Author

Dan CuiFollow

Date of Award

8-8-2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Margo Brinton

Second Advisor

Richard Plemper

Third Advisor

Casonya Johnson

Abstract

Upon viral infection, host cells produce type I interferon (IFN), which activates the JAK-STAT signaling pathway and induces the expression of hundreds of interferon-stimulated genes (ISGs) to establish an antiviral state. In West Nile virus (WNV)-infected cells, the JAK-STAT signaling pathway is blocked by viral proteins. However, the expression of a subset of ISGs, which includes 2¢-5¢-oligoadenylate synthetase 1a (Oas1a), Oas1b, interferon regulatory factor 7 (Irf7), Mx1, and interferon-induced proteins with tetratricopeptide repeats 1 (Ifit1), is still upregulated by an IFN-independent mechanism in WNV-infected mouse embryonic fibroblasts (MEFs). Studies in cells with one or more components of RNA-sensing pathway knocked out showed that the alternative ISG upregulation is activated through RIG-I or MDA5, and the downstream adaptor IPS-1. In cells with IRF3, 5 and 7 knocked out, the alternative ISG upregulation by WNV infection is reduced but not eliminated. As an initial means of discovering the transcription factors involved in this non-canonical ISG upregulation, the critical regulatory regions in the promoters of two representative ISGs, Oas1b and Ifit1, were mapped using a dual luciferase assay system with a NanoLuc luciferase promoter reporter in WNV-infected Ifnar1-/- MEFs. The region from -299 to -28 in the Oas1b promoter, and the region from -192 to -50 in the Ifit1 promoter were identified as being important for upregulating non-canonical gene expression after WNV infection. Fine mapping identified enhancer and repressor sub-regions as well as transcription factor binding sites (TFBSs) putatively involved in the IFN-independent antiviral mechanism. Mutation of one identified TFBS in the ISG promoters reduced Oas1b and Ifit1 promoter activities. In electrophoretic mobility shift assays (EMSAs), a unique band, which was detected in WNV-infected but not in mock-infected Ifnar1-/- MEF nuclear extracts, was not observed when a probe with the identified TFBS mutated was used, suggesting that a unique complex forms at the identified TFBS when it is in the context of the adjacent flanking regions. The unique complex appears to contain NF-κB components and IRF3, IRF5 or IRF7. Our findings provide new insights into the mechanism involved in non-canonical upregulation of ISGs after WNV infection.

Available for download on Sunday, July 21, 2019

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