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Journal of Virology, November 2006, p. 10847-10857, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.00789-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Characterization of Membrane Association Domains within the Tomato Ringspot Nepovirus X2 Protein, an Endoplasmic Reticulum-Targeted Polytopic Membrane Protein{triangledown} ,{dagger}

Guangzhi Zhang1 and Hélène Sanfaçon2*

Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada,1 Pacific Agri-Food Research Centre, Box 5000, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada2

Received 18 April 2006/ Accepted 9 August 2006

Replication of nepoviruses (family Comoviridae) occurs in association with endoplasmic reticulum (ER)-derived membranes. We have previously shown that the putative nucleoside triphosphate-binding protein (NTB) of Tomato ringspot nepovirus is an integral membrane protein with two ER-targeting sequences and have suggested that it anchors the viral replication complex (VRC) to the membranes. A second highly hydrophobic protein domain (X2) is located immediately upstream of the NTB domain in the RNA1-encoded polyprotein. X2 shares conserved sequence motifs with the comovirus 32-kDa protein, an ER-targeted protein implicated in VRC assembly. In this study, we examined the ability of X2 to associate with intracellular membranes. The X2 protein was fused to the green fluorescent protein and expressed in Nicotiana benthamiana by agroinfiltration. Confocal microscopy and membrane flotation experiments suggested that X2 is targeted to ER membranes. Mutagenesis studies revealed that X2 contains multiple ER-targeting domains, including two C-terminal transmembrane helices and a less-well-defined domain further upstream. To investigate the topology of the protein in the membrane, in vitro glycosylation assays were conducted using X2 derivatives that contained N-glycosylation sites introduced at the N or C termini of the protein. The results led us to propose a topological model for X2 in which the protein traverses the membrane three times, with the N terminus oriented in the lumen and the C terminus exposed to the cytoplasmic face. Taken together, our results indicate that X2 is an ER-targeted polytopic membrane protein and raises the possibility that it acts as a second membrane anchor for the VRC.

* Corresponding Author. Present address: Pacific Agri-Food Research Centre, Box 5000, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada. Phone: (250) 494-6393. Fax: (250) 494-0755. E-mail: SanfaconH{at}agr.gc.ca.

{triangledown} Published ahead of print on 23 August 2006.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

Journal of Virology, November 2006, p. 10847-10857, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.00789-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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