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qi ke etd.pdf (1.88 MB)
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Negative Regulation of Host Innate Immune Signaling and Response Pathways by Viral and Host Regulatory Factors.
Author Info
Ke, Qi
Permalink:
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1470185159
Abstract Details
Year and Degree
2016, Doctor of Philosophy, University of Toledo, Biology (Cell-Molecular Biology).
Abstract
Eukaryotes have evolved intricate innate immune systems that allow rapid response to pathogens such as viruses. The initiation of innate immune responses depends on the recognition of pathogen associated molecular patterns (PAMPs) by host germ-line encoded pattern-recognition receptors (PRRs). Activation of antiviral responses by these pathways are intended to slow or contain viral replication until the adaptive immune system can clear the infection. Viral hemorrhagic septicemia virus (VHSv) is a deadly fish rhabdovirus that infects over 50 species of freshwater and marine fishes around the world. In 2003, a new substrain of VHSv (IVb) was found in the Great Lakes region when it caused a massive die-off of many freshwater species. The VHSv genome is about 11-kb long containing six genes, and replication occurs entirely in the cytoplasm using a combination of virally encoded and host-derived factors. As with other viruses, VHSv must neutralize or evade the host innate immune response in order to survive. We have found that VHSv can inhibit IFN-stimulated antiviral responses. Interestingly, the matrix (M) protein of VHSV IVb alone can potently suppress MAVS- and IFN-induced gene expression in a dose-dependent manner. The inhibition of constitutive SV40 promoter-driven gene expression by M implicated a general effect on transcription or translation. Our study showed decreased nascent RNA levels in both VHSv-infected cells and M-transfected cells. Co-transfection of M with a tetracycline inducible reporter gene (mouse secreted embryonic alkaline phosphatase - mSEAP) resulted in potent inhibition of tet-induced mSEAP mRNA synthesis. These results suggested that M inhibited protein expression by shutting down host transcription. Indeed, ChIP studies illustrated M-dependent inhibition of RNA polymerase II (RNAP II) recruitment to a gene promoter, and decreased RNAP II CTD Ser2 phosphorylation, an indicator of transcript elongation, during VHSv infection. Therefore we hypothesize that VHSv M inhibits host transcription by preventing RNAP II recruitment or by disrupting its association with target genes. M inhibited pol I, II and III transcription in cell-based luciferase studies, but blocked RNAP II-dependent transcription most potently. When M proteins from a variety of VHSv strains and related fish rhabdoviruses were tested for potency in cell-based luciferase inhibition assays, a VHSv F1 substrain M variant was significantly less potent than M from the IVb substrain. Among the four amino acid differences between the two M protein, two of them (D62A and E181A) were demonstrated to be crucial for the transcriptional inhibitory effect of M. Reverse genetics studies to introduce these amino acid changes into the VHSv IVb backbone have been initiated to determine how they impact virulence within the context of an intact virus. These studies should enhance our understanding of M’s role in host inhibition and, ultimately, viral replication. Type I IFNs play important roles in both innate and adaptive immune responses and are highly regulated to limit tissue damage and prevent autoimmunity. A second dissertation project focused on mammalian RNF114, an E3 ubiquitin ligase, which we propose regulates dsRNA-induced antiviral responses. Our work has revealed that the RNF114 RING finger domain and ubiquitin interacting motif (UIM) are both crucial for its ubiquitination activity. When ectopically expressed, RNF114 negatively impacted cellular dsRNA responsiveness, down-regulated the RLH adaptor molecule MAVS expression, potentially through ubiquitination-dependent degradation, and also suppressed RIG-I, MDA5 and MAVS signaling. In contrast, knocking down RNF114 robustly promoted cellular dsRNA responses. RNF114 mRNA was expressed in many different tissues in the mouse, but was predominant in spleen. We have successfully generated a RNF114 knockout mouse which exhibited elevated basal and dsRNA induced IFN and ISG56 mRNA levels, as compared to wild type mice. Mouse bone marrow-derived macrophage (BMDM) studies also suggested RNF114 KO leads to augmented mRNA levels of ISG and IL-10 induced by dsRNA treatment. Thus, we hypothesize that RNF114 may function as a regulatory E3 ubiquitin ligase to inhibit uninduced IFN production by regulating MAVS steady state levels through ubiquitination. Our overall hypothesis is that RNF114 functions to prevent uncontrolled inflammatory signal. Overall, the combined results of our studies emphasize the critical roles of factors within the innate immune response system, as well as external forces that can alter normal innate immune pathway function to the detriment of the host, and reiterate the need for integrated studies from both the host’s and pathogen’s perspective. Better understanding of type I IFN regulation, in particular, is important to understand a variety of normal, pathogenic and disease states.
Committee
Douglas Leaman, Ph.D. (Committee Chair)
Malathi Krishnamurthy, Ph.D. (Committee Member)
Travis Taylor, Ph.D. (Committee Member)
Carol Stepien, Ph.D. (Committee Member)
Scott Leisner, Ph.D. (Committee Member)
Pages
127 p.
Subject Headings
Biology
Keywords
VHSV
;
IFN
;
ZNF313
;
RNF114
;
Recommended Citations
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Citations
Ke, Q. (2016).
Negative Regulation of Host Innate Immune Signaling and Response Pathways by Viral and Host Regulatory Factors.
[Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1470185159
APA Style (7th edition)
Ke, Qi.
Negative Regulation of Host Innate Immune Signaling and Response Pathways by Viral and Host Regulatory Factors.
2016. University of Toledo, Doctoral dissertation.
OhioLINK Electronic Theses and Dissertations Center
, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1470185159.
MLA Style (8th edition)
Ke, Qi. "Negative Regulation of Host Innate Immune Signaling and Response Pathways by Viral and Host Regulatory Factors." Doctoral dissertation, University of Toledo, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1470185159
Chicago Manual of Style (17th edition)
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Document number:
toledo1470185159
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601
Copyright Info
© 2016, all rights reserved.
This open access ETD is published by University of Toledo and OhioLINK.