Year of Award

2022

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Cellular, Molecular and Microbial Biology

Other Degree Name/Area of Focus

Molecular Biology and Biochemistry

Department or School/College

Division of Biological Sciences

Committee Chair

Stephen Lodmell

Commitee Members

Ekaterina Voronina, Scott Samuels, Brent Ryckman, Monica Serban

Keywords

rift valley fever virus, innate immunity, splicing, virology, riok3, tra2-beta

Subject Categories

Biochemistry | Cell Biology | Molecular Biology | Virology

Abstract

Rift Valley fever virus (RVFV) is a mosquito-borne RNA virus that infects humans and livestock in sub-Saharan Africa and the Arabian peninsula, causing disease ranging from a mild flu-like illness to liver damage, blindness, hemorrhagic fever, death, and, especially in livestock animals, high rates of abortive pregnancies. There is no approved vaccine for RVFV, and as a disease with a high rate of spread that causes severe illness, it is listed as a Category A pathogen by the USA CDC. A better understanding of RVFV’s molecular virology will be instrumental to combating RVFV as climate change causes its mosquito host range to expand. RVFV infection causes global changes to host transcriptional activities, including host alternative splicing. Untangling these changes in transcription and alternative splicing will be key to a detailed understanding of viral infection and host responses to viral infection. Host cellular immunity, which is activated as soon as a viral incursion is detected, is multifaceted and complex, and many members of the innate immune response also encode alternatively spliced mRNA isoforms to regulate their activity. In this dissertation, RVFV’s wide-ranging effects on host transcription and splicing programs is described, and focus is placed on one particular alternative splicing event in the mRNA for host innate immune protein RIOK3. Chapter 1 reviews relevant aspects of cellular innate immunity and alternative splicing of innate immune genes, especially during RNA virus infection, and emphasizes recent evidence for RNA virus interference of host splicing mechanisms. Chapter 2 highlights our RNAseq work on RVFV-infected cells that shows the widespread changes in host transcription and splicing during infection, including in RIOK3. In Chapter 3, RIOK3’s antiviral role in the innate immune response to RVFV infection is demonstrated along with the observation that expression of the alternatively spliced isoform, RIOK3 X2, correlates with a diminished innate immune response, which indicates that this alternative splicing observed in RVFV infection may be important for regulation of innate immunity. Chapter 4 describes studies to more deeply characterize RIOK3’s alternatively spliced isoforms, and elucidates the importance of host splicing factor TRA2-b for the constitutive splicing of RIOK3. Of particular interest, we observed that in RVFV-infected cells TRA2-b mRNA is mostly alternatively spliced to diminish stability and translation, while conversely, TRA2-b overexpression and concomitant enhanced expression of constitutively spliced RIOK3 mRNA significantly limits RVFV replication. These data show that RVFV infection benefits from decreased RIOK3 via alternative splicing, and that RIOK3 is an essential member of the antiviral response. This work also contributes to an emerging story that mRNA splicing is vitally important for regulation of the innate immune response against viral infection.

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