Date of Completion

10-18-2019

Embargo Period

10-15-2024

Keywords

PKR, analytical ultracentrifugation, MDA5

Major Advisor

James L. Cole

Associate Advisor

Carolyn M. Teschke

Associate Advisor

Victoria L. Robinson

Associate Advisor

Eric May

Associate Advisor

Charles Giardina

Field of Study

Biochemistry

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

PKR is an important protein of the innate immune pathway that binds to viral dsRNA, resulting in dimerization-induced autophosphorylation. During viral infection, activated PKR blocks protein synthesis by phosphorylating eukaryotic initiation factor 2a (eIF2a). Epstein Barr virus-encoded RNA 1 (EBER1) and Adenovirus virus-associated RNA I (VAI), produced by Epstein Barr Virus and Adenovirus, respectively are non-coding, highly-structured RNAs that bind to PKR and have been characterized as inhibitors. Here we show that EBER1 weakly activates PKR in vitro, but VAI does not. Both RNAs bind two PKR monomers and promote PKR dimerization. We generated a low-resolution model of EBER1 that rationalizes PKR activation by EBER1, and show that the presence of a pseudoknot in the central domain and Loop 2 of VAI are important for PKR inhibition.

Two homologous innate immune receptors classified as RIG-I like receptors (RLRs), RIG-I and MDA5, are protein surveyors of the cytoplasm that recognize viral RNA. Upon RLR activation by RNA, the 2CARD signaling domain associates with the CARD domain of MAVS, leading to the oligomerization of MAVS, downstream signaling, and interferon induction. K63-linked polyubiquitin chains (polyUb) play a role in RLR signaling by interacting with the 2CARD domain. PolyUb binding to RIG-I 2CARD induces the formation and stabilization of a 2CARD tetramer which forms a scaffold to induce MAVS polymerization. However, the nature of the signaling complex of MDA5 2CARD and the role of polyUb binding in MDA5 2CARD activation are not well understood. Here, we characterize the oligomerization of MDA5 2CARD in the presence and absence of K63-linked polyubiquitin chains. We show that K63-linked polyubiquitin chains do not induce oligomerization of MDA5 2CARD, but polyUb chains bind to MDA5 with 2:1 stoichiometry. Oligomerization of MDA5 2CARD occurs in a concentration-dependent manner, and complexes with sizes of 7-18mer 2CARD form readily in solution.

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