Interferon regulatory factor 7 mediates activation of Tap-2 by Epstein-Barr virus latent membrane protein 1

Transporter associated with antigen processing 2 (Tap-2) is responsible for ATP-dependent transport of peptides from the cytosol to the endoplasmic reticulum, where peptides bind to newly synthesized human leukocyte antigen (HLA) class I molecules, which are essential for cellular immune responses. Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) has been shown to induce the expression of Tap-2. In this study, the induction of endogenous Tap-2 by LMP-1 is shown to be associated with and requires the expression of interferon regulatory factor 7 (IRF-7). In DG75 Burkitt's lymphoma (BL) cells, in which LMP-1 induces the expression of IRF-7, LMP-1 induced endogenous Tap-2, and ectopic expression of IRF-7 could enhance the induction. In Akata BL cells, in which LMP-1 could not induce IRF-7, LMP-1 could not induce Tap-2. Addition of IRF-7, which complements the defect in Akata cells, could stimulate the expression of Tap-2. Furthermore, LMP-1 and IRF-7A but not other IRF-7 splicing variants could activate endogenous Tap-2. A Tap-2 promoter reporter construct could be activated by the overexpression of IRF-7A. The activation could be specifically enhanced by LMP-1 and was dependent on an intact interferon-stimulated response element (ISRE) present in the Tap-2 promoter. Also, IRF-7 can bind to the Tap-2 promoter under physiological conditions in vivo, as shown by formaldehyde cross-linking, as well as to the Tap-2 ISRE in vitro, as shown by gel mobility shift assays. Furthermore, LMP-1 facilitates the phosphorylation and nuclear translocation of IRF-7. These data point to the role of IRF-7 as a secondary mediator of LMP-1-activated signal transduction for Tap-2 as follows: LMP-1 stimulates the expression of IRF-7 and facilitates its phosphorylation and nuclear translocation, and then the activated IRF-7 mediates the activation of the cellular Tap-2 gene. The induction of Tap-2 by IRF-7 and LMP-1 may have an important implication for the immune response to EBV and its persistence in vivo.     

Fas-associated death domain-containing protein-mediated antiviral innate immune signaling involves the regulation of Irf7

The induction of type I (alphabeta) IFN following virus infection is necessary for the stimulation of effective antiviral host defense. In fibroblasts, a subset of primary genes (including those encoding IFN-beta and IFN-alpha4) are induced directly by intracellular dsRNA generated by the virus during its replication. These primary type I IFNs induce expression of IFN regulatory factor (IRF)-7, required for production of a second cascade of IFN-alpha subtypes and the further establishment of a complete antiviral state. Previously, we had reported on a role for Fas-associated death domain-containing protein (FADD) in the control of TLR-independent innate immune responses to virus infection. Our data in this study demonstrate that FADD is not only required for efficient primary gene induction, but is also essential for induction of Irf7 and effective expression of secondary IFN-alphas and other antiviral genes. Ectopic overexpression of IRF-7 partially rescued dsRNA responsiveness and IFN-alpha production, and a constitutively active variant of IRF-7 displayed normal activity in Fadd(-/-) murine embryonic fibroblasts. MC159, a FADD-interacting viral protein encoded by the molluscum contagiosum poxvirus was found to inhibit dsRNA-activated signaling events upstream of IRF-7. These data indicate that FADD's antiviral activity involves regulation of IRF-7-dependent production of IFN-alpha subtypes and consequent induction of secondary antiviral genes.     

Inhibition of interferon signaling by the Kaposi's sarcoma-associated herpesvirus full-length viral interferon regulatory factor 2 protein

Interferon (IFN) signal transduction involves interferon regulatory factors (IRF). Kaposi's sarcoma-associated herpesvirus (KSHV) encodes four IRF homologues: viral IRF 1 (vIRF-1) to vIRF-4. Previous functional studies revealed that the first exon of vIRF-2 inhibited alpha/beta interferon (IFN-alpha/beta) signaling. We now show that full-length vIRF-2 protein, translated from two spliced exons, inhibited both IFN-alpha- and IFN-lambda-driven transactivation of a reporter promoter containing the interferon stimulated response element (ISRE). Transactivation of the ISRE promoter by IRF-1 was negatively regulated by vIRF-2 protein as well. Transactivation of a full-length IFN-beta reporter promoter by either IRF-3 or IRF-1, but not IRF-7, was also inhibited by vIRF-2 protein. Thus, vIRF-2 protein is an interferon induction antagonist that acts pleiotropically, presumably facilitating KSHV infection and dissemination in vivo.     

Interleukin-29 functions cooperatively with interferon to induce antiviral gene expression and inhibit hepatitis C virus replication

The interferon (IFN)-related cytokine interleukin (IL)-29 (also known as IFN-lambda1) inhibits virus replication by inducing a cellular antiviral response similar to that activated by IFN-alpha/beta. However, because it binds to a unique receptor, this cytokine may function cooperatively with IFN-alpha/beta or IFN-gamma during natural infections to inhibit virus replication, and might also be useful therapeutically in combination with other cytokines to treat chronic viral infections such as hepatitis C (HCV). We therefore investigated the ability of IL-29 and IFN-alpha or IFN-gamma to cooperatively inhibit virus replication and induce antiviral gene expression. Compared with the individual cytokines alone, the combination of IL-29 with IFN-alpha or IFN-gamma was more effective at blocking vesicular stomatitis virus and HCV replication, and this cooperative antiviral activity correlated with the magnitude of induced antiviral gene expression. Although the combined effects of IL-29 and IFN-alpha were primarily additive, the IL-29/IFN-gamma combination synergistically induced multiple genes and had the greatest antiviral activity. Two different mechanisms contributed to the enhanced gene expression induced by the cytokine combinations: increased activation of ISRE promoter elements and simultaneous activation of both ISRE and GAS elements within the same promoter. These findings provide new insight into the coregulation of a critical innate immune response by functionally distinct cytokine families.     

Selective DNA binding and association with the CREB binding protein coactivator contribute to differential activation of alpha/beta interferon genes by interferon regulatory factors 3 and 7

Recent studies implicate the interferon (IFN) regulatory factors (IRF) IRF-3 and IRF-7 as key activators of the alpha/beta IFN (IFN-alpha/beta) genes as well as the RANTES chemokine gene. Using coexpression analysis, the human IFNB, IFNA1, and RANTES promoters were stimulated by IRF-3 coexpression, whereas the IFNA4, IFNA7, and IFNA14 promoters were preferentially induced by IRF-7 only. Chimeric proteins containing combinations of different IRF-7 and IRF-3 domains were also tested, and the results provided evidence of distinct DNA binding properties of IRF-3 and IRF-7, as well as a preferential association of IRF-3 with the CREB binding protein (CBP) coactivator. Interestingly, some of these fusion proteins led to supraphysiological levels of IFN promoter activation. DNA binding site selection studies demonstrated that IRF-3 and IRF-7 bound to the 5'-GAAANNGAAANN-3' consensus motif found in many virus-inducible genes; however, a single nucleotide substitution in either of the GAAA half-site motifs eliminated IRF-3 binding and transactivation activity but did not affect IRF-7 interaction or transactivation activity. These studies demonstrate that IRF-3 possesses a restricted DNA binding site specificity and interacts with CBP, whereas IRF-7 has a broader DNA binding specificity that contributes to its capacity to stimulate delayed-type IFN gene expression. These results provide an explanation for the differential regulation of IFN-alpha/beta gene expression by IRF-3 and IRF-7 and suggest that these factors have complementary rather than redundant roles in the activation of the IFN-alpha/beta genes.     

Inhibitory activity of constitutive nitric oxide on the expression of alpha/beta interferon genes in murine peritoneal macrophages.

We investigated the role of the constitutive nitric oxide (NO) in the expression of interferon (IFN) genes in mouse peritoneal macrophages (PM). The treatment of PM with L-arginine-N(G)-amine (AA), a potent inhibitor of NO-producing enzymes, resulted in a marked accumulation of IFN-alpha4 mRNA and, to a minor extent, of IFN-beta mRNA. In contrast, the expression of IFN-gamma mRNA, as well as tumor necrosis factor alpha and interleukin-6 mRNA, was not affected. Furthermore, a remarkable increase in the expression of the IFN regulating factor 1 (IRF-1), but not of IRF-2, mRNA was detected in AA-treated PM. To investigate whether the AA-induced activation of the IFN system correlates with the production and antiviral activity of IFN, the extent of encephalomyocarditis virus (EMCV) replication was monitored in AA-treated PM with respect to control cultures. AA treatment strongly inhibited, in a dose-dependent manner, EMCV yields in PM. Likewise, similar results were obtained by the addition of the NO-scavenger carboxyphenyl-tetramethylimidazoline-oxyl-oxide. In addition, inhibition of NO synthesis by N(G)-mono-methyl-L-arginine in PM strongly decreased virus replication in coculture of PM and EMCV-infected L929 cells, whereas no antiviral effect was observed in L929 cells alone. Moreover, the AA-mediated antiviral activity was abrogated in the presence of antibody to IFN-alpha/beta, whereas antibody to IFN-gamma was completely ineffective. Taken together, these results indicate that low levels of NO, constitutively released by resting PM, negatively regulate the expression and activity of IFN-alpha/beta in PM. We suggest that NO acts as a homeostatic agent in the regulation of IFN pathway expression in macrophages.     

Genetic control directed toward spontaneous IFN-alpha/IFN-beta responses and downstream IFN-gamma expression influences the pathogenesis of a murine psoriasis-like skin disease

Psoriasis is an inflammatory skin disease, onset and severity of which are controlled by multiple genetic factors; aberrant expression of and responses to several cytokines including IFN-alpha/IFN-beta and IFN-gamma are associated with this "type 1" disease. However, it remains unclear whether genetic regulation influences these cytokine-related abnormalities. Mice deficient for IFN regulatory factor-2 (IRF-2) on the C57BL/6 background (IRF-2(-/-)BN mice) exhibited accelerated IFN-alpha/IFN-beta responses leading to a psoriasis-like skin inflammation. In this study, we found that this skin phenotype disappeared in IRF-2(-/-) mice with the BALB/c or BALB/c x C57BL/6 F(1) backgrounds. Genome-wide scan revealed two major quantitative trait loci controlled the skin disease severity. Interestingly, these loci were different from that for the defect in CD4(+) dendritic cells, another IFN-alpha/IFN-beta-dependent phenotype of the mice. Notably, IFN-gamma expression as well as spontaneous IFN-alpha/IFN-beta responses were up-regulated several fold spontaneously in the skin in IRF-2(-/-)BN mice but not in IRF-2(-/-) mice with "resistant" backgrounds. The absence of such IFN-gamma up-regulation in IRF-2(-/-)BN mice lacking the IFN-alpha/IFN-beta receptor or beta(2)-microglobulin indicated that accelerated IFN-alpha/IFN-beta signals augmented IFN-gamma expression by CD8(+) T cells in the skin. IFN-gamma indeed played pathogenic roles as skin inflammation was delayed and was much more infrequent when IRF-2(-/-)BN mice lacked the IFN-gamma receptor. Our current study thus revealed a novel genetic mechanism that kept the skin immune system under control and prevented skin inflammation through regulating the magnitude of IFN-alpha/IFN-beta responses and downstream IFN-gamma production, independently of CD4(+) dendritic cells.