Two Tomato bushy stunt virus (TBSV) proteins, P19 and P22, have been found to interact with the Nicotiana benthamiana host proteins Hin19 and HFi22 in yeast two,hybrid assays. To determine functional roles of these interacting host proteins, viral induced gene silencing (VIGS) was employed to knock,down their expression. TBSV has been demonstrated to activate a virus,specific antiviral response pathway in N. benthamiana. To characterize this pathway, the antiviral RNAi induced silencing complex (RISC) was isolated from TBSV-infected plants. Additionally, putative RISC-associated proteins were identified in silico and suggested roles for these have been identified through literature and database searches. A further aim was the identification of proteins that coimmunoprecipitate with the TBSV-induced RISC following RISC isolation. A primary aim of this investigation was to identify functional roles for host proteins that interact with the two TBSV 3-terminal encoded proteins, P22 and P19. Each of these has functional roles in viral movement and pathogenicity. In yeast two-hybrid assays, P22 has been shown to interact with HFi22 while P19 interacts with Hin19. VIGS was utilized in attempts to silence the expression of these two host proteins in order to determine their functional roles. VIGS-mediated suppression of the TBSV-interacting proteins Hin19 and HFi22 has not been accomplished. Despite multiple attempts and multiple approaches, these proteins have not been amenable to silencing. In light of this finding, it is proposed that rather than utilizing VIGS to down-regulate protein levels for Hin19 and HFi22, other approaches should be utilized. To characterize the TBSV-mediated RNAi pathway, functionally active antiviral RISC was purified from TBSV-infected N. benthamiana plants using ion-exchange chromatography. This RISC was found to be active only in the degradation of TBSV transcripts, indicating the specificity expected from a programmed RISC. Characterization and identification of proteins that copurify with RISC has not yet been accomplished, though in silico analysis has yielded over 150 putative RISC-associated proteins. Of these, a subset has been identified as highly likely candidates based upon function and/or homology to RISC-associated proteins in non-plant organisms, and a model for the TBSV-induced antiviral pathway has been proposed.

Positive strand RNA viruses are intracellular parasites, and their genome replication and infection involves complex virus-host interactions. Therefore, identification of host factors and dissection of their functions during virus replication could facilitate our understanding of the mechanism of virus infection. Those host factors may also provide new targets for viral disease control. Tomato bushy stunt virus (TBSV) has recently become one of the model viruses to study positive strand RNA virus replication and hostvirus interactions. To identify host factors involved in TBSV replication we used yeast as a model host. Co-expression of the replication proteins and a replicon RNA (DI RNA) via plasmids in yeast resulted in robust replication of the viral RNA. Previous work using a yeast single gene deletion library (YKO) revealed 96 yeast genes affecting virus replication. The essential yeast genes could not be deleted so we used the Yeast Tet Promoters Hughes Collection (yTHc) where the original promoter was replaced by Tetracyclin-titratable promoter. I tested the 800 essential host genes available in yTHc. In total, we found 30 new host genes whose down-regulated expression either increased or decreased the accumulation of a TBSV repRNA. The identified essential yeast genes fall into different categories on the basis of the cellular processes they are involved in, such as RNA transcription/metabolism, protein metabolism/transport etc. Detailed analysis of the effects of some of the identified yeast genes revealed that they might affect RNA replication by altering (i) the amounts of p33 and p92(pol) viral replication proteins, (ii) the activity of the tombusvirus replicase complex, and (iii) the ratio of plus- versus minus-stranded RNA replication products. Altogether, this and previous YKO screening of yeast led to the identification of 126 host genes (out of ~5,600 genes that represent ~95% of all the known and predicted yeast genes) that affected the accumulation of tombusvirus RNA. In the YKO screening, we found NSR1 (homologous to plant nucleolin) gene, whose deletion led to increased TBSV repRNA accumulation. Nucleolin is an abundant RNA binding protein, which shuttles between the nucleolus, the nucleoplasm and the cytoplasm. This protein is involved in rRNA maturation, ribosome assembly and regulation of cellular RNA metabolism.We found that over-expression of Nsr1p in yeast or nucleolin in Nicotiana benthamiana inhibited the accumulation of tombusvirus RNA by ~10-fold. Temporal regulation of Nsr1p over-expression revealed that the inhibitory effect of Nsr1p was more profound when it was expressed at early stages of viral replication. In vitro binding experiments showed that Nsr1p binds preferably to the RIII in the repRNA (which is derived from 3’ UTR of viral genome). Consistent with its RIII specific binding, over-expression of Nsr1p only reduced 40% of the accumulation of TBSVΔRIII repRNA in yeast. The purified recombinant Nsr1p inhibited the in vitro replication of the viral RNA in a yeast cell-free assay when pre-incubated with the viral RNA before the in vitro replication assay. Our data suggest that Nsr1p/nucleolin inhibits tombusvirus replication by interfering with the recruitment of the viral RNA for replication.

Tomato bushy stunt virus (TBSV) provides a good model system to investigate molecular virus-host interactions in plants. P22 and P19 proteins encoded by TBSV contribute to multiple invasion-associated functions. Green fluorescence-mediated visualization of TBSV invasion in this study suggests that virus exit from inoculated epidermal cells is a crucial event. Close examination of one P22 mutant showed that it had lost the capacity to move between epidermis and mesophyll which was possibly due to an altered subcellular localization. P19 is a potent suppressor of RNA interference (RNAi) in various systems by forming dimers that bind 21-nucleotide (nt) duplex siRNAs (short interfering RNAs), to affect the programming of the RNA-induced silencing complex (RISC). P19 is attractive for biotechnological and research purposes to prevent RNAi of certain value-added genes in plants. To obtain a good plant-based expression platform, a suppression-active mutant P19 was expressed in transgenic N. benthamiana lines. This is the first example of P19 accumulating to detectable levels in a transgenic plant and initial results suggest it is actively suppressing RNAi. Furthermore, to investigate the correlation between siRNA binding of P19 and its various biological roles, predicted siRNA-interacting sites of TBSV P19 were modified, and the corresponding TBSV mutants were used to inoculate plants. Substitutions on siRNA-contact sites on the central domain of P19 resulted in more severe symptoms in N. benthamiana compared to those affecting peripheral regions. All tested combinations of siRNA-binding mutations were associated with reduced accumulation of total TBSV-derived siRNAs, and loss of siRNA sequestration by P19. Additionally, some modifications were found to cause RNAi-mediated disappearance of viral and host materials in N. benthamiana but not in spinach. In conclusion, exit out of epidermal cells is a key host range determinant for TBSV and particular amino acids on P22 may influence this by regulating the proper subcellular localization. Mutant P19 transgenic plants were successfully established with minor physiological effects to be applied as a platform to study RNAi and to over-express proteins. Finally, a compromised P19-siRNA binding impacts symptom development, systemic invasion, integrity of virus plus host RNA and proteins, and that all in a hostdependent manner.

Peroxisome biogenesis is complex, involving a diverse array of intracellular pathways and mechanisms that mediate their biogenesis and cellular functions. Relevant to our understanding of peroxisome biogenesis is the utilization of peroxisomal membranes for viral genome replication as observed in plant cells infected by several members of the Tombusviridae family of positive-strand RNA viruses. Tomato Bushy Stunt Virus (TBSV), for instance, usurps an array of host factors that facilitate the transformation of peroxisomes into peroxisomal multivesicular bodies (pMVB) the sites of viral RNA replication. In this study, pMVB topology and biogenesis was investigated using transmission electron and epifluorescence microscopy of tobacco and wildtype or mutant budding yeast that were transformed with TBSV replicase proteins and a defective interfering viral RNA. Overall, the results suggest that host-virus interactions specifically associated with Endosomal Sorting Complex Required for Transport (ESCRT) and lipid metabolism are involved in TBSV replication and pMVB biogenesis.