CHARACTERIZATION OF VIRAL AND HOST PROTEINS AND RNA ELEMENTS IN TOMBUSVIRUS REPLICATION

Two thirds of plant viruses are positive-strand RNA viruses including the family Tombusviridae. One of the best-studied members of this family is Tomato bushy stunt virus (TBSV). Like many other viruses, TBSV has much fewer genes when compared to its hosts’ genome. Nevertheless, TBSV utilizes its genome very judiciously. To compensate for a lack of many proteins of its own, it codes for multi-functional replication protein p33 and also co-opts host factors to facilitate its replication. By using recombinant replication proteins p33 and p92 containing single amino acid changes in protein-protein interaction domains (S1 and S2), I demonstrated that the replication proteins are required in sequential steps during virus replication. The in vitro cell-free extract(CFE) based TBSV replication assays revealed that mutations in S1 and S2 domains affected RNA template selection, recruitment and assembly of replicase complex. TBSV replicates on the cytosolic surface of peroxisomal membranes. To identify the host factor involved in this process of transporting viral replication proteins to peroxisome, I tested the peroxisomal transporter proteins for their ability to bind to p33 in vitro, which led to the discovery of Pex19p. Pull-down and co-purification experiments revealed transient nature of p33-Pex19p binding as expected from a transporter. When pex19p was retargeted to mitochondria, a large fraction of p33 was also re-distributed to the mitochondria validating the importance of Pex19p in p33 localization. TBSV also utilizes its genomic RNA for non-template activities during its replication. Accordingly, TBSV RNA serves as a platform for the assembly of replicase complex. To further characterize the regulatory cis-elements involved in this process, I utilized CFE and different TBSV RNA mutants together with recombinant p33 and p92 in vitro replication assays. These experiments revealed the role of RNA recruitment element [RIISL(+)] and 3’ non-coding regions as minimal cis-elements required to assemble functional replicase complex. The experiments also indicated that the RIISL(+) and 3’ non coding regions could be physically separated on two different RNA molecules to assemble TBSV replicase, suggesting insights into viral evolution

Role of Viral RNA and Co-opted Cellular ESCRT-I and ESCRT-III Factors in Formation of Tombusvirus Spherules Harboring the Tombusvirus Replicase

Plus-stranded RNA viruses induce membrane deformations in infected cells in order to build viral replication complexes (VRCs). Tomato bushy stunt virus (TBSV) co-opts cellular ESCRT (endosomal sorting complexes required for transport) proteins to induce the formation of vesicle (spherule)-like structures in the peroxisomal membrane with tight openings toward the cytosol. In this study, using a yeast (Saccharomyces cerevisiae) vps23Δ bro1Δ double-deletion mutant, we showed that the Vps23p ESCRT-I protein (Tsg101 in mammals) and Bro1p (ALIX) ESCRT-associated protein, both of which bind to the viral p33 replication protein, play partially complementary roles in TBSV replication in cells and in cell extracts. Dual expression of dominant-negative versions of Arabidopsis homologs of Vps23p and Bro1p inhibited tombusvirus replication to greater extent than individual expression in Nicotiana benthamiana leaves. We also demonstrated the critical role of Snf7p (CHMP4), Vps20p, and Vps24p ESCRT-III proteins in tombusvirus replication in yeast and in vitro. Electron microscopic imaging of vps23Δ yeast revealed the lack of tombusvirus-induced spherule-like structures, while crescent-like structures are formed in ESCRT-III deletion yeasts replicating TBSV RNA. In addition, we also showed that the length of the viral RNA affects the sizes of spherules formed in N. benthamiana cells. The 4.8-kb genomic RNA is needed for the formation of spherules 66 nm in diameter, while spherules formed during the replication of the ∼600-nucleotide (nt)-long defective interfering RNA in the presence of p33 and p92 replication proteins are 42 nm. We propose that the viral RNA serves as a “measuring string” during VRC assembly and spherule formation