design of MDCK suspension cells with further increase in influenza virus yields will be performed.

Group 2 Innate Lymphoid Cells Directly Induce B Cell Activation in Humans

Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by type 2 inflammation and increased group 2 innate lymphoid cells (ILC2). We have also found elevated B cells and overexpression of the extrafollicular B cell marker Epstein-Barr virus-induced protein 2 (EBI2). We investigated whether B cells in polyps expressed EBI2, indicating an extrafollicular phenotype, and whether ILC2s could regulate B cell activation. EBI2 expression on cells from polyps of CRSwNP patients or tonsils from non-CRS patients was assessed by flow cytometry. Antibody production by tissue B cells was assessed by Luminex assay. In other experiments, B cells and ILC2s were isolated from peripheral blood by magnetic bead- and flow cytometry-based methods, respectively. B cells were cultured for 5 days under IgG-promoting conditions (IL-2 and R848 (a TLR7/8 agonist)), or IgE-promoting conditions (IL-4 and anti-CD40), or with autologous ILC2s at a 1:1 ratio, or with IL-2 alone. Polyps contained elevated levels of plasmablasts (PB) and EBI2+ PB compared to tonsil (>5 fold, p<0.001 and >2.5 fold, p<0.01, respectively). In vitro, polyps also produced significantly higher levels of antibodies (p<0.01). IL-4 and anti-CD40 increased B cell survival and EBI2 expression (>5 fold), indicating in vitro development of extrafollicular PB, while IL-2 and R848 had no effect on EBI2. Co-culture of B cells with ILC2s increased B cell survival and promoted a significant increase of EBI2+ B cells (>5 fold, p<0.05). These findings suggest that type 2 inflammation, particularly ILC2s, may play an important role in B cell activation during chronic airway inflammation.

ID: 113: STING, regulated by RIG-I signaling, contributes to 5’ppp RNA restriction of HSV via sustaining IFN response

RIG-I is essential for recognizing RNA viruses with a 5’ triphosphate (ppp) signature in the cytoplasm. Upon viral RNA recognition, RIG-I recruits adaptor protein MAVS to trigger the activation of IRF3 and NF-kB transcription factors, leading to activation of antiviral response. STING has been identified as an RIG-I signaling cofactor and a critical adaptor protein required for cytosolic DNA and cyclic dinucleotides (CDNs) triggered immune responses. We have identified STING among a plethora of differentially expressed genes induced by the 5’ppp RNA; in the present study, we further detail the mechanism of STING regulation. Our data shows that virus infection induces STING expression at both the mRNA and protein levels. Furthermore STING induction is shown to be dependent on functional RIG-I signaling. STING induction by the RIG-I agonist 5’ppp RNA was recognized as a delayed event resulting from an autocrine/paracrine mechanism. Indeed, co-treatment with TNF α and IFN α has a synergistic effect on the regulation of STING expression. Depletion of STAT1 and RELA, either individually or combined, significantly diminished virus-induced STING protein expression compared with the cells. In A549 cells, the expression of interferon-response/inflammatory response genes was not affected in the absence of STING at early time points, but was significantly reduced at late time points. Furthermore, our results also unveiled an essential contribution of STING in the establishment of the 5’pppRNA induced antiviral responses during HSV1 infection. Taken together, these observations demonstrate that the STING is induced via RIG-I signaling and up-regulated STING is essential for 5’ppp RNA mediated HSV restriction.

The virus-induced protein APOBEC3G inhibits anoikis by activation of Akt kinase in pancreatic cancer cells.

Pancreatic cancer is one of the more common cancers with a poor prognosis. Some varieties of cancer are related to virus infection. As a virus-induced protein, APOBEC3G (A3G) presents extensive anti-virus ability, but the role of A3G in pancreatic cancer was previously unknown. The expression of A3G in pancreatic cancer was examined using TaqMan real-time qPCR, immunohistochemical and immunofluorescent staining. Subsequently, the role of A3G in pancreatic cancer was evaluated in vivo using the tumor xenograft model. Anoikis was detected by colony formation assay and flow cytometry in vitro. The Akt kinase activity and target protein PTEN were examined by co-immunoprecipitation and immunoblot. The virus-induced protein A3G was significantly up-regulated in pancreatic cancer, and the up-regulation of A3G promoted xenograft tumor formation. A3G inactivated PTEN by binding to the C2 tensin-type and PDZ domains, thereby inducing anoikis resistance through Akt activation. Our results demonstrate that the up-regulation of A3G in pancreatic cancer cells induces anoikis resistance, and they provide novel insight into the mechanism by which A3G affects the malignant behavior of pancreatic cancer cells.

The biology of interleukin-27 reveals unique pro- and anti-inflammatory functions in immunity

Interleukin (IL)-27 is a multifaceted heterodimeric cytokine with pronounced pro- and anti-inflammatory as well as immunoregulatory functions. It consists of the two subunits p28/IL-30 and Epstein Bar virus-induced protein 3 (EBI3). EBI3 functions as a soluble α-receptor, and IL-27 can therefore directly activate its target cells through a heterodimer of glycoprotein 130 (gp130) and WSX-1. Being a heterodimeric cytokine that signals through gp130, IL-27 is either grouped into the IL-6 or the IL-12 family of cytokines. Originally identified as an IL-12-like cytokine that induces proliferation of CD4+ T cells and production of IFN-γ more than ten years ago, subsequent research revealed a much broader role of IL-27 in inflammation, cancer development and regulation and differentiation of immune cells. In this review, we summarize the current biochemical and molecular knowledge about the signal transduction of IL-27. Based on this, we highlight functional overlaps and plasticity with other cytokines and cytokine receptors of the IL-6/IL-12 superfamily, and describe the important role of IL-27 with regard to the differentiation of T cells, infections and cancer development. We further discuss IL-27 as a therapeutic target and how specific blockade of this cytokine could be achieved.

Chronic upper airway inflammation is characterized by an extrafollicular B cell response and elevated antibody production in humans (HUM1P.260)

Abstract Chronic rhinosinusitis (CRS) is an upper airway disease that affects 30 million Americans and a subgroup of CRS patients also has nasal polyps (CRSwNP). In CRSwNP patients, inflamed sinus tissues can be removed as part of treatment, which provides a unique model system to investigate mechanisms of human mucosal inflammation. We have previously reported local elevations in NP of B cells, antibodies (Ab), and expression of Epstein-Barr virus-Induced protein 2 (EBI2), a marker of extrafollicular plasma cells (PC). To characterize the mechanisms mediating these responses, we assessed B cells from fresh tissue (d0) or after 4 days of culture (d4) from NP, uncinate tissue (UT) or tonsils from CRS and control subjects by flow cytometry. We measured Ab production by multiplex array and assessed formation of B cell follicles by immunohistochemistry. We assessed the Ab repertoire by DNA deep sequencing and by analysis of Ab genes from single cells. We found that NP uniquely supported the accumulation of PC (p&amp;lt;0.01) and Ab production (p&amp;lt;0.05) in vitro. Plasmablasts (PB) and PC were Ki67+, and EBI2 expression was highest on PB. We found no evidence for increased B cell follicles in NP compared to control UT (n&amp;gt;10/group). Ab from NP had fewer CDR3 mutations than Ab from UT (p&amp;lt;0.05), but more mutations than Ab from peripheral blood (p&amp;lt;0.05). These data suggest that there is a strong extrafollicular B cell response in NP that may drive Ab production during chronic airway inflammation.