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Characterizing human Nogo-66 receptor 1 interactions with reovirus by cryo-EM and receptor mutagenesis

Welsh, Olivia L. (2023) Characterizing human Nogo-66 receptor 1 interactions with reovirus by cryo-EM and receptor mutagenesis. Master's Thesis, University of Pittsburgh. (Unpublished)

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Human Nogo-66 receptor 1 (NgR1) modulates axonal plasticity in the brain and serves as a receptor for mammalian orthoreovirus (reovirus). To elucidate the mechanism by which this receptor engages reovirus, we conducted cryo-electron microscopy (cryo-EM) analyses of soluble NgR1 bound to reovirus. A three-dimensional (3D) cryo-EM reconstruction reveals that NgR1 engages two protomers of the σ3 viral capsid protein. However, the resolution of the map is insufficient to confirm critical contact residues. We placed known reovirus and NgR1 crystal structures into the 3D reconstruction and used this reovirus-NgR1 model to guide mutagenesis. We engineered a panel of σ3 mutants within the proposed NgR1-binding region and assessed σ3 binding to NgR1, however no σ3 residue substitution was sufficient to disrupt NgR1 binding. We also engineered NgR1 mutants in which NgR1 residues in high-proximity or low-proximity to reovirus σ3 were exchanged with arginine or glutamate. Mutation of most high-proximity residues disrupted reovirus binding and infection of NgR1-expressing cells. However, mutation of low-proximity residues infrequently diminished binding and infectivity. Placement of residues required for reovirus binding onto the NgR1 structure revealed a binding footprint on both the concave and convex surfaces of NgR1. Although human NgR1 shares sequence similarities with family member NgR2 and homolog murine NgR1, only human NgR1 is a functional reovirus receptor. We therefore sought to identify key residues at the reovirus-binding interface of human NgR1 that also may serve as polymorphic sites that impede reovirus binding to NgR2 or murine NgR1. We engineered a panel of NgR1-to-NgR2 mutants and also murine-to-human NgR1 mutants within the convex, non-conserved interface proximal to reovirus σ3. Mutation of these residues was not sufficient to diminish reovirus binding in loss-of-function studies, or confer receptor functionality in gain-of-function studies. Collectively, these findings validate the 3D cryo-EM reconstruction and provide insight into the mechanism used by reovirus to engage NgR1.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Welsh, Olivia L.olw13@pitt.eduolw130000-0002-8039-7807
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairHartman, Amyhartman2@pitt.eduhartman2
Committee MemberMailliard, Robbie B.rbm19@pitt.edurbm19
Thesis AdvisorDermody, Terence
Date: 28 June 2023
Date Type: Publication
Defense Date: 5 May 2023
Approval Date: 28 June 2023
Submission Date: 16 June 2023
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 57
Institution: University of Pittsburgh
Schools and Programs: School of Public Health > Infectious Diseases and Microbiology
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: Reovirus, virology, cryo-EM, mutagenesis, viral receptor
Date Deposited: 28 Jun 2023 15:03
Last Modified: 28 Jun 2023 15:03


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