Type I IFN is a major component of both innate and adaptive immune responses, critical for generation of an effective defense to infection of pathogen. Interferon-β (IFN-β) is the first type I IFN cytokine produced in response to viral infection and plays a major role in activation of the downstream antiviral factors. Production of IFN-β is dependent on the primary recognition of viral infection by cellular sensors and the signaling cascade leading to its expression. The importance of the IFN-β pathway in the antiviral response is highlighted by the multiple strategies that viruses use to block the pathway activation. Efforts to understand the regulation of IFN-β induction pathways are crucial for development of strategies to modulate the antiviral response.
Our work focuses on two key steps in IFN-β induction; (1) RIG-I regulation responsible mechanisms related to IFN production; and (2) the formation of the enhanceosome, a pre-transcriptional complex required for production of IFN-β messenger RNA. Negative regulation of components involved in IFN induction is well documented and appears to be important for the “off” state of innate immunity in the absence of infection as well as for shutting down of IFN production at later time points in infection. We focus our work on the negative regulation of one of the key viral sensors, the retinoic acid inducible gene I (RIG-I), and show that phosphorylation of its amino acid serine 8 plays a role in modulating the IFN-β pathway activation (chapter II). In chapter III, we characterize the natural human polymorphism arginine/cysteine 7 in RIG-I and describe its effect on the expression of IFNB1 gene in various cell types. In chapter IV, we describe the study of IFN-β enhanceosome formation, specifically focusing on the mechanism by which interferon regulatory factor-3 (IRF-3) interacts with the enhancer DNA sequence of the IFN-β gene. We show that this binding controls the formation of the IFN-β enhanceosome pre-transcriptional complex and subsequent levels of IFN-β production.
From the above work we have unraveled two important aspects in the control of IFN-β pathway activation. First, we identified phosphorylation of RIG-I at serine 8 as an important posttranslational modification that serves as a negative regulator of the IFN-β induction pathway. We showed that this phosphorylation negatively affects the interaction of RIG-I with TRIM25, an E3 ubiquitin ligase that ubiquitinates RIG-I in its amino terminal CARD domain. Through inhibition of TRIM25 binding, the phosphorylation prevents the ubiquitination of CARD, a step required for optimal binding of RIG-I to MAVS. MAVS is a key downstream adaptor protein required for transmission of the signaling cascade that finally induces IFN-β enhanceosome formation. The importance of serine 8 is highlighted by the IFN-β induction phenotype associated to the proximal polymorphic arginine/cysteine 7 residue. Second, we determined the structure of DNA binding domain of IRF-3 bound to the IFN-β enhanceosome. We found that four IRF-3 DNA binding domain molecules bind to a single DNA molecule and are accommodated in different ways. IRF-3 binding to consensus and non-consensus DNA sites allows us to understand the suboptimal mechanism that controls the IFN-β production. These suboptimal IRF binding sites lead to the requirement for other transcription factors (AP1 and NF-κB) for the expression of IFN-β at physiological levels.
Overall, our findings contribute to a detailed understanding of the intricate mechanisms that govern production of IFN-β and ultimately to a better comprehension of the cellular response to virus infection. This knowledge will eventually lead to development of strategies to manipulate the cellular machinery in order to control viral infectious diseases.
|Commitee:||Aggarwal, Aneel K., Gale, Michael, Jr., Palese, Peter, TenOever, Benjamin, Tortorella, Domenico|
|School:||Mount Sinai School of Medicine of New York University|
|School Location:||United States -- New York|
|Source:||DAI-B 71/04, Dissertation Abstracts International|
|Subjects:||Molecular biology, Microbiology, Virology|
|Keywords:||Innate immunity, Interferon, RNA viruses, Signal transduction|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be