Advisor(s)
Chad Petit
Committee Member(s)
Aaron Lucius
David Schneider
Jun Zhang
Todd Green
School
College of Arts and Sciences
Document Type
Dissertation
Department (new version)
Chemistry
Date of Award
9-11-2025
Abstract
The influenza A nuclear export protein (NEP) is a multi-functional protein that mediates the export of progeny viral ribonucleoprotein (vRNP) complexes from the host nucleus, regulates the activity of the RNA-dependent RNA polymerase (RdRp), and is implicated in zoonosis. It is therefore critical to understand the mechanisms of NEP’s functions at the molecular level to inform its roles in the viral lifecycle and influenza zoonosis. In this dissertation, we report the structure of NEP in complex with the host nuclear export holo-complex as well as investigate the biophysical effects of the adaptive M16I mutation on NEP. We solved the cryo-electron microscopy structure of NEP in complex with chromosome regional maintenance 1 (CRM1) as well as two cofactors, Ras-associated nucleoprotein (Ran) and Ran-binding protein 1 (RanBP1). The structure shows that NEP’s N-terminal nuclear export signal (NES1) binds CRM1 preferentially to its C-terminal nuclear export signal (NES2). The conformation of NES1 matches the class 1a NES consensus pattern when bound to CRM1 and includes stabilizing intramolecular hydrogen bonds in its extended loop conformation. These observations are unique to the CRM1-bound conformation of NEP and were used to identify key residues which were not previously implicated in the NEP-CRM1 interaction. We analyzed models of wildtype NEP as well as mutant NEP incorporating the adaptive M16I mutation (NEPM16I) which were predicted by AlphaFold2. Analysis of these predicted structures revealed a network of interactions between methionine 16 of NEP and multiple aromatic amino acids (also known as Met-Aro interactions) that are abrogated in NEPM16I. We further probed the structural and dynamic changes to NEP resulting from the M16I mutation through the use of nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, and hydrogen-deuterium exchange mass spectrometry (HDX-MS). Our results indicate that the M16I mutation alters the conformational exchange of the protein as a whole, and more specifically results in changes to the stability of peptides in NEP which participate in the predicted Met-Aro interactions. Together, these studies inform the mechanisms by which NEP is involved in nuclear export and influenza zoonosis.
ProQuest ID
Recommended Citation
Staup, Andrew James, "Structural And Functional Investigation Of The Influenza Nuclear Export Protein" (2025). All ETDs from UAB. 7403.
https://digitalcommons.library.uab.edu/etd-collection/7403
