UC Riverside

Positive sense RNA viruses play a central role in the field of virology in general and agriculture in particular. Cucumber mosaic virus (CMV) is a positive sense RNA virus, playing an important role in agriculture because of its broad host range, as it can infect over 1200 species of plants. CMV is sometime accompanied by satellite RNA, a 336 nucleotides long noncoding RNA, which can either ameliorate or intensify symptom expression by the virus. CMV is a multipartite virus, requiring a constellation of three virion particles to initiate a successful infection. Chapter 1 of this dissertation focuses on optimization the Agrobacterium cell concentration of three genomic RNA agroconstructs of Brome mosaic virus (BMV), a widely used model system to study replication, recombination and packaging in positive sense RNA viruses. In this chapter, the focus is made on understanding effect of optical density (O.D600) of Agrobacterium cell cultures on synchronized infection followed viral gene expression. Chapter 2 is largely focused on understanding the mechanism regulating nuclear import of satellite RNA (satRNA) in the presence and absence of CMV. It was identified that Bromodomain containing RNA binding protein (BRP1), an ortholog of which has been previously documented to play an important role in the life cycle of Potato Spindle Tuber Viroid (PSTVd), plays a crucial role in the nuclear import of satRNA. Information garnered in this chapter helped bridging the evolutionary gap between satellite RNA and PSTVd. In Chapter 3, an attempt is made to understand the proteome interacting with BRP1, whose functionality in a plant is yet unknown. By using proteomic approaches, we were able to understand proteome interacting with BRP1 by itself, or in the presence of CMV or satellite RNA in Nicotiana benthamiana plants. Chapter 4 is an extension of chapter 3, where single gene knockout lines of Arabidopsis thaliana of short listed host proteins from chapter 3 were tested for their role in CMV replication, and specificaly the role of Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) in replicase complex assembly/stability was studied. Understanding the shift in proteome of host in case of a satRNA is a daunting task (due to the inability of satRNA to code for proteins), hence, we employed riboproteomic approach in Chapter 5 to short list host proteins interacting with satellite RNA in plus- or minus- sense, in the presence or absence of CMV. Viral protein-protein interactions play an important role in the replication of positive sense RNA viruses. Chapter 6, and 7 focus on comparative study of protein protein interactions for two important members of Bromoviridae family. In chapter 6, live cell imaging using BiFC analysis helped us visualize protein protein interactions and subcellular localization of BMV proteins.. Finally, in Chapter 7, we provide an extensive protein-protein interaction study of CMV viral proteins in vivo using Bimolecular Fluorescence complementation (BiFC) assay.