Graduation Date

Spring 5-7-2022

Document Type


Degree Name

Doctor of Philosophy (PhD)


Immunology, Pathology & Infectious Disease

First Advisor

St. Patrick Reid, PhD


Ebola virus (EBOV) causes a severe hemorrhagic fever in humans that is oftentimes lethal, emerging as a global public health threat in recent years. Understanding the host factors that contribute to the dynamic states and functions of the EBOV ribonucleoprotein (RNP) complex could potentially advance the development of therapeutics. The viral architecture of the EBOV RNP complex has been defined but host protein interactions that modulate viral replication have largely gone unaddressed. EBOV nucleoprotein (NP) and viral protein 35 (VP35) play critical roles in viral replication and capsid assembly, comprising two major components of the RNP complex. Elucidation of host proteins and post-translational modifications (PTMs) that impact the function of the EBOV RNP complex will provide a deeper understanding of RNP complex dynamics during infection that may lead to new opportunities to target and manipulate crucial host-pathogen interactions.

Here, novel NP cellular interactors were identified and functionally assessed during EBOV replication. Mass spectrometry (MS)-based proteomics was used to identify candidate NP cellular interactors. First, RuvB-like 1 (RUVBL1) and RuvB-like 2 (RUVBL2), ATPase constituents of the heat shock protein 90 (HSP90) co-chaperone R2TP complex, were validated as NP interactors. Further studies suggest the R2TP complex, which consists of RUVBL1, RUVBL2, PIH1 domain containing 1 (PIH1D1), and RNA polymerase II-associated protein 3 (RPAP3), plays a role in capsid assembly but not viral replication. Next, NP was evaluated for interaction with N6-methyladenosine (m6a) machinery. m6A writer components were confirmed to interact with NP. Functional studies revealed dose-dependent inhibition and enhancement of EBOV replication upon m6A writer and m6A eraser overexpression, respectively. These effects were dependent on m6A writer/eraser catalytic activity. These data suggest m6A modification negatively regulates EBOV replication and may require NP-mediated viral inhibition for a productive EBOV infection. Finally, we aimed to further elucidate the regulation of VP35 function by evaluating its phosphorylation sites. MS-based PTM analysis predicted phosphorylation at the highly conserved Serine (Ser) 129 residue. Functional evaluation of VP35 Ser129 mutants revealed that modulation of Ser129 impacts its function in viral replication but not its interferon inhibitory activity. Studies are underway to identify the host kinase(s) responsible for phosphorylation-dependent regulation of VP35.

Available for download on Tuesday, February 27, 2024