Flavivirus induces interferon-beta gene expression through a pathway involving RIG-I-dependent IRF-3 and PI3K-dependent NF-kappaB activation

In this study, we found that infection with flaviviruses, such as Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2), leads to interferon-beta (IFN-beta) gene expression in a virus-replication- and de novo protein-synthesis-dependent manner. NF-kappaB activation is essential for IFN-beta induction in JEV- and DEN-2-infected cells. However, these two viruses seem to preferentially target different members of the interferon regulatory factor (IRF) family. The activation of constitutively expressed IRF-3, characterized by slower gel mobility, dimer formation, and nuclear translocation, is more evident in JEV-infected cells. Other members of the IRF family, such as IRF-1 and IRF-7 are also induced by DEN-2, but not by JEV infection. The upstream molecules responsible for IRF-3 and NF-kappaB activation were further studied. Evidently, a cellular RNA helicase, retinoic acid-inducible gene I (RIG-I), and a cellular kinase, phosphatidylinositol-3 kinase (PI3K), are required for flavivirus-induced IRF-3 and NF-kappaB activation, respectively. Therefore, we suggest that JEV and DEN-2 initiate the host innate immune response through a molecular mechanism involving RIG-I/IRF-3 and PI3K/NF-kappaB signaling pathways.     

Central role of interferon regulatory factor-1 (IRF-1) in controlling retinoic acid inducible gene-I (RIG-I) expression

Retinoic acid inducible gene-I (RIG-I) functions as the first line of defense against viral infection by sensing dsRNA and inducing type I interferon (IFN) production. The expression of RIG-I itself is induced by IFN-alpha/beta and dsRNA. To comprehend the molecular mechanism of expression regulation, we cloned the RIG-I promoter and analyzed its activity upon IFN-beta and dsRNA treatment. Under basal condition, RIG-I mRNA level and promoter activity were significantly higher in normal cells versus their tumor counterparts. In both normal and cancer cells, RIG-I expression was induced by IFN-beta and dsRNA. A single IRF-1 binding site in the proximal promoter functioned as a crucial regulator of basal, IFN-beta- and dsRNA-mediated induction of the RIG-I promoter. IFN-beta and dsRNA treatment increased IRF-1 binding to the RIG-I promoter. IRF-1 expression was also higher in normal cells than in cancer cells and it was induced by IFN-beta with similar kinetics as RIG-I. These results confirm that by controlling RIG-I expression, IRF-1 plays an essential role in anti-viral immunity. IRF-1 is a tumor suppressor and the expression profile of RIG-I together with its regulation by IRF-1 and the presence of a caspase-recruitment domain in RIG-I suggest that RIG-I might also possess tumor suppressor properties.     

Repression of IFN-gamma induction of class II transactivator: a role for PRDM1/Blimp-1 in regulation of cytokine signaling

MHC class II is expressed in restricted lineages and is modulated in response to pathogens and inflammatory stimuli. This expression is controlled by MHC CIITA, which is transcribed from multiple promoters. Although factors required for induction of CIITA are well characterized, less is known about the mechanisms leading to repression of this gene. During plasma cell differentiation, B lymphocyte-induced maturation protein-1 (PRDM1/Blimp-1) represses promoter (p)III of CIITA, responsible for constitutive expression in B cells. pIV is inducible by IFN-gamma in epithelia, macrophages and B cells. An IFN regulatory factor-element (IRF-E) in CIITA-pIV, which is bound by IRF-1 and IRF-2, is necessary for this response. This site matches the PRDM1/Blimp-1 consensus binding site, and PRDM1/Blimp-1 is expressed in cell lineages in which this promoter is operative. We, therefore, investigated whether PRDM1 regulates CIITA-pIV and found that PRDM1 bound to CIITA-pIV in vivo and the IRF-E in vitro. PRDM1 repressed IFN-gamma-mediated induction of a CIITA-pIV luciferase reporter in a fashion dependent on an intact consensus sequence and competes with IRF-1/IRF-2 for binding to the IRF-E and promoter activation. In human myeloma cell lines that express IRFs, PRDM1 occupancy of CIITA-pIV was associated with resistance to IFN-gamma stimulation, while short interfering RNA knockdown of PRDM1 led to up-regulation of CIITA. Our data indicate that PRDM1 is a repressor of CIITA-pIV, identifying a target of particular relevance to macrophages and epithelia. These findings support a model in which PRDM1/Blimp-1 can modulate the cellular response to IFN-gamma by competing with IRF-1/IRF-2 dependent activation of target promoters.     

The N- and C-terminal domains of the NS1 protein of influenza B virus can independently inhibit IRF-3 and beta interferon promoter activation

The NS1 proteins of influenza A and B viruses (A/NS1 and B/NS1 proteins) have only approximately 20% amino acid sequence identity. Nevertheless, these proteins show several functional similarities, such as their ability to bind to the same RNA targets and to inhibit the activation of protein kinase R in vitro. A critical function of the A/NS1 protein is the inhibition of synthesis of alpha/beta interferon (IFN-alpha/beta) during viral infection. Recently, it was also found that the B/NS1 protein inhibits IFN-alpha/beta synthesis in virus-infected cells. We have now found that the expression of the B/NS1 protein complements the growth of an influenza A virus with A/NS1 deleted. Expression of the full-length B/NS1 protein (281 amino acids), as well as either its N-terminal RNA-binding domain (amino acids 1 to 93) or C-terminal domain (amino acids 94 to 281), in the absence of any other influenza B virus proteins resulted in the inhibition of IRF-3 nuclear translocation and IFN-beta promoter activation. A mutational analysis of the truncated B/NS1(1-93) protein showed that RNA-binding activity correlated with IFN-beta promoter inhibition. In addition, a recombinant influenza B virus with NS1 deleted induces higher levels of IRF-3 activation, as determined by its nuclear translocation, and of IFN-alpha/beta synthesis than wild-type influenza B virus. Our results support the hypothesis that the NS1 protein of influenza B virus plays an important role in antagonizing the IRF-3- and IFN-induced antiviral host responses to virus infection.