UBC Theses and Dissertations
Characterization of two tomato ringspot virus replication proteins : the NTB protein and the RNA-dependent RNA polymerase Wei, Ting
Tomato ringspot virus (ToRSV) replicates in large protein complexes that are associated with modified endoplasmic reticulum membranes. The ToRSV RNA-dependent RNA polymerase (Pol) and integral membrane protein NTB-VPg are essential components of these complexes. Membrane-associated modifications of NTB-VPg (N-glycosylation and a putative signal peptidase cleavage) were previously observed in vitro but were not well characterized. Two forms of the polymerase were detected in infected plants: the full-length Pol, which accumulates at low concentration and the VPg-Pro-Pol' polyprotein, which includes a 15kDa C-terminal truncation of Pol and accumulates to higher levels. My specific objectives were to characterize the signal peptidase cleavage in NTB-VPg and investigate the stability and function of various forms of the polymerase. Using in vitro translation assays and plant transient expression assays, I detected signal peptidase processing in the NTB-VPg of three ToRSV isolates. Using site-directed mutagenesis, I mapped a suboptimal GAAGG cleavage site (Rasp2 isolate) and identified key amino acids that regulate the efficiency of cleavage. Compared to typical signal peptides, the NTB-VPg sequence has an unusually long distance between the end of the hydrophobic region and the cleavage site, indicating that it adopts a unique topology in the membrane. This is the first detailed characterization of signal peptidase cleavage of a plant virus replication protein. This cleavage may alter the conformation of NTB-VPg in the membrane and influence the architecture of the replication complexes. Using agroinfiltration assays, I show that the full-length Pol and VPg-Pro-Pol are unstable when ectopically expressed in N. benthamiana. Truncation of the C-terminal 15 kDa from Pol or VPg-Pro-Pol increased their stability in plants, which is consistent with the accumulation of VPg-Pro-Pol' in infected plants. In spite of repeated attempts, I was unable to establish an in vitro assays to compare the activity of Pol and VPg-Pro-Pol', possibly because an essential plant factor is missing. The instability of VPg-Pro-Pol and Pol may regulate the rate of virus replication, allowing the virus to keep its genome integrity and reducing the chance of being recognized by host defense responses.
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