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Date of Award

Spring 5-15-2018

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Biochemistry)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Filoviruses are among the deadliest viruses that cause severe and often fatal hemorrhagic fever in humans during outbreaks. Marburg (MARV) and Ebola (EBOV) viruses are non-segmented, negative-sense RNA viruses that are clinically and symptomatically indistinguishable, but antigenically distinct. It is becoming increasing evident that two employ unique strategies to bypass antiviral responses. However, the molecular determinants of filovirus pathogenesis and important distinguishing characteristics between the two viruses are not completely defined. The work presented in this thesis elucidates mechanisms of immune modulation by viral proteins encoded by MARV and highlights distinguishing molecular determinants compared to EBOV-encoded viral proteins. We describe the mechanism by which MARV Viral Protein 24 (mVP24) activates the Antioxidant Response Element (ARE) pathway to express cytoprotective genes and reduce cell death associated with viral replication. We describe a direct interaction between mVP24 and Kelch-like ECH-associated protein 1 (Keap1), a regulator of the cytoprotective response pathway. The interaction between mVP24 and Keap1 relieves Keap1-mediated repression of the transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Resulting ARE pathway stimulation leads to the induction of cytoprotective genes, preventing cell death and enhancing MARV replication. In contrast, EBOV VP24 (eVP24) does not activate the ARE pathway. Structural, biochemical, and genetic studies reveal an insertion in mVP24, termed the K-Loop that is critical for Keap1 binding. eVP24 does not contain K-Loop residues, but corresponding residues are important for inhibition of interferon-stimulated gene (ISG) expression.

The host interferon response and subsequent induction of ISGs contribute to the control of viral infection. Thus, it is not surprising that MARV and EBOV have established strategies to inhibit interferon signaling. Additional studies presented in this thesis evaluate the importance of Interferon-induced protein with tetratricopeptide repeats (IFIT) heterocomplexes in restriction of RNA viruses and describes the first known study to characterize an IFIT1-IFIT3 interaction that is critical for optimal antiviral activity. The proposed basis for the antiviral activity is the binding of IFIT1 to the exposed 5’-ppp on the genome of filoviral RNA. However, recent studies by Pinto et al demonstrate that IFIT1 binds 5’-ppp RNA with low affinity compared to Cap 0 (m7GpppN) RNA. To corroborate these findings, we further describe the structural basis by which IFIT3 binds IFIT1 and thereby allosterically regulates the IFIT1 RNA-binding channel and recognition of Cap 0 but not Cap 1 (m7GpppNm) or 5'-ppp RNA. Thus, the IFIT3 interaction with IFIT1 is important for restricting infection of viruses lacking 2’-O methylation in their RNA cap structures, but not filoviruses.

In addition, MARV and EBOV encode the nucleoprotein (NP) which closely associates with the viral RNA to form the nucleocapsid (NC), minimizing recognition by IFIT proteins and other host factors. MARV and EBOV NC are distinct in morphology and differentially associate with accessory viral proteins to carry out transcription and replication. However, the molecular basis by which MARV and EBOV NC structures assemble and associate with replication complex proteins, including Viral Protein 35 (VP35), is not well understood. To this end, studies presented within this thesis determine the molecular basis for four mNP states (unbound, mNP-, RNA-, and VP35-bound). We report an X-ray crystal structure of mNP core (17-361) and critical residues for mNP oligomerization, including mNP N-terminal residues and the extended mNP alpha helix-20 (alpha 20), which is structurally distinct from known EBOV NP structures. mNP 20-mediated oligomerization excludes an mVP35 1-25 (NPBP) binding site but enhances ssRNA binding to a highly basic cleft between mNP Head and Foot Lobes. At high concentrations, mVP35 NPBP disrupts mNP oligomerization and facilitates the release of ssRNA. In contrast, our recent efforts to elucidate eNP oligomerization and nucleocapsid-like structure assembly reveals an alternative eNP conformation important for eNP-eNP binding. eNP alpha 22-23 do not extend from the eNP core, but alternatively adopts a helix-turn-helix conformation to bind the hydrophobic eVP35 binding site. Although the VP35 and NP interfaces are conserved, our studies determine key distinctions between eNP and mNP oligomerization, and the role in VP35 and ssRNA binding critical for transcription, replication, and NC assembly. Taken together, this thesis enhances our understanding of distinguishing molecular determinants for multifunctional MARV and EBOV viral proteins and provides additional frameworks for the development of much-needed therapeutics.

Language

English (en)

Chair and Committee

Gaya K. Amarasinghe

Committee Members

Thomas J. Brett, Peter M. Burgers, Michael L. Gross, Deborah J. Lenschow,

Comments

Permanent URL: https://doi.org/10.7936/K77W6BMV

Available for download on Sunday, May 15, 2118

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