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Characterization of two tomato ringspot virus replication proteins : the NTB protein and the RNA-dependent RNA polymerase

University of British Columbia

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.

Text

2015-10-24

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/54840

Botany

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/