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Interaction between two tomato ringspot virus replication proteins and endoplasmic reticulum membranes Zhang, Guangzhi


Genome replication of Tomato ringspot virus (ToRSV) occurs in association with the endoplasmic reticulum (ER) and/or ER-derived membranes. To identify the viral membrane anchor proteins for the replication complex, two hydrophobic proteins encoded by ToRSV RNA1, termed NTB-VPg and X2, were studied for their ability to associate with intracellular membranes in planta and in vitro. NTB-VPg and X2 were fused to the green fluorescent protein (GFP) and the fusion proteins were transiently expressed in Nicotiana benthamiana (N. benthamiana) epidermal cells. Subcellular localization of the fusion proteins was examined by confocal microscopy and subcellular fractionation and immunobloting. Mutagenesis was conducted to identify the membrane targeting domains within each protein. In addition, in vitro membrane association assays and glycosylation site mapping were used to investigate the membrane topology of the two proteins. Confocal images showed that both NTB-VPg and X2 directed GFP to the ER membranes, and both proteins co-fractionated with an ER marker (Bip) when expressed in planta. In the NTB VPg protein, two distinct ER-binding domains were identified: an N-terminal amphipathic helix and a C-terminal transmembrane domain. The C-terminal transmembrane domain was sufficient to direct GFP to the ER and translocate the downstream VPg domain into the ER lumen, resulting in ER-specific glycosylation at the naturally occurring glycosylation site in the VPg domain; the N-terminal amphipathic helix was also necessary and sufficient to direct GFP to intracellular membranes in planta and translocate the N-terminus of NTB into the ER lumen at least in vitro. In X2, three ER-targeting domains were identified by mutagenesis in planta : two C-terminal transmembrane domains and a less-well-defined domain further upstream. In vitro glycosylation mapping studies indicated that all three domains within X2 were able to traverse the membranes, resulting in an overall N[sub lumen] /C[sub cytosol] topology. Taken together, the results indicate NTB-VPg and X2 are polytopic ER membrane proteins. ER-association of both proteins in the absence of other viral components suggests they are the membrane anchor proteins for the viral replication complex.

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