Year of Award


Document Type


Degree Type

Doctor of Philosophy (PhD)

Degree Name

Biochemistry & Biophysics

Department or School/College

Department of Chemistry and Biochemistry

Committee Chair

J. Stephen Lodmell

Commitee Members

Brooke Martin, Brent Ryckman, Ekaterina Voronina, Scott Wetzel


CLIP-seq, Mass spectrometry, Nucleocapsid, Rift Valley fever virus, RNA, RT-qPCR


University of Montana


Rift Valley fever virus (RVFV) is a mosquito-borne virus that infects humans and livestock and exhibits potential for global spread. No vaccine or treatment is currently available for human use. The nucleocapsid protein (N) in RVFV is a multifunctional RNA-binding protein that is necessary for viral replication and dissemination. Because of its essential roles, N is a promising candidate to be targeted for anti-RVFV therapy. The goal of this dissertation is to characterize N-RNA interactions in RVFV infected cells. To achieve this goal, two methods, strand-specific reverse transcription and quantitative PCR (RT-qPCR) as well as multiple reaction monitoring mass spectrometry (MRM-MS) for N, were developed. Using strand-specific RT-qPCR, we were able to discriminate expression levels of viral genomic, antigenomic, and protein coding mRNAs. We determined that the relative expression levels of viral protein-coding RNAs increased according to previously published viral promoter strength determinations. However, expression levels of antigenomic RNAs were independent of the RVFV promoter strength. Moreover, the presence of NSs protein, the virulence factor for RVFV, positively correlated with increased viral RNA (vRNA) synthesis. Combining RT-qPCR and MRM-MS methods with crosslinking immunoprecipitation and RNA sequencing (CLIP-seq), it was found that N interacts not only with viral but with host RNAs during infection. The interactions between N and vRNAs increased with infection time, which correlated with an increase in the specific infectivity of virus particles produced later in infection. The results provided in this dissertation add important knowledge pertaining to vRNA synthesis, encapsidation, and packaging in RVFV infection and will be useful in the development of N-targeting anti-RVFV therapeutics in the future.



© Copyright 2021 Miyuki Hayashi