Year of Award

2019

Document Type

Dissertation - Campus Access Only

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Cellular, Molecular and Microbial Biology

Department or School/College

Division of Biological Sciences

Committee Chair

J. Stephen Lodmell

Commitee Members

Ekaterina Voronina, Brent Ryckman, Scott Samuels, Elizabeth Putnam

Keywords

nucleocapsid, protein-RNA interactions, Rift Valley fever virus, RIOK3, splicing, transcriptome

Publisher

University of Montana

Abstract

Rift Valley fever virus (RVFV) is an emerging pathogen that causes severe disease in humans and domestic livestock. There is no licensed treatment or vaccine to combat RVFV infection available for human use. RVFV is endemic to sub-Saharan Africa, but in recent years outbreaks have extended throughout Africa and onto the Arabian Peninsula. The virus has been shown to be transmitted by a diversity of mosquito species, including those that are present in the Western Hemisphere. This demonstrated potential for spread and the potential health and economic impacts of outbreaks compels a greater understanding of RVFV molecular virology and development of therapeutic options. The virally encoded nucleocapsid protein (N) has several known functions at critical stages of the viral replication cycle. Each of these important roles involves the ability of N to bind RNA. Thus the N-RNA interface is an intriguing molecular target for antiviral therapeutic intervention. The work described here focuses on characterization of N-RNA interactions, inhibition of these interactions, and the possible role of N or other viral proteins in diminishing the cellular antiviral response. We carried out a small molecule screen and identified a compound that diminished the ability of N to interact with RNA. We also executed a crosslinking and immunoprecipitation with high-throughput sequencing experiment to investigate N-RNA interactions in infected cells. This study allowed us to propose specific sequence motifs as putative N recognition sites. This work further indicated that N protein is not only a critical component of the viral replication machinery, but its extensive interaction with host RNAs could be a previously unappreciated viral mechanism for disrupting host RNA metabolism and processing. The unexpected affinity of N for host RNAs led us to perform transcriptome profiling in RVFV infected cells, which highlighted pervasive changes in gene expression and alternative splicing that occur as a result of infection. We examined alternative splicing during RVFV infection on both a global scale and in detail with a particular host gene, RIOK3, which we discovered to be an important antiviral protein whose expression and processing are altered upon RVFV infection. This work adds to a new but growing body of knowledge that recognizes splicing as an important yet understudied mechanism that influences host-pathogen interactions.

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© Copyright 2019 Katherine Elizabeth Havranek