Epstein-Barr virus BRLF1 inhibits transcription of IRF3 and IRF7 and suppresses induction of interferon-β

Activation of interferon regulatory factors (IRFs) 3 and 7 is essential for the induction of Type I interferons (IFN) and innate antiviral responses, and herpesviruses have evolved mechanisms to evade such responses. We previously reported that Epstein-Barr virus BZLF1, an immediate-early (IE) protein, inhibits the function of IRF7, but the role of BRLF1, the other IE transactivator, in IRF regulation has not been examined. We now show that BRLF1 expression decreased induction of IFN-β, and reduced expression of IRF3 and IRF7; effects were dependent on N- and C-terminal regions of BRLF1 and its nuclear localization signal. Endogenous IRF3 and IRF7 RNA and protein levels were also decreased during cytolytic EBV infection. Finally, production of IFN-β was decreased during lytic EBV infection and was associated with increased susceptibility to superinfection with Sendai virus. These data suggest a new role for BRLF1 with the ability to evade host innate immune responses.     

Hepatitis B virus X protein enhances activation of nuclear factor κB through interaction with valosin-containing protein

Hepatitis B virus X protein (HBx protein) is a multifunctional regulatory protein. The transactivation of nuclear factor kappa B (NF-κB) by HBx protein has been shown to be of importance in the pathogenesis of HBV-related diseases. However, the mechanism involved remains largely unclear. In this study, a CytoTrap yeast two-hybrid system was employed to screen binding partners of the HBx protein; 29 cellular proteins, including valosin-containing protein (VCP), were identified. The interaction between HBx protein and VCP was further confirmed in vitro and in vivo using a glutathione S-transferase pull-down assay and co-immunoprecipitation, respectively. It was also shown that this interaction is mediated by amino acid residues 51–120 of the HBx protein. In Huh-7 hepatoma cells, HBx protein enhanced the VCP-mediated activation of NF-κB. Our findings provide new insights into the molecular mechanisms that lead to the activation of NF-κB by HBx protein.     

Porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-β production by inhibiting IRF3 activation in immortalized porcine alveolar macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) infection appears to elicit a weak innate immune response suppressing type 1 interferon (IFN) production. Recent studies have revealed that several nonstructural proteins encoded by the PRRSV genome independently antagonize the type 1 IFN system. The present study sought to identify the structural proteins that possess the immune evasion properties in immortalized porcine alveolar macrophages (PAM). Each structural protein gene was stably expressed in a porcine monocyte-derived macrophage cell line, PAM-pCD163, and tested for its potential to inhibit IFN-β induction. We then focused on the nucleocapsid (N) protein, which has a strong inhibitory effect on dsRNA-induced IFN-β production. Upon dsRNA stimulation, IFN-β production was shown to decrease proportionally with increasing levels of N expression. Furthermore, the PRRSV N protein was found to down-regulate IFN-dependent gene production by dsRNA. Taken together, these results indicate the ability of N to modulate the dsRNA-mediated IFN induction pathways. In addition, the N protein significantly interfered with dsRNA-induced phosphorylation and nuclear translocation of IRF3. Our data suggest that the PRRSV N protein is a responsible component, independent of other nonstructural elements, for evading the IFN response by antagonizing IRF3 activation.     

SARS-CoV nucleocapsid protein antagonizes IFN-β response by targeting initial step of IFN-β induction pathway,and its C-terminal region is critical for the antagonism

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a highly basic nucleocapsid (N) protein which can inhibit the synthesis of type I interferon (IFN), but the molecular mechanism of this antagonism remains to be identified. In this study, we demonstrated that the N protein of SARS-CoV could inhibit IFN-beta (IFN-β) induced by poly(I:C) or Sendai virus. However, we found that N protein could not inhibit IFN-β production induced by overexpression of downstream signaling molecules of two important IFN-β induction pathways, toll-like receptor 3 (TLR3)- and RIG-I-like receptors (RLR)-dependent pathways. These results indicate that SARS-CoV N protein targets the initial step, probably the cellular PRRs (pattern recognition receptors)-RNAs-recognition step in the innate immune pathways, to suppress IFN expression responses. In addition, co-immunoprecipitation assays revealed that N protein did not interact with RIG-I or MDA5. Further, an assay using truncated mutants revealed that the C-terminal domain of N protein was critical for its antagonism of IFN induction, and the N deletion mutant impaired for RNA-binding almost completely lost the IFN-β antagonist activity. These results contribute to our further understanding of the pathogenesis of SARS-CoV.     

Interleukin-32 expression induced by hepatitis B virus protein X is mediated through activation of NF-κB

HBV replicates noncytopathically in hepatocytes, but HBV or proteins encoded by HBV genome could induce cytokines, chemokines expression by hepatocytes. Moreover, liver damage in patients with HBV infection is immune-mediated and cytokines play important roles in immune-mediated liver damage after HBV infection. Interleukin-32 (IL-32) is a proinflammatory cytokine and plays a critical role in inflammation. However, the role of HBV in IL-32 expression remains unclear. In the present study, we demonstrate that hepatitis B virus protein X (HBx) increases IL-32 expression through the promoter of IL-32 at positions from -746 to +25 and in a dose-dependent manner. Furthermore, we demonstrate that increase of NF-κB subunits p65 and p50 in Huh7 cells also augments IL-32 expression, and the NF-κB inhibitor blocks the effect of HBx on IL-32 induction. These results indicate that NF-κB activation is required for HBx-induced IL-32 expression. In conclusion, IL-32 is induced by HBx in Huh7 cells. Our results suggest that IL-32 might play an important role in inflammatory response after HBV infection.     

The NS1 protein of H5N6 feline influenza virus inhibits feline beta interferon response by preventing NF-κB and IRF3 activation

Despite the apparent lack of a feline influenza virus lineage, cats are susceptible to infection by influenza A viruses. Here, we characterized in vitro A/feline/Guangdong/1/2015, an H5N6 avian influenza virus recently isolated from cats. A/feline/Guangdong/1/2015 replicated to high titers and caused CPE in feline kidney cells. We determined that infection with A/feline/Guangdong/1/2015 did not activate the IFN-β promoter, but inhibited it by blocking the activation of NF-κB and IRF3. We also determined that the viral NS1 protein mediated the block, and that the dsRNA binding domain of NS1 was essential to perform this function. In contrast to treatment after infection, cells pretreated with IFN-β suppressed viral replication. Our findings provide an example of an H5N6 influenza virus suppressing IFN production, which might be associated with interspecies transmission of avian influenza viruses to cats.     

Nonstructural protein 1α subunit-based inhibition of NF-κB activation and suppression of interferon-β production by porcine reproductive and respiratory syndrome virus

Induction of type I interferon (IFN-α/β) is an early antiviral response of the host, and porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to downregulate the IFN response during infection in cells and pigs. We report that the PRRSV nonstructural protein 1α (Nsp1α) subunit of Nsp1 is a nuclear-cytoplasmic protein distributed to the nucleus and contains a strong suppressive activity for IFN-β production that is mediated through the retinoic acid-inducible gene I (RIG-I) signaling pathway. Nsp1α suppressed the activation of nuclear factor (NF)-κB when stimulated with dsRNA or tumor necrosis factor (TNF)-α, and NF-κB suppression was RIG-I-dependent. The suppression of NF-κB activation was associated with the poor production of IFN-β during PRRSV infection. The C-terminal 14 amino acids of the Nsp1α subunit were critical in maintaining immunosuppressive activity of Nsp1α for both IFN-β and NF-κB, suggesting that the newly identified zinc finger configuration comprising of Met180 may be crucial for inhibitory activities. Nsp1α inhibited IκB phosphorylation and as a consequence NF-κB translocation to the nucleus was blocked, leading to the inhibition of NF-κB stimulated gene expression. Our results suggest that PRRSV Nsp1α is a multifunctional nuclear protein participating in the modulation of the host IFN system.     

TLR3-mediated IFN-β gene induction is negatively regulated by the TLR adaptor MyD88 adaptor-like

There is limited insight into the mechanisms involved in the counterregulation of TLR. Given the important role of TLR3/TIR domain-containing adaptor-inducing IFN-β (TRIF)-dependent signalling in innate immunity, novel insights into its modulation is of significance in the context of many physiological and pathological processes. Herein, we sought to perform analysis to definitively assign a mechanistic role for MyD88 adaptor-like (Mal), an activator of TLR2/4 signalling, in the negative regulation of TLR3/TRIF signalling. Biochemical and functional analysis demonstrates that Mal negatively regulates TLR3, but not TLR4, mediated IFN-β production. Co-immunoprecipitation experiments demonstrate that Mal associates with IRF7 (IRF, IFN regulatory factor), not IRF3, and Mal specifically blocks IRF7 activation. In doing so, Mal impedes TLR3 ligand-induced IFN-β induction. Interestingly, Mal does not affect the induction of IL-6 and TNF-α upon TLR3 ligand engagement. Together, these data show that the TLR adaptor Mal interacts with IRF7 and, in doing so, impairs IFN-β induction through the positive regulatory domains I-III enhancer element of the IFN-β gene following poly(I:C) stimulation. Our findings offer a new mechanistic insight into TLR3/TRIF signalling through a hitherto unknown mechanism whereby Mal inhibits poly(I:C)-induced IRF7 activation and concomitant IFN-β production. Thus, Mal is essential in restricting TLR3 signalling thereby protecting the host from unwanted immunopathologies associated with excessive IFN-β production.     

Pellino3 ligase negatively regulates influenza B dependent RIG-I signalling through downregulation of TRAF3-mediated induction of the transcription factor IRF3 and IFNβ production

Viral infection activates the innate immune system, which recognizes viral components by a variety of pattern recognition receptors and initiates signalling cascades leading to the production of pro-inflammatory cytokines. To date, signalling cascades triggered after virus recognition are not fully characterized and are investigated by many research groups. The critical role of the E3 ubiquitin ligase Pellino3 in antibacterial and antiviral response is now widely accepted, but the precise mechanism remains elusive. In this study, we sought to explore Pellino3 role in the retinoic acid-inducible gene I (RIG-I)-dependent signalling pathway. In this work, the molecular mechanisms of the innate immune response, regulated by Pellino3, were investigated in lung epithelial cells during influenza B virus infection. We used wild-type and Pellino3-deficient A549 cells as model cell lines to examine the role of Pellino3 ligase in the type I interferon (IFN) signalling pathway. Our results indicate that Pellino3 is involved in direct ubiquitination and degradation of the TRAF3, suppressing interferon regulatory factor 3 (IRF3) activation and interferon beta (IFNβ) production.     

Vitamin K2 stimulates osteoblastogenesis and suppresses osteoclastogenesis by suppressing NF-κB activation

Several bone protective factors are reported to exhibit stimulatory activities on bone formation coupled with inhibitory effects on bone resorption; one such factor is vitamin K2. Vitamin K species [K1 (phylloquinone) and K2 (menaquinone)] have long been associated with bone protective activities and are receiving intense interest as nutritional supplements for the prevention or amelioration of bone disease in humans. However, the mechanisms of vitamin K action on the skeleton are poorly defined. Activation of the nuclear factor κB (NF-κB) signal transduction pathway is essential for osteoclast formation and resorption. By contrast, NF-κB signaling potently antagonizes osteoblast differentiation and function, prompting us to speculate that NF-κB antagonists may represent a novel class of dual anti-catabolic and pro-anabolic agents. We now show that vitamin K2 action on osteoblast and osteoclast formation and activity is accomplished by down-regulating basal and cytokine-induced NF-κB activation, by increasing IκB mRNA, in a γ-carboxylation-independent manner. Furthermore, vitamin K2 prevented repression by tumor necrosis factor α (TNFα) of SMAD signaling induced by either transforming growth factor ? (TGF?) or bone morphogenetic protein-2 (BMP-2). Vitamin K2 further antagonized receptor activator of NF-κB (RANK) ligand (RANKL)-induced NF-κB activation in osteoclast precursors. Our data provide a novel mechanism to explain the dual pro-anabolic and anti-catabolic activities of vitamin K2, and may further support the concept that pharmacological modulation of NF-κB signal transduction may constitute an effective mechanism for ameliorating pathological bone loss and for promoting bone health.     

Attaching and effacing bacterial effector NleC suppresses epithelial inflammatory responses by inhibiting NF-κB and p38 mitogen-activated protein kinase activation

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli are noninvasive attaching and effacing (A/E) bacterial pathogens that cause intestinal inflammation and severe diarrheal disease. These pathogens utilize a type III secretion system to deliver effector proteins into host epithelial cells, modulating diverse cellular functions, including the release of the chemokine interleukin-8 (IL-8). While studies have implicated the effectors NleE (non-locus of enterocyte effacement [LEE]-encoded effector E) and NleH1 in suppressing IL-8 release, by preventing NF-κB nuclear translocation, the impact of these effectors only partially replicates the immunosuppressive actions of wild-type EPEC, suggesting another effector or effectors are involved. Testing an array of EPEC mutants, we identified the non-LEE-encoded effector C (NleC) as also suppressing IL-8 release. Infection by ΔnleC EPEC led to exaggerated IL-8 release from infected Caco-2 and HT-29 epithelial cells. NleC localized to EPEC-induced pedestals, with signaling studies revealing NleC inhibits both NF-κB and p38 mitogen-activated protein kinase (MAPK) activation. Using Citrobacter rodentium, a mouse-adapted A/E bacterium, we found that ΔnleC and wild-type C. rodentium-infected mice carried similar pathogen burdens, yet ΔnleC strain infection led to worsened colitis. Similarly, infection with ΔnleC C. rodentium in a cecal loop model induced significantly greater chemokine responses than infection with wild-type bacteria. These studies thus advance our understanding of how A/E pathogens subvert host inflammatory responses.     

Porcine deltacoronavirus nucleocapsid protein antagonizes IFN-β production by impairing dsRNA and PACT binding to RIG-I

Virus Genes - Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that causes watery diarrhea, vomiting and mortality in newborn piglets. Previous studies have...     

Roles of TRAF2 and TRAF3 in Epstein-Barr virus latent membrane protein 1-induced alternative NF-κB activation

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1)-induced NF-κB activation is essential for EBV-transformed B cell survival. LMP1 has two C-terminal cytoplasmic domains referred to as C-Terminal Activation Regions (CTAR) 1 and 2 that activate the alternative and canonical NF-κB pathways, respectively. While CTAR2 activates TRAF6, IKKβ and IKKγ-dependent canonical NF-κB pathway, CTAR1 interacts with TRAF2 and TRAF3 and activates NIK and IKKα-dependent alternative NF-κB pathway involving p100 processing into functional p52. Using IKKα^−/−, IKKβ^−/−, IKKγ^−/−, TRAF2^−/−, TRAF3^−/−, TRAF6^−/−, and NIK^aly/aly mouse embryonic fibroblasts (MEFs), potential roles of these proteins in LMP1-induced alternative NF-κB activation were investigated. Deficiency in IKKα or functional NIK, but not in IKKβ, IKKγ, or TRAF6, severely impaired LMP1-induced p100 processing. Notably, p100 was constitutively processed in TRAF2^−/− or TRAF3^−/− MEFs independently of LMP1 suggesting that TRAF2 or TRAF3 may play a regulatory role in p100 processing. Subsequently, TRAF2 or TRAF3 over-expression in HEK293 cells significantly blocked LMP1-induced p100 processing. The LMP1 CTAR1 expression in 293HEK cells activated the alternative p65/p52 complex while CTAR2 failed to do so. Taken together, LMP1 activates alternative NF-κB pathway through functional NIK and IKKα that is regulated by TRAF2 or TRAF3.