Inhibition of interferon regulatory factor 7 (IRF7)-mediated interferon signal transduction by the Kaposi's sarcoma-associated herpesvirus viral IRF homolog vIRF3

Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway that is mediated by IFN regulatory factors (IRFs). In order to complete their life cycle, viruses must modulate the host IFN-mediated immune response. Kaposi's sarcoma-associated herpesvirus (KSHV), a human tumor-inducing herpesvirus, has developed a unique mechanism for antagonizing cellular IFN-mediated antiviral activity by incorporating viral homologs of the cellular IRFs, called vIRFs. Here, we report a novel immune evasion mechanism of KSHV vIRF3 to block cellular IRF7-mediated innate immunity in response to viral infection. KSHV vIRF3 specifically interacts with either the DNA binding domain or the central IRF association domain of IRF7, and this interaction leads to the inhibition of IRF7 DNA binding activity and, therefore, suppression of alpha interferon (IFN-alpha) production and IFN-mediated immunity. Remarkably, the central 40 amino acids of vIRF3, containing the double alpha helix motifs, are sufficient not only for binding to IRF7, but also for inhibiting IRF7 DNA binding activity. Consequently, the expression of the double alpha helix motif-containing peptide effectively suppresses IRF7-mediated IFN-alpha production. This demonstrates a remarkably efficient means of viral avoidance of host antiviral activity.     

Epstein-Barr Virus BGLF4 Kinase Suppresses the Interferon Regulatory Factor 3 Signaling Pathway

The BGLF4 protein kinase of Epstein-Barr virus (EBV) is a member of the conserved family of herpesvirus protein kinases which, to some extent, have a function similar to that of the cellular cyclin-dependent kinase in regulating multiple cellular and viral substrates. In a yeast two-hybrid screening assay, a splicing variant of interferon (IFN) regulatory factor 3 (IRF3) was found to interact with the BGLF4 protein. This interaction was defined further by coimmunoprecipitation in transfected cells and glutathione S-transferase (GST) pull-down in vitro. Using reporter assays, we show that BGLF4 effectively suppresses the activities of the poly(I:C)-stimulated IFN-β promoter and IRF3-responsive element. Moreover, BGLF4 represses the poly(I:C)-stimulated expression of endogenous IFN-β mRNA and the phosphorylation of STAT1 at Tyr701. In searching for a possible mechanism, BGLF4 was shown not to affect the dimerization, nuclear translocation, or CBP recruitment of IRF3 upon poly(I:C) treatment. Notably, BGLF4 reduces the amount of active IRF3 recruited to the IRF3-responsive element containing the IFN-β promoter region in a chromatin immunoprecipitation assay. BGLF4 phosphorylates GST-IRF3 in vitro, but Ser339-Pro340 phosphorylation-dependent, Pin1-mediated downregulation is not responsible for the repression. Most importantly, we found that three proline-dependent phosphorylation sites at Ser123, Ser173, and Thr180, which cluster in a region between the DNA binding and IRF association domains of IRF3, contribute additively to the BGLF4-mediated repression of IRF3(5D) transactivation activity. IRF3 signaling is activated in reactivated EBV-positive NA cells, and the knockdown of BGLF4 further stimulates IRF3-responsive reporter activity. The data presented here thus suggest a novel mechanism by which herpesviral protein kinases suppress host innate immune responses and facilitate virus replication.     

Role of Interferon Regulatory Factor 7 in T Cell Responses during Acute Lymphocytic Choriomeningitis Virus Infection

Type I interferons (IFNs), predominantly IFN-α and -β, play critical roles in both innate and adaptive immune responses against viral infections. Interferon regulatory factor 7 (IRF7), a key innate immune molecule in the type I IFN signaling pathway, is essential for the type I IFN response to many viruses, including lymphocytic choriomeningitis virus (LCMV). Here, we show that although IRF7 knockout (KO) mice failed to control the replication of LCMV in the early stages of infection, they were capable of clearing LCMV infection. Despite the lack of type I IFN production, IRF7 KO mice generated normal CD4⁺ T cell responses, and the expansion of naïve CD8⁺ T cells into primary CD8⁺ T cells specific for LCMV GP_33–41 was relatively normal. In contrast, the expansion of the LCMV NP₃₉₆-specific CD8⁺ T cells was severely impaired in IRF7 KO mice. We demonstrated that this defective CD8⁺ T cell response is due neither to an impaired antigen-presenting system nor to any intrinsic role of IRF7 in CD8⁺ T cells. The lack of a type I IFN response in IRF7 KO mice did not affect the formation of memory CD8⁺ T cells. Thus, the present study provides new insight into the impact of the innate immune system on viral pathogenesis and demonstrates the critical contribution of innate immunity in controlling virus replication in the early stages of infection, which may shape the quality of CD8⁺ T cell responses.     

TRIM56 Is an Essential Component of the TLR3 Antiviral Signaling Pathway

Members of the tripartite motif (TRIM) proteins are being recognized as important regulators of host innate immunity. However, specific TRIMs that contribute to TLR3-mediated antiviral defense have not been identified. We show here that TRIM56 is a positive regulator of TLR3 signaling. Overexpression of TRIM56 substantially potentiated extracellular dsRNA-induced expression of interferon (IFN)-β and interferon-stimulated genes (ISGs), while knockdown of TRIM56 greatly impaired activation of IRF3, induction of IFN-β and ISGs, and establishment of an antiviral state by TLR3 ligand and severely compromised TLR3-mediated chemokine induction following infection by hepatitis C virus. The ability to promote TLR3 signaling was independent of the E3 ubiquitin ligase activity of TRIM56. Rather, it correlated with a physical interaction between TRIM56 and TRIF. Deletion of the C-terminal portion of TRIM56 abrogated the TRIM56-TRIF interaction as well as the augmentation of TLR3-mediated IFN response. Together, our data demonstrate TRIM56 is an essential component of the TLR3 antiviral signaling pathway and reveal a novel role for TRIM56 in innate antiviral immunity.     

Implication of PMLIV in Both Intrinsic and Innate Immunity

PML/TRIM19, the organizer of nuclear bodies (NBs), has been implicated in the antiviral response to diverse RNA and DNA viruses. Several PML isoforms generated from a single PML gene by alternative splicing, share the same N-terminal region containing the RBCC/tripartite motif but differ in their C-terminal sequences. Recent studies of all the PML isoforms reveal the specific functions of each. The knockout of PML renders mice more sensitive to vesicular stomatitis virus (VSV). Here we report that among PML isoforms (PMLI to PMLVIIb), only PMLIII and PMLIV confer resistance to VSV. Unlike PMLIII, whose anti-VSV activity is IFN-independent, PMLIV can act at two stages: it confers viral resistance directly in an IFN-independent manner and also specifically enhances IFN-β production via a higher activation of IRF3, thus protecting yet uninfected cells from oncoming infection. PMLIV SUMOylation is required for both activities. This demonstrates for the first time that PMLIV is implicated in innate immune response through enhanced IFN-β synthesis. Depletion of IRF3 further demonstrates the dual activity of PMLIV, since it abrogated PMLIV-induced IFN synthesis but not PMLIV-induced inhibition of viral proteins. Mechanistically, PMLIV enhances IFN-β synthesis by regulating the cellular distribution of Pin1 (peptidyl-prolyl cis/trans isomerase), inducing its recruitment to PML NBs where both proteins colocalize. The interaction of SUMOylated PMLIV with endogenous Pin1 and its recruitment within PML NBs prevents the degradation of activated IRF3, and thus potentiates IRF3-dependent production of IFN-β. Whereas the intrinsic antiviral activity of PMLIV is specific to VSV, its effect on IFN-β synthesis is much broader, since it affects a key actor of innate immune pathways. Our results show that, in addition to its intrinsic anti-VSV activity, PMLIV positively regulates IFN-β synthesis in response to different inducers, thus adding PML/TRIM19 to the growing list of TRIM proteins implicated in both intrinsic and innate immunity.     

Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

The baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) has been widely used to achieve a high level of foreign gene expression in insect cells, as well as for efficient gene transduction into mammalian cells without any replication. In addition to permitting efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. In this study, we examined the effects of the innate immune responses on gene expression by recombinant baculoviruses in cultured cells. The reporter gene expression in IRF3-deficient mouse embryonic fibroblasts (MEFs) infected with the recombinant baculovirus was shown to be enhanced in accordance with the suppression of beta interferon (IFN-β) production. Furthermore, efficient gene transduction by the recombinant baculovirus was achieved in MEFs deficient for stimulator of interferon genes (STING), TANK binding kinase 1 (TBK1), IFN regulatory factor 3 (IRF3), or IFN-β promoter stimulator 1 (IPS-1), but not in those deficient for IRF7, MyD88, or Z-DNA binding protein 1 (ZBP1)/DAI. Enhancement of gene expression by the recombinant baculovirus was also observed in human hepatoma cell lines replicating hepatitis C virus (HCV), in which innate immunity was impaired by the cleavage of IPS-1 by the viral protease. In addition, infection with the recombinant baculovirus expressing the BH3-only protein, BIM_S, a potent inducer of apoptosis, resulted in a selective cell death in the HCV replicon cells. These results indicate that innate immune responses induced by infection with baculovirus attenuate transgene expression, and this characteristic might be useful for a selective gene transduction into cells with impaired innate immunity arising from infection with various viruses.     

HSV-2 Increases TLR4-Dependent Phosphorylated IRFs and IFN-β Induction in Cervical Epithelial Cells

Our previous studies demonstrated that HSV-2 infection up-regulates TLR4 expression and induces NF-kB activity, thereby facilitating innate immune response in human cervical epithelial cells. This process requires involvement of TLR4 adaptors, Mal and MyD88. In the current study, we found that HSV-2 infection increases levels of phosphoryalted IRF3 and IRF7, then regulating expression of type I IFN. As expected, these changes induced by HSV-2 infection depended upon TLR4. Knockdown of TRIF and/or TRAM by siRNAs indicated that TRIF/TRAM might be involved in expression of IFN-β. Our results demonstrate for the first time that IRF3 and IRF7 are both involved in inducing TLR4-dependent IFN-β expression in response to HSV-2 in its primary infected genital epithelial cells. Thus, TLR4-Mal/MyD88 and TLR4-TRIF/TRAM signaling may synergize and/or cooperate in innate immune response of cervical epithelial cells to HSV-2 infection.     

Hepatitis B Virus Polymerase Blocks Pattern Recognition Receptor Signaling via Interaction with DDX3: Implications for Immune Evasion

Viral infection leads to induction of pattern-recognition receptor signaling, which leads to interferon regulatory factor (IRF) activation and ultimately interferon (IFN) production. To establish infection, many viruses have strategies to evade the innate immunity. For the hepatitis B virus (HBV), which causes chronic infection in the liver, the evasion strategy remains uncertain. We now show that HBV polymerase (Pol) blocks IRF signaling, indicating that HBV Pol is the viral molecule that effectively counteracts host innate immune response. In particular, HBV Pol inhibits TANK-binding kinase 1 (TBK1)/IκB kinase-ε (IKKε), the effector kinases of IRF signaling. Intriguingly, HBV Pol inhibits TBK1/IKKε activity by disrupting the interaction between IKKε and DDX3 DEAD box RNA helicase, which was recently shown to augment TBK1/IKKε activity. This unexpected role of HBV Pol may explain how HBV evades innate immune response in the early phase of the infection. A therapeutic implication of this work is that a strategy to interfere with the HBV Pol-DDX3 interaction might lead to the resolution of life-long persistent infection.     

Human cytomegalovirus blocks canonical TGFβ signaling during lytic infection to limit induction of type I interferons

Human cytomegalovirus (HCMV) microRNAs (miRNAs) significantly rewire host signaling pathways to support the viral lifecycle and regulate host cell responses. Here we show that SMAD3 expression is regulated by HCMV miR-UL22A and contributes to the IRF7-mediated induction of type I IFNs and IFN-stimulated genes (ISGs) in human fibroblasts. Addition of exogenous TGFβ interferes with the replication of a miR-UL22A mutant virus in a SMAD3-dependent manner in wild type fibroblasts, but not in cells lacking IRF7, indicating that downregulation of SMAD3 expression to limit IFN induction is important for efficient lytic replication. These findings uncover a novel interplay between SMAD3 and innate immunity during HCMV infection and highlight the role of viral miRNAs in modulating these responses.     

Respiratory Syncytial Virus Induced Type I IFN Production by pDC Is Regulated by RSV-Infected Airway Epithelial Cells,RSV-Exposed Monocytes and Virus Specific Antibodies

Innate immune responses elicited upon virus exposure are crucial for the effective eradication of viruses, the onset of adaptive immune responses and for establishing proper immune memory. Respiratory syncytial virus (RSV) is responsible for a high disease burden in neonates and immune compromised individuals, causing severe lower respiratory tract infections. During primary infections exuberant innate immune responses may contribute to disease severity. Furthermore, immune memory is often insufficient to protect during RSV re-exposure, which results in frequent symptomatic reinfections. Therefore, identifying the cell types and pattern recognition receptors (PRRs) involved in RSV-specific innate immune responses is necessary to understand incomplete immunity against RSV. We investigated the innate cellular response triggered upon infection of epithelial cells and peripheral blood mononuclear cells. We show that CD14⁺ myeloid cells and epithelial cells are the major source of IL-8 and inflammatory cytokines, IL-6 and TNF-α, when exposed to live RSV Three routes of RSV-induced IFN-α production can be distinguished that depend on the cross-talk of different cell types and the presence or absence of virus specific antibodies, whereby pDC are the ultimate source of IFN-α. RSV-specific antibodies facilitate direct TLR7 access into endosomal compartments, while in the absence of antibodies, infection of monocytes or epithelial cells is necessary to provide an early source of type I interferons, required to engage the IFN-α,β receptor (IFNAR)-mediated pathway of IFN-α production by pDC. However, at high pDC density infection with RSV causes IFN-α production without the need for a second party cell. Our study shows that cellular context and immune status are factors affecting innate immune responses to RSV. These issues should therefore be addressed during the process of vaccine development and other interventions for RSV disease.     

pMGF505-7R determines pathogenicity of African swine fever virus infection by inhibiting IL-1β and type I IFN production

Inflammatory factors and type I interferons (IFNs) are key components of host antiviral innate immune responses, which can be released from the pathogen-infected macrophages. African swine fever virus (ASFV) has developed various strategies to evade host antiviral innate immune responses, including alteration of inflammatory responses and IFNs production. However, the molecular mechanism underlying inhibition of inflammatory responses and IFNs production by ASFV-encoded proteins has not been fully understood. Here we report that ASFV infection only induced low levels of IL-1β and type I IFNs in porcine alveolar macrophages (PAMs), even in the presence of strong inducers such as LPS and poly(dA:dT). Through further exploration, we found that several members of the multigene family 360 (MGF360) and MGF505 strongly inhibited IL-1β maturation and IFN-β promoter activation. Among them, pMGF505-7R had the strongest inhibitory effect. To verify the function of pMGF505-7R in vivo, a recombinant ASFV with deletion of the MGF505-7R gene (ASFV-Δ7R) was constructed and assessed. As we expected, ASFV-Δ7R infection induced higher levels of IL-1β and IFN-β compared with its parental ASFV HLJ/18 strain. ASFV infection-induced IL-1β production was then found to be dependent on TLRs/NF-κB signaling pathway and NLRP3 inflammasome. Furthermore, we demonstrated that pMGF505-7R interacted with IKKα in the IKK complex to inhibit NF-κB activation and bound to NLRP3 to inhibit inflammasome formation, leading to decreased IL-1β production. Moreover, we found that pMGF505-7R interacted with and inhibited the nuclear translocation of IRF3 to block type I IFN production. Importantly, the virulence of ASFV-Δ7R is reduced in piglets compared with its parental ASFV HLJ/18 strain, which may due to induction of higher IL-1β and type I IFN production in vivo. Our findings provide a new clue to understand the functions of ASFV-encoded pMGF505-7R and its role in viral infection-induced pathogenesis, which might help design antiviral agents or live attenuated vaccines to control ASF.