Interferon regulatory factor 3 (IRF3) plays an important role in activating the innate immune response in a variety of conditions, including viral infection. As well as regulating the immune response to viruses, IRF3 is involved in regulating cellular functions including apoptosis. Apoptosis and the inflammatory response to viral infection are very different; therefore, it is obvious that IRF3 plays dramatically different roles in the cell depending on the conditions. We previously identified a non-activating phosphorylation of IRF3 in response to adenovirus (Ad) in which Serine-173 is phosphorylated. In addition to Ad infection, IRF3- S173 is phosphorylated in response to genotoxic stresses including ultraviolet (UV) irradiation and etoposide. In this study, I show that this phosphorylation event is involved in a variety of processes including protein stability, cell survival and IRF3 regulation. Thus, phosphorylation of IRF3-S173 is a novel and important event in a complex regulatory pathway of an integral protein.

Interferon regulatory factor 3 (IRF3) plays an important role in activating the innate immune response in a variety of conditions, including viral infection. As well as regulating the immune response to viruses, IRF3 is involved in regulating cellular functions including apoptosis. Apoptosis and the inflammatory response to viral infection are very different; therefore, it is obvious that IRF3 plays dramatically different roles in the cell depending on the conditions. We previously identified a non-activating phosphorylation of IRF3 in response to adenovirus (Ad) in which Serine-173 is phosphorylated. In addition to Ad infection, IRF3- S173 is phosphorylated in response to genotoxic stresses including ultraviolet (UV) irradiation and etoposide. In this study, I show that this phosphorylation event is involved in a variety of processes including protein stability, cell survival and IRF3 regulation. Thus, phosphorylation of IRF3-S173 is a novel and important event in a complex regulatory pathway of an integral protein.

Cardiovascular disease, and myocardial infarction (MI) in particular, remains a major burden in the developed world today. In fact, the remodeling process, which follows the initial ischemic episode of MI, is a major determinant of heart failure. Although several key mechanistic pathways involving cell growth and death have been identified, there is limited knowledge surrounding the role of the innate immune response as a positive or negative regulator of cardiac remodeling. Recent data strongly support a role for key regulatory components within the toll-like receptor (TLR) family as potent modulators of cardiac remodeling post-MI. It has been demonstrated that targeted gene knockdown of TLR4, as well as downstream adaptor proteins and kinases, significantly improve cardiac function and overall survival. While the well-known NF-κB transcriptional factor that is downstream to TLR4 signaling has been linked to remodeling, there has been no evidence thus far describing a role of the parallel interferon regulatory factor-3 (IRF3) signaling cascade in any facet of this process. Several key findings suggest that IRFs contribute to both cell growth and apoptosis, thus providing an appealing, and novel target for interrogation. In this thesis I describe how IRF3 contributes to maladaptive remodeling post-MI. In my first set of experiments, I demonstrate that IRF3 is acutely upregulated within the cardiomyocyte following MI and that this response contributes to excessive apoptosis post-MI. A targeted deletion of the IRF3 gene enhances cardiac function, decreases infarct size, and improves survival following MI. In the second set of experiments I demonstrate that IRF3 attenuates angiogenesis at the ischemic border zone by upregulating the expression of thrombospondins. I have shown that IRF3 deficiency, which liberates endogenous anti-angiogenic signals, promotes angiogenesis following ischemic injury. These data suggest that IRF3 is a potent regulator of cardiac remodeling and may be an effective therapeutic target to ameliorate maladaptive cardiac repair post-MI.

Influenza A viruses can cause a highly contagious respiratory illness in humans. Immediately after virus infection the innate immune response is initiated by binding of viral RNA species to RIG-I that leads to activation of IRF3 and NF-κB transcription factors and activation of interferon (IFN) transcription. LGP2 is a member of the RIG-I like receptor (RLR) family and is induced after virus infection. The role of LGP2 in virus infection is controversial: it has been reported to either positively or negatively affect RIG-I mediated signaling. The goal of this study was to determine whether LGP2 has a role during infection with influenza A viruses that have circulated in humans. We focused on two viruses expressing NS1 proteins that differ in their ability to inhibit IRF3 activation and IFN transcription; a H1N1 virus (Tx91) that inhibits IRF3 activation and a H3N2 virus (Ud) that does not. This study revealed that LGP2 has strikingly different roles during infection of mouse embryonic fibroblasts and human cells with these viruses. Specifically, LGP2 has no detectable role in H1N1 virus-infected cells, whereas it downregulates IFN synthesis in H3N2 virus-infected cells. Our results indicate that LGP2 acts as a negative regulator of the IFN response in influenza A viruses that activate IRF3. The NS1 protein also binds the 30kDa-subunit of the cleavage and polyadenylation specificity factor-CPSF30, a protein required for 3′-end processing of cellular pre-mRNAs, thereby inhibiting production of mature IFN-β mRNA. The NS1 proteins of pathogenic 1997 H5N1 viruses lack two highly conserved residues (F103 and M106) that are needed to stabilize the NS1-CPSF30 complex. Instead their NS1 proteins have L at 103 and I at 106, resulting in non-optimal CPSF30 binding in infected cells. We demonstrated that strengthening CPSF30 binding by changing L and I to the consensus residues (F and M respectively) leads to a dramatic (300-fold) increase in lethality of the virus in mice. This increased virulence is associated with faster systemic spread of the virus. Microarray analyses revealed increased cytokine levels in extrapulmonary tissues, particularly the brain. These results highlight the importance of NS1:CPSF30 binding in modulating virulence in H5N1 viruses. / text

Cardiovascular disease, and myocardial infarction (MI) in particular, remains a major burden in the developed world today. In fact, the remodeling process, which follows the initial ischemic episode of MI, is a major determinant of heart failure. Although several key mechanistic pathways involving cell growth and death have been identified, there is limited knowledge surrounding the role of the innate immune response as a positive or negative regulator of cardiac remodeling. Recent data strongly support a role for key regulatory components within the toll-like receptor (TLR) family as potent modulators of cardiac remodeling post-MI. It has been demonstrated that targeted gene knockdown of TLR4, as well as downstream adaptor proteins and kinases, significantly improve cardiac function and overall survival. While the well-known NF-κB transcriptional factor that is downstream to TLR4 signaling has been linked to remodeling, there has been no evidence thus far describing a role of the parallel interferon regulatory factor-3 (IRF3) signaling cascade in any facet of this process. Several key findings suggest that IRFs contribute to both cell growth and apoptosis, thus providing an appealing, and novel target for interrogation. In this thesis I describe how IRF3 contributes to maladaptive remodeling post-MI. In my first set of experiments, I demonstrate that IRF3 is acutely upregulated within the cardiomyocyte following MI and that this response contributes to excessive apoptosis post-MI. A targeted deletion of the IRF3 gene enhances cardiac function, decreases infarct size, and improves survival following MI. In the second set of experiments I demonstrate that IRF3 attenuates angiogenesis at the ischemic border zone by upregulating the expression of thrombospondins. I have shown that IRF3 deficiency, which liberates endogenous anti-angiogenic signals, promotes angiogenesis following ischemic injury. These data suggest that IRF3 is a potent regulator of cardiac remodeling and may be an effective therapeutic target to ameliorate maladaptive cardiac repair post-MI.

Thesis (Ph. D.)--University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Includes bibliographical references (p. 170-180).

Thesis (M.Sc.). / Written for the Dept. of Microbiology and Immunology. Title from title page of PDF (viewed 2008/05/14). Includes bibliographical references.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA tumor virus that has been associated with all epidemiological forms of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). Like other herpesviruses, KSHV undergoes two phases of life cycle (latent and lytic replication). During latency, the viral genome persists as a circular episome in the nucleus of the host cell and only a few viral genes are expressed, namely LANA (latency- associated nuclear antigen), Kaposin, vFLIP (viral FLICE inhibitory protein), vCyclin, and vIRF3/LANA2 (viral interferon regulatory factor 3). These viral genes are responsible for regulation of host cell proliferation, prevention of apoptosis, facilitation of immune evasion, and maintenance of the extrachromosomal viral genome during cell divisions. vIRF3 is a multifunctional nuclear protein that is constitutively expressed in KSHV positive PEL cells and Castleman's disease tumors, which expression causes dramatic changes of critical host pathways that are involved in the regulation of apoptosis, cell cycle, antiviral immunity, and tumorigenesis. In our study, we have demonstrated and elucidated predicted mechanism, by which vIRF3 enhances transcription activity of c-Myc. Moreover, we have clarified the previously unappreciated...

Central driving forces in the pathogenesis of liver disease are hepatocyte death and immune cell-driven inflammation. The interplay between outcomes, stemming from these two major cell types, is present from the earliest ethanol exposure, and are both determinants in advanced stages of liver disease particularly in alcoholic liver disease (ALD). The complexities associated with advanced ALD are many and therapies are limited. Due to the liver’s role in ethanol metabolism and filtering gut-derived products, it is becoming increasingly clear that innate immunity plays a central role in triggering activation of cell death and inflammatory pathways in ALD. We identified interferon regulatory factor 3 (IRF3) activation as a mediator of hepatocyte death as the first event after ethanol exposure, and the inflammasome as a protein complex responsible for the subsequent inflammatory cascade, driven by the NLRP3 inflammasome. Our novel findings in murine samples and human patients with alcoholic hepatitis demonstrate that ethanol-induced inflammasome activity results in Caspase-1-mediated pyroptosis and extracellular ASC aggregates in the liver and circulation. Pyroptosis can be abrogated by therapeutic inhibition of inflammasome components, NLRP3 or Caspase-1. Taken together, the event leading to mtDNA release into the cytoplasm is the inception of the pathogenesis of ALD, triggering hepatocyte death, culminating in a pro-inflammatory cascade driven by the NLRP3 inflammasome and pyroptotic release of ASC.