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.

Satellite tobacco mosaic virus (STMV) as the name implies is the satellite virus of the well characterized helper Tobacco mosaic virus (TMV). This satellite virus is 18nm in diameter having a T1 symmetry encapsidating a positive sense genomic RNA of 1058nt in size whose 3' 150nt shares 65% homology with that of its Helper virus. Extensive Structural studies on STMV suggested that its coat protein interacts extensively with the stem-loops of the packaged genomic RNA and hence would be unstable to form virions without interacting with its genomic RNA internally. However invivo experiments to prove the same were not performed. Host range effects and the role of the positively charged amino acids at the N-terminal of its capsid protein on replication and packaging were not been studied yet on this Satellite virus (or in any other satellite virus). Chapter 1 of this dissertation describes T-DNA based agrotransformants of STMV to synthesize its genomic RNA in either (+) or (-) orientation when expressed ectopically in Nicotiana benthamiana plants. The biological activity of ectopically expressed agrotransformants of STMV (+) or (-) transcripts was confirmed when co-expressed with TMV. Furthermore, progeny virions of STMV recovered from agroinfiltrations were indistinguishable from those recovered from mechanically inoculated plants. However, STMV virions assembled in N. benthamiana, but not in tobacco, efficiently packaged a variant form of the genomic RNA. Chapter 2 describes Individual substitution of the positively charged amino acids at the N-terminal region of its Coat protein to a neutrally charged amino acid Alanine affecting dramatically the replication and packaging of these mutant viruses. Chapter 3 describes Agrobacterium mediated transient expression of the three genomic RNA of Tomato Aspermy virus in N.benthamiana and by utilizing this system we demonstrate that expressed genomic RNA were biologically active and also induced symptoms in N.benthamiana.

Viruses with positive-sense single stranded RNA genomes comprise the majority of viruses pathogenic to humans, animals and plants like Poliovirus, West Nile Virus, Equine arteritis virus, Cucumber Mosaic Virus and Brome Mosaic Virus. Brome Mosaic Virus (BMV) belongs to this group and is in the family Bromoviridae, it is the type member of the family. BMV is used as a model to study replication, packaging and recombination of positive-sense RNA viruses. It is a tripartite virus with three RNA molecules each is packaged independently. For a successful infection, all three particles must infect the same cell for efficient replication of the genome and packaging. Chapter one of this dissertation will focus on proteome analysis of Nicotiana benthamiana infected with BMV to study N. benthamiana proteome that interact with three virally encoded proteins: the replicase proteins p1a and p2a, and the coat protein. N. benthamiana is an experimental plant host that is widely used in research. For that Co-immunoprecipitation was performed to recovered host proteins that interact with the tested virally encoded proteins. Bioinformatics analyses to predict the subcellular localization and functions of the recovered proteins is discussed. Chapter two, similarly to chapter one, will focus on proteome analysis of the natural host for BMV Hordeum vulgare infected with BMV to study its proteome that interact with the three virally encoded proteins: p1a, p2a, and the coat protein. In Chapter three the effect of silencing one of the proteins that interact with BMV replicase protein on BMV accumulation will be examined. Specifically, the protein S-phase kinase related protein-1 was silenced via Virus Induced Gene Silencing (VIGS) and further infection with BMV to examine if SKP1 is important for BMV replication.

The N-proximal region of cowpea chlorotic mottle virus (CCMV) capsid protein (CP) contains an arginine-rich RNA binding motif (ARM) that is also found in the CPs of other members of Bromoviridae and in other RNA binding proteins such as the Tat and Rev proteins of human immunodeficiency virus. To assess the critical role played by this motif during encapsidation, a variant of CCMV RNA3 (C3) precisely lacking the ARM region (C3/Delta919) of its CP gene was constructed. The biology and the competence of the matured CP derived in vivo from C3/Delta919 to assemble and package progeny RNA was examined in whole plants. Image analysis and computer-assisted three-dimensional reconstruction of wild-type and mutant virions revealed that the CP subunits bearing the engineered deletion assembled into polymorphic virions with altered surface topology. Northern blot analysis of virion RNA from mutant progeny demonstrated that the engineered mutation down-regulated packaging of all four viral RNAs; however, the packaging effect was more pronounced on genomic RNA1 and RNA2 than genomic RNA3 and its CP mRNA. In vitro assembly assays with mutant CP subunits and RNA transcripts demonstrated that the mutant CP is inherently not defective in packaging genomic RNA1 (53%) and RNA2 (54%), but their incorporation into virions was competitively inhibited by the presence of other viral RNAs. Northern blot analysis of RNA encapsidation in vivo of two distinct bromovirus RNA3 chimeras, constructed by exchanging CPs having the Delta919 deletion, demonstrated that the role of the conserved N-terminal ARM in recognizing and packaging specific RNA is distinct for each virus.