Characterization of the membrane association of Tomato ringspot virus proteins containing the NTB binding domain

Replication of all known positive-strand RNA viruses occurs in replication complexes associated with intracellular membranes. The putative Nucleoside Triphosphate Binding (NTB) protein of Tomato ringspot virus (ToRSV) contains a stretch of hydrophobic residues at its C-terminus suggesting that it may act as a membrane anchor for the replication complex. Anti-NTB antibodies detected two predominant proteins in membrane-enriched fractions (the 66 kDa NTB and the 69 kDa NTB-VPg) along with other larger proteins. The proteins containing NTB co-fractionated with markers of the endoplasmic reticulum (ER) and with ToRSV specific RNA-dependent RNA polymerase activity in sucrose gradients. ToRSV infection induced severe changes in the morphology of the ER in plants expressing an ER-targeted green fluorescent protein (ER-GFP), and proteins containing NTB co-localized with the ER-GFP in indirect immunofluorescence assays. The proteins containing NTB have properties of integral membrane proteins. Proteinase K protection assays using purified membranes from infected plants revealed that although the central portion of NTB is exposed to the cytoplasmic face of the membranes, an 8 kDa fragment, recognized by anti- VPg antibodies, is protected by the membranes. This fragment probably consists of the 3 kDa VPg and the 5 kDa stretch of hydrophobic residues at the C-terminus of NTB, suggesting a lumenal location for the VPg in at least a portion of the molecules. To study whether NTB-VPg could associate with membranes, an in vitro membrane association assay was used. A truncated NTB-VPg protein containing the transmembrane domain could associate with canine microsomal membranes. The NTB-VPg and cNTB-VPg were glycosylated in the VPg domain in vitro suggesting a topology similar to that observed in infected plants. The cNTB-VPg protein was also processed by a membrane-associated proteinase in vitro at a site immediately upstream of the VPg domain. Expression of the cNTB-VPg protein in protoplasts revealed that this processing might also take place in vivo. Taken together, our results provide evidence that proteins containing the NTB domain are transmembrane proteins associated with ER-derived membranes and support the hypothesis that one or several of the proteins containing NTB anchor the replication complex to the ER.Science, Faculty ofBotany, Department ofGraduat