All ETDs from UAB

Advisory Committee Chair

Todd Green

Advisory Committee Members

Terje Dokland

Elliot Lefkowitz

Chad Petit

Mark Walter

Document Type

Dissertation

Date of Award

2020

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Vesicular stomatitis virus (VSV) is a member of the order Mononegavirales, which is comprised of viruses with genomes of non-segmented negative sense RNA. Many fundamental insights into the molecular biology of mononegaviruses were first made in VSV, such as the unique viral RNA synthesis template comprised of the nucleocapsid (N) protein and genomic RNA and the distinctive large (L) protein which contributes all enzymatic activities of RNA synthesis and transcript maturation. The L protein performs its enzymatic functions in concert with the phosphoprotein (P), a flexible, charged, and phosphorylated cofactor. The goal of the research presented in this dissertation was to better characterize the protein-protein interactions involving L and P. The first portion of the presented research involves the connector domain of VSV L. Though known to be structured and functionally significant, what role it played in RNA synthesis was unknown. A multi-technique approach including nuclear magnetic resonance (NMR), hydrogen-deuterium exchange mass spectrometry (HDX-MS), and functional analyses allowed us to demonstrate that the connector domain binds a fragment of the phosphoprotein cofactor in a manner that positively regulates RNA synthesis. Building upon this work, we turned our attention to the interaction between P and the C-terminal domain of L (LCTD). Using an NMR and electron microscopy (EM) approach, we found that an N-terminal segment of P which contains known phosphorylation sites interacts with the C-terminal domain. We went on to demonstrate that phosphorylation weakens the interaction of these two proteins, and in doing so brings about a more structurally heterogeneous state of L. Taken as a whole, our research has provided the first functional assignment for a connector domain in any mononegavirus and has also provided a role for phosphorylation that had not been characterized previously. Combining our results with those of other recent studies in mononegavirus polymerase structure, we suggest a model in which a balancing act between conformational compaction and mobility of the methyltransferase and C-terminal domains is mediated by phosphorylated P, allowing for optimal RNA synthesis by VSV.

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