Date of Award

Fall 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Xiong, Yong

Abstract

Among the betacoronaviruses (?-CoV) family, there are three viruses which cause severe disease in humans: SARS-CoV, SARS-CoV-2, and MERS-CoV. Key to their viral lifecycle is Non-structural protein 1 (Nsp1), an essential pathogenicity factor that potently restricts the cell’s antiviral response. Despite poor protein sequence conservation of Nsp1 between evolutionarily distant ?-CoVs, these proteins have proven to be highly conserved both functionally and structurally. Nsp1 functions by inhibiting host gene expression through contacts made with two mRNA ribonucleoprotein (mRNP) complexes: the initiating ribosome and the Nxf1/Nxt1 mRNA nuclear export complex. It is believed that two Nsp1 functions occur on the ribosome: Nsp1 is able to coordinate cleavage of the host mRNA and Nsp1 is able to sterically occupy the mRNA entry channel to inhibit translation initiation. In this thesis, I’ve investigated Nsp1’s ability to engage both of these mRNP complexes and probed into all three of its purported functions for divergent ?-CoV Nsp1 proteins. We report a high-resolution cryo electron microscopy structure that shows the divergent MERS Nsp1 binds the mRNA entry channel of the ribosome in a highly analogous fashion to the sarbecovirus Nsp1 proteins. I also show that SARS-CoV-2 Nsp1 is able to efficiently cut mRNA bound to the 40S ribosome and less efficiently cut free mRNA in vitro. I put forth a model where Nsp1 is an inefficient independent endonuclease whose activity is enhanced by binding mRNA binding complexes such as initiating ribosome. Further, Nsp1 from divergent coronaviruses may utilize a disparate set of host factors to enhance their ability to cut host mRNA. Collectively, I report that binding the ribosome is a conserved mechanistic strategy of ?-CoV Nsp1 proteins but divergent Nsp1 proteins may co-opt different host factors to enhance their endonuclease activities.

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