Honors College Thesis
 

Liquid-liquid Phase Separation of the Nucleocapsid of SARS-CoV-2

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  • The nucleocapsid of SARS-CoV-2 is critical for viral genome packaging through binding to viral genomic RNA. Liquid-liquid phase separation, or LLPS, is the formation of microscopic, rapidly reversible, liquid-like immiscible droplets. There is increasing scientific evidence that LLPS underlies the formation of membraneless compartments within cells and that this process has a significant role in human health and the development of disease. Prior research suggests that the nucleocapsid of SARS-CoV-2 undergoes LLPS with different sequences of viral RNA and that this mechanism may facilitate viral assembly. In this study, the full-length nucleocapsid (FL-N), the C- and N-terminal domain (CTD and NTD), and Linker, in addition to segments of viral genomic RNA, were grown, purified, and fluorescently labeled. Through microscopic analysis, droplet formation was observed for FL-N and CTD with all RNA constructs tested, with increased droplet formation after overnight incubation at 37° C, suggesting that RNA sequence specificity does not drive phase separation under these conditions and that the dimerization domain assists in LLPS. The WT Linker was observed to form spherical condensates after shorter incubation times that would shift to aggregates after overnight incubation. Phosphorylation of S188 was observed to have decreased droplet formation at lower concentrations of FL-N.
  • Key Words: Liquid-liquid phase separation, SARS-CoV-2, nucleocapsid, virus, pandemic, protein, RNA.
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  • National Science Foundation EAGER MCB 2034446 to Elisar Barbar. Additionally, research funding was awarded from URSA Engage 2020, and SURE 2021.
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