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Journal of Virology, January 2003, p. 523-534, Vol. 77, No. 1
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.1.523-534.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Tomato Ringspot Virus Proteins Containing the Nucleoside Triphosphate Binding Domain Are Transmembrane Proteins That Associate with the Endoplasmic Reticulum and Cofractionate with Replication Complexes

Department of Botany, University of British Columbia, Vancouver, British Columbia V6T 1Z4,¹ Pacific Agri-Food Research Centre, Summerland, British Columbia V0H 1Z0, Canada²

Received 8 August 2002/ Accepted 1 October 2002

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 69-kDa NTB-VPg proteins) along with other, larger proteins. The proteins containing the NTB domain cofractionated 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 the NTB domain colocalized with ER-GFP in indirect immunofluorescence assays. The proteins containing the NTB domain have properties of integral membrane proteins. Proteinase K protection assays using purified membranes from infected plants revealed that although the central portion of the NTB domain 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 the NTB protein, suggesting a luminal location for the VPg in at least a portion of the molecules. These 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 the NTB domain anchor the replication complex to the ER.

Contribution no. 2175 of the Pacific Agri-Food Research Centre.