Date of Award
Summer 8-24-2024
Document Type
Dissertation
Degree Name
Ph.D.
Organizational Unit
College of Natural Science and Mathematics, Chemistry and Biochemistry
First Advisor
Michelle Knowles
Second Advisor
Scott Horowitz
Third Advisor
Todd Wells
Fourth Advisor
Gareth Eaton
Fifth Advisor
Maria Calbi
Copyright Statement / License for Reuse
All Rights Reserved.
Keywords
AlphaFold3, Non-structural protein 15, SARS-CoV-2, Sequence specificity
Abstract
The SARS-CoV-2 virus was recently able to cause a global pandemic, largely due to the asymptomatic period that occurs during initial infection. During this period, patients are infectious but do not exhibit symptoms, resulting in the unintentional spread of the virus. This occurs because the virus effectively avoids detection by the immune system during the early stages of infection, and as symptoms are the result of immune system activation, they never manifest. Non-structural Protein 15 (Nsp15) is one of the tools the virus uses to remain undetected, via its degradation of Pathogen-Associated Molecular Patterns (PAMPs) that would otherwise be recognized by the immune system. Specifically, the PAMP targeted by Nsp15 is the double-stranded RNA (dsRNA) intermediate that necessarily results from replication of the viral genome in positive sense single-stranded RNA ((+)-sense ssRNA) viruses like SARS-CoV-2. Nsp15 preferentially cleaves the (+)-sense strand of this dsRNA complex, although the exact sequences targeted by Nsp15 within that strand have been difficult to identify. In this work, the dinucleotide sequence specificity of Nsp15 is investigated using small, single stranded RNA substrates in which two nucleotides have been systematically altered across all 16 possible dinucleotide combinations, thus covering the entire sequence space for dinucleotides. Denaturing polyacrylamide gel electrophoresis (dPAGE) was used to generate the primary experimental dataset, providing a systematic ranking of the dinucleotide specificity of Nsp15. Trends and anomalies that were observed in these results were further investigated by employing AlphaFold3 (AF3) as a tool, where structures of all 16 substrates individually bound to Nsp15 were generated. Correlations between the predicted models and experimental results were then used to form molecular hypotheses about Nsp15 sequence specificity.
Because Nsp15’s role is intimately tied to the nature of the SARS-CoV-2 virus, a general background on viruses will first be provided. A more in-depth discussion of Nsp15 biology and its defining characteristics will then follow, after which the results of experimentation and its subsequent AlphaFold3 analysis will be presented and discussed.
Copyright Date
8-2024
Publication Statement
Copyright is held by the author. User is responsible for all copyright compliance.
Rights Holder
John Vincent McGuire
Provenance
Received from Author
File Format
application/pdf
Language
English (eng)
Extent
108 pgs
File Size
3.9 MB
Recommended Citation
McGuire, John Vincent, "Sequence Specificity of Non-Structural Protein 15 from SARS-CoV-2" (2024). Electronic Theses and Dissertations. 2467.
https://digitalcommons.du.edu/etd/2467
