Date of Award

2025

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

Committee Chair and Members

Joseph Morin, Chair

Cecilia Kovac

Timothy Leslie  

Keywords

COVID-19, Microbiology, Pandemic, SARS-Cov-2, Virology

Abstract

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, has resulted in over 774 million cases and 7 million deaths worldwide, establishing it as one of the most devastating pandemics in recorded history. While current therapeutic approaches primarily target viral entry mechanisms and specific enzymes, the limited understanding of non-structural protein functions hinders the development of comprehensive antiviral strategies. This research focuses on Nsp10 (non-structural protein 10), a highly conserved 139-amino acid protein essential for viral mRNA capping and replication. Nsp10 serves as a critical cofactor in the formation of methyltransferase complexes with Nsp14 and Nsp16, playing a vital role in viral immune evasion and pathogenesis. Given its conservation across coronavirus species and indispensable function in viral replication, Nsp10 represents a promising therapeutic target for broad-spectrum antiviral development. This study employed yeast two-hybrid (Y2H) screening methodology to identify and characterize potential interactions between SARS-CoV-2 Nsp10 and human cellular proteins. The research utilized systematic molecular cloning techniques, including yeast colony PCR verification, plasmid DNA isolation, bacterial transformation, and protein expression analysis. Through comprehensive screening of yeast clones containing the Nsp10 activation domain construct, we aimed to identify host cellular proteins that interact with Nsp10, potentially revealing novel mechanisms of viral pathogenesis and host cell manipulation. Through the use of Yeast-2-Hybrid screening and molecular characterization, we identified a potential interaction between SARS-CoV-2 Nsp10 and human Galectin-1 (Gal-1), a carbohydrate-binding protein known to play critical roles in immune regulation and viral pathogenesis. Sequencing analysis revealed that the interacting clone contained a 528 base pair sequence corresponding to Exon-1 of Galectin-1, though the sequence was found to be out of frame with the activation domain vector. This interaction is particularly significant given Galectin-1's documented involvement in viral attachment, immune evasion, and host-pathogen interactions with other viral pathogens, suggesting a potential novel mechanism by which SARS-CoV-2 may manipulate host cellular functions. The findings from this research contribute to the fundamental understanding of SARS-CoV-2 host-pathogen interactions and provide insights for the development of targeted antiviral therapeutics that could be effective against current and future coronavirus variants.

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