Date of Award
Doctor of Philosophy (PhD)
Darnell James Laboratory
The interaction of type I interferon (IFN) with a specific cell surface receptor elicits a number of physiological changes, including the attainment of a state in which viral replication and cellular proliferation is inhibited. The response to IFN is mediated by a group of IPN-induced proteins which are regulated at the transcriptional level. Therefore, a central event in the cellular response to IFN is the coordinate transcriptional induction of a specific set of interferon stimulated genes (ISGs). In this thesis, I have addressed the cellular strategies employed to transmit specific cell surface signals across compartmental boundaries, ultimately resulting in specific transcriptional modulation in the nucleus. The analysis of the 5' regulatory sequences of two ISGs has identified a conserved interferon stimulated response element (ISRE) present in all ISGs, which is necessary and sufficient for transcriptional responsiveness to IFN. Three nuclear DNA-binding factors were identified that specifically bind the ISRE, one which was constitutive and two whose presence was dependent of IFN treatment. The analysis of the kinetics, protein synthesis-dependence, and precise sequence requirements for ISRE binding of these interferon stimulated gene factors (lSGFs) implicated a single IFN-inducible factor, ISGF3, as the positive regulator of ISGs. ISGF3 was induced rapidly in the absence of ongoing protein synthesis, characteristics identical to transcriptional activation of ISGs. The study of IFN-resistant variant cell lines which do not respond to IFN physiologically, but do express normal IFN receptors, supported the role of ISGF3 as the ISG activator. In these resistant cell lines the lack of ISG induction correlated with a defect in ISGF3 activation. Furthermore, it was found that ISGF3 activation occurred in the cytoplasm of stimulated cells through a series of events involving post-translational activation of a latent cytoplasmic protein, and subsequent association with a second cytoplasmic protein forming a heteromeric complex which accumulated in the nucleus. Additional characterization of the ISGF3 complex revealed a heterotetromeric complex (48, 84, 91, and 113 kD subunits). The activation and formation of ISGF3 required IFN-dependent post-translational activation of at least one of the high molecular weight subunits (84, 91, or 113 kD), leading to facilitated nuclear translocation of this group of three proteins. Upon nuclear accumulation these proteins associated with the 48 kD ISRE-binding subunit resulting in an enhancement of DNA binding affinity and formation of the stable, transcriptionally active ISGF3- ISRE complex. In the type I IFN system, specific transcriptional regulation in response to a cell surface ligand is mediated by activation of a latent cytoplasmic transcription factor which is a highly specific intracellular messenger.
Kessler, Daniel Solomon, "Signal Transduction and Transcriptional Regulation in the Response to Type I Interferon" (1990). Student Theses and Dissertations. 371.