Interferon establishes an antiviral state in numerous cell types through the induction of a set of immediateearly response genes. Activation of these genes is mediated by phosphorylation of latent transcription factors of the STAT family. We found that infection of primary foreskin fibroblasts with human cytomegalovirus (HCMV) causes selective transcriptional activation of the alpha/beta-interferon-responsive ISG54 gene. However, no activation or nuclear translocation of STAT proteins was detected. Activation of ISG54 occurs independent of protein synthesis but is prevented by protein tyrosine kinase inhibitors. Further analysis revealed that HCMV infection induced the DNA binding of a novel complex, tentatively called cytomegalovirus-induced interferonstimulated response element binding factor (CIF). CIF is composed, at least in part, of the recently identified interferon regulatory factor 3 (IRF3), but it does not contain the STAT1 and STAT2 proteins that participate in the formation of interferon-stimulated gene factor 3. IRF3, which has previously been shown to possess no intrinsic transcriptional activation potential, interacts with the transcriptional coactivator CREB binding protein, but not with p300, to form CIF. Activating interferon-stimulated genes without the need for prior synthesis of interferons might provide the host cell with a potential shortcut in the activation of its antiviral defense.Alpha interferon (IFN-␣) and IFN-␤ are unique among the continuously growing superfamily of cytokines in their ability to confer resistance to viral infection (20,36). The synthesis of IFN-␣ and IFN-␤ is induced at the transcriptional level after a cell encounters virus or double-stranded RNA (dsRNA) (16,46). The subsequent secretion of the newly produced interferons and their binding to a common cell surface receptor results in the induction of a set of immediate-early response genes (12, 21, 24-26, 32, 44, 47). The activation of these interferon-stimulated genes (ISGs) represents the first step towards the development of an antiviral state. Control over ISGs is exerted by an IFN-␣/␤-activated transcription factor complex termed interferon-stimulated gene factor 3 (ISGF3), which binds to a common enhancer element referred to as the interferon-stimulated response element (ISRE) (10,14,23,27,43). ISGF3 is formed through the interaction of the DNA binding subunit ISGF3␥ (p48) and the regulatory component ISGF3␣ (14,28,48), which itself is composed of two members of the STAT (signal transducers and activators of transcription) family of transcription factors, STAT1 and STAT2 (14,15,43). Both STAT proteins become tyrosine phosphorylated in response to IFN-␣/␤ stimulation, which enables their nuclear translocation and DNA binding (8,10,13,41). Transcriptionally active STAT1 has been shown to be a requirement for the antiviral and antiproliferative effects of IFN-␣/␤ (5, 11, 31). The phosphorylation of STAT1 and STAT2 is mediated through the action of two related tyrosine kinases, Jak1 and Tyk2, which are enzymatic...

No abstract

Pathogenic bacteria of the genus Yersinia employ a type III secretion system to inject bacterial effector proteins directly into the host cytosol. One of these effectors, the Yersinia serine/threonine protein kinase YpkA, is an essential virulence determinant involved in host actin cytoskeletal rearrangements and in inhibition of phagocytosis. Here we report that YpkA inhibits multiple Galphaq signaling pathways. The kinase activity of YpkA is required for Galphaq inhibition. YpkA phosphorylates Ser47, a key residue located in the highly conserved diphosphate binding loop of the GTPase fold of Galphaq. YpkA-mediated phosphorylation of Ser47 impairs guanine nucleotide binding by Galphaq. Y. pseudotuberculosis expressing wild-type YpkA, but not a catalytically inactive YpkA mutant, interferes with Galphaq-mediated signaling pathways. Identification of a YpkA-mediated phosphorylation site in Galphaq sheds light on the contribution of the kinase activity of YpkA to Yersinia pathogenesis.

Interferon regulatory factor 3 (IRF3) is known to participate in the transcriptional induction of interferon (IFN) ␣ and IFN␤ genes, as well as of a number of interferon-stimulated genes (ISGs), as a result of viral infection. In the present study we demonstrate the activation of IRF3 followed by ISG induction after exposure of cells to the bacterial cell wall component lipopolysaccharide. Engagement of Toll-like receptors by lipopolysaccharide triggered the nuclear translocation of IRF3, followed by its DNA binding and the subsequent induction of several interferon-regulated genes. Transcriptional activation of ISGs occurred in a protein synthesis independent manner, but was sensitive to inhibition of the stress-activated protein kinase, p38. The activation of IRF3 by viral particles or bacterial membrane components suggests that this signaling pathway might contribute to the evolutionary conserved innate immune response.Interferons ␣/␤ (IFN␣/␤) 1 induce gene expression by activating members of the signal transducers and activators of transcription (STAT) family of protein via their tyrosine phosphorylation, a process that involves the tyrosine kinases Jak1 and Tyk2 (1). Cooperative binding of STATs 1 and 2 in conjunction with p48 ISGF3␥ to the interferon-stimulated response element (ISRE), an IFN␣/␤ inducible enhancer, is necessary and sufficient for the induction of IFN␣/␤-stimulated gene expression (2-4).Numerous IFN␣/␤-induced genes (ISGs) were identified that contain an ISRE. They represent components of the antiviral defense such as the 2Ј-5Ј poly(A) synthase (5, 6) and the doublestranded RNA activated protein kinase (7), cell surface proteins such as the immunoglobulin (Ig) superfamily cell adhesion molecule (8, 9) or the major histocompatibility complex class I and II molecules (10), genes encoding chemokines such as the ISG15 and the IP10 gene (2, 11), as well as many other genes of yet unknown functions such as ISG54, ISG56 (4), GBP (12), or 6-16 (13).The DNA binding adapter p48 ISGF3␥ shares strong sequence homology in its DNA interaction domain with members of the interferon regulatory factor (IRF) family. Indeed, these proteins are able to bind to the ISRE and activate a specific subset of the genes typically activated by IFN␣/␤ (14, 15). The ubiquitously expressed IRF3 has been found to be an important cellular response factor to viral infection. We have previously demonstrated that infection of fibroblasts with human cytomegalovirus causes nuclear translocation of IRF3 and cooperative DNA binding with the transcriptional co-activator CBP/ p300 (16). This is followed by subsequent induction of a distinct subset of ISRE containing genes. Other labs reported similar observations after infection of cells with Newcastle disease virus or . Surprisingly, even transfection of cells with double-stranded RNA is able to trigger the formation of an IRF3 containing DNA binding complex (20). While the connection between viral infection and the interferon system is well established, it is largely unclear if or ...

Viruses utilize numerous mechanisms to counteract the host's immune response. Interferon production is a major component of the host antiviral response. Many viruses, therefore, produce proteins or RNA molecules that inhibit interferon-induced signal transduction pathways and their associated antiviral effects. Surprisingly, some viruses directly induce expression of interferon-induced genes. SM, an early lytic Epstein-Barr virus (EBV) nuclear protein, was found to specifically increase the expression of several genes (interferonstimulated genes) that are known to be strongly induced by alpha/beta interferons. SM does not directly stimulate alpha/beta interferon secretion but instead induces STAT1, an intermediate step in the interferon signaling pathway. SM is a posttranscriptional activator of gene expression and increases STAT1 mRNA accumulation, particularly that of the functionally distinct STAT1␤ splice variant. SM expression in B lymphocytes is associated with decreased cell proliferation but does not decrease cell viability or induce cell cycle arrest. These results indicate that EBV can specifically induce cellular genes that are normally physiological targets of interferon by inducing components of cytokine signaling pathways. Our findings therefore suggest that some aspects of the interferon response may be positively modulated by infecting viruses. Epstein-Barr virus (EBV), a human gammaherpesvirus, is the agent of infectious mononucleosis and is associated withBurkitt lymphoma, nasopharyngeal carcinoma, and lymphomas in immunosuppressed hosts (for a review, see reference 32). Infection by human herpesviruses of all classes specifically modulates cellular-gene expression. Because herpesviruses establish lifelong infections in the face of a competent immune system, many of the cellular genes affected are components of the innate or adaptive immune response. For example, an EBV immediate-early gene product inhibits gamma interferon (IFN-␥) signaling and down-regulates expression of the IFN-␥ receptor (42).The EBV SM protein is a posttranscriptional gene regulatory protein expressed early during lytic replication (9,12,14,53,66). Homologues of SM are found in herpes simplex virus (HSV), human cytomegalovirus (CMV), varicella-zoster virus, and Kaposi's sarcoma-associated virus (human herpesvirus 8) and act as transcriptional and posttranscriptional regulators (2,10,17,26,29,33,40). During lytic EBV replication, SM is expressed prior to other early genes but after the immediateearly genes BRLF1 and BZLF1. SM enhances the expression of several EBV genes and heterologous genes in cotransfection assays (30,31,39,52,55). Its ability to activate expression of cotransfected genes in a promoter-independent fashion has led to it being described as a promiscuous transactivator. Further studies demonstrated that several genes containing introns were inhibited by SM, whereas intronless genes were activated by SM (52). The majority of cellular genes and latent EBV genes are spliced, whereas most lytic EBV genes are n...

The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type Ⅲ secretion system (T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia effector proteins and their contribution to Yersinia pathogenesis.

IFN-regulatory factor 3 (IRF3) is known to participate in the transcriptional induction of chemokines and cytokines, including IFNs, as a result of viral or bacterial infection. In this study, we demonstrate that the LPS-mediated activation of IRF3 and subsequent induction of chemokine genes or IRF3-responsive reporter constructs are inhibited after exposure of human or murine macrophages to the Bacillus anthracis toxin lethal factor. The inhibitory effect is caused by interference with the activation of the stress-activated protein kinase, p38, due to a proteolytic cleavage of mitogen-activated protein kinase kinase 6, and can be overcome by the ectopic expression of a cleavage-resistant mutant of mitogen-activated protein kinase kinase 6 or a constitutively active IRF3. The lethal factor-mediated inhibition of IRF3 activation and subsequent cytokine production through bacterial membrane components offers Bacillus anthracis an efficient mechanism to evade the innate immune response.

Interferon regulatory factor 3 (IRF3) mediates the transcriptional induction of interferon-stimulated genes (ISGs) in response to viral and bacterial infections. Here we show that the hydroxystilbene piceatannol inhibits the LPS-mediated activation of IRF3 and subsequent ISG induction. Consequently, piceatannol blocks the LPS-induced up-regulation of critical mediators of the inflammatory response such as interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), intercellular adhesion molecule 1 (ICAM-1), and macrophage chemoattractant protein (MCP-1). Furthermore, the LPS-mediated induction of tissue factor (TF), a cell surface protein responsible for initiating the coagulation cascade, is also inhibited by piceatannol. The effectiveness of piceatannol in blocking both the inflammatory response and the coagulation pathway is evidenced by its ability to confer protection against LPS-induced septic shock in a murine model. Thus, IRF3 appears to be a promising target for pharmacologic intervention in the prevention or treatment of septic shock syndrome.