Defense Date

10-31-2024

Graduation Date

Fall 12-20-2024

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Chemistry and Biochemistry

School

School of Science and Engineering

Committee Chair

Mihaela Rita Mihailescu

Committee Member

Jeffrey Evanseck

Committee Member

Michael Cascio

Committee Member

Michael Jensen-Seaman

Keywords

SARS-CoV-2, Biophysics, Bioinformatics, RNA, Virus, microRNA, Immune System

Abstract

This dissertation focuses on the characterization of RNA-RNA interactions within the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome and with host microRNAs. As the causative agent of coronavirus disease 2019 (COVID-19), SARS-CoV-2 has evolved rapidly since its appearance. This has warranted prompt characterization of the virus particularly of its single stranded RNA (ssRNA) genome. By using a combination of bioinformatics, biophysics, and/or biological assays, we analyzed the SARS-CoV-2 viral genomic RNA and uncovered interactions of genomic RNA with host RNAs, highlighting an underutilized method of targeting RNA viruses. We showed here that the conserved elements in the viral genomic RNA, particularly within the 3’-untranslated region (3’-UTR), are involved in intramolecular and intermolecular RNA-RNA interactions that can have key roles in the viral life cycle. The stem-loop II-like motif (s2m) was one of these conserved elements. We show here that s2m, which is known for its role in genome dimerization, underwent a stepwise mutation that eliminates its interactions with host miR-1307-3p alongside its ability to dimerize. We showed that this interaction can inhibit viral translation, suggesting the loss of binding to be a mechanism of immune evasion. Additionally, we identified other microRNA interactions which could mediate ACE2 expression in infected and nearby cells, allowing hijacking of the immune system through JAK/STAT3 manipulation. Our work also demonstrated that these interactions can inhibit viral translation, similar to the miR-1307-3p interactions. We then implemented 2’-fluoro-D-arabinonucleic acids as effective competitive inhibitors for these microRNA binding interactions. Finally, we also characterized a second dimerization site, the stem-loop III-like motif (s3m), which utilizes a kissing dimer intermediate like s2m and can bind miR-1236-3p with the conserved octanucleotide motif. Taken together, this work elucidated a set of RNA-RNA interactions with SARS-CoV-2 viral RNAs that allow for specific interactions with immune-related regulatory microRNAs. While we suggest here that these microRNAs could be hijacked for benefit of the virus, we also suggest that these interactions could be an antiviral response targeting SARS-CoV-2. Through the identification of these interactions, and the implementation of antisense oligonucleotide competitors, we establish a foundation for therapeutic intervention against SARS-CoV-2 aimed at the virus-host interface.

Language

English

Additional Citations

Frye, C. J.; Shine, M.; Makowski, J. A.; Kensinger, A. H.; Cunningham, C. L.; Milback, E. J.; Evanseck, J. D.; Lackey, P. E.; Mihailescu, M. R. Bioinformatics Analysis of the S2m Mutations within the SARS‐CoV‐2 Omicron Lineages. Journal of Medical Virology 2023, 95 (1). https://doi.org/10.1002/jmv.28141. Frye, C. J.; Cunningham, C. L.; Mihailescu, M. R. Characterization of the SARS-CoV-2 Genome 3′-Untranslated Region Interactions with Host MicroRNAs. ACS Omega 2024, 9 (34), 36148–36164. https://doi.org/10.1021/acsomega.4c01050. Cunningham, C. L.; Frye, C. J.; Makowski, J. A.; Kensinger, A. H.; Shine, M.; Milback, E. J.; Lackey, P. E.; Evanseck, J. D.; Mihailescu, M.-R. Effect of the SARS-CoV-2 Delta-Associated G15U Mutation on the S2m Element Dimerization and Its Interactions with miR-1307-3p. RNA 2023, 29 (11), 1754–1771. https://doi.org/10.1261/rna.079627.123.

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