Downloaded from www.microbiologyresearch.org by IP: 54.145.26.59 On: Sat, 05 Mar 2016 21:07:45 Topogenesis in membranes of the NTB–VPg protein of Tomato ringspot nepovirus: definition of the C-terminal transmembrane domain Aiming Wang, 1 3 Sumin Han 1 4 and He ´ le ` ne Sanfac ¸on 2 Correspondence He ´le ` ne Sanfac ¸on SanfaconH@agr.gc.ca 1 Department of Botany, University of British Columbia, Vancouver, BC, Canada 2 Pacific Agri-Food Research Centre, 4200 Highway 97, Summerland, BC, Canada V0H 1Z0 Received 29 August 2003 Accepted 22 October 2003 The putative NTP-binding protein (NTB) of Tomato ringspot nepovirus (ToRSV) contains a hydrophobic region at its C terminus consisting of two adjacent stretches of hydrophobic amino acids separated by a few amino acids. In infected plants, the NTB–VPg polyprotein (containing the domain for the genome-linked protein) is associated with endoplasmic reticulum-derived membranes that are active in ToRSV replication. Recent results from proteinase K protection assays suggested a luminal location for the VPg domain in infected plants, providing support for the presence of a transmembrane domain at the C terminus of NTB. In this study, we have shown that NTB–VPg associates with canine microsomal membranes in the absence of other viral proteins in vitro and adopts a topology similar to that observed in vivo in that the VPg is present in the lumen. Truncated proteins containing 60 amino acids at the C terminus of NTB and the entire VPg exhibited a similar topology, confirming that this region of the protein contains a functional transmembrane domain. Deletion of portions of the C-terminal hydrophobic region of NTB by mutagenesis and introduction of glycosylation sites to map the luminal regions of the protein revealed that only the first stretch of hydrophobic amino acids traverses the membrane, while the second stretch of hydrophobic amino acids is located in the lumen. Our results provide additional evidence supporting the hypothesis that the NTB–VPg polyprotein acts as a membrane-anchor for the replication complex. INTRODUCTION Replication of the genome of positive-strand RNA plant viruses involves the formation of a replication complex composed of several viral proteins and host plant proteins, associated with specific intracellular membranes (Buck, 1996). The replication complexes of picornaviruses and several plant picorna-like viruses (including potyviruses, comoviruses and nepoviruses) have been shown to be associated with membranes having properties of the endoplasmic reticulum (ER) (Carette et al., 2000; Han & Sanfac ¸on, 2003; Ritzenthaler et al., 2002; Schaad et al., 1997; Suhy et al., 2000). Virus infection results in a massive modification and proliferation of the ER membranes, and de novo lipid synthesis has been shown to be required for the replication of Poliovirus (PV), Cowpea mosaic virus (CPMV, genus Comovirus), and Grapevine fanleaf nepovirus (GFLV) (Carette et al., 2000; Guinea & Carrasco, 1990; Ritzenthaler et al., 2002). Specialized viral proteins are thought to play a role as membrane anchors, either by directly associating with intracellular membranes as integral membrane proteins or by interacting with membrane proteins from the host. The viral proteins that act as membrane anchors may in turn interact with other viral proteins (such as the RNA- dependent RNA polymerase, Pol) to redirect them towards the membranes and induce the formation of the replica- tion complexes. In picorna-like viruses, mature proteins as well as intermediate precursor proteins are released from large polyproteins by virus-encoded proteinases. The membrane anchors may therefore associate with mem- branes as mature proteins or as larger polyprotein precursors. For example, in PV, 3AB is a membrane- associated intermediate polyprotein that forms a complex with 3CD (proteinase–polymerase) and with the viral RNA (Porter, 1993; Xiang et al., 1997). Tomato ringspot virus (ToRSV, genus Nepovirus, family Comoviridae) has a bipartite, single-stranded, positive-sense RNA genome (Mayo & Robinson, 1996; Sanfac ¸on, 1995). Each molecule of RNA is covalently linked to a protein at its 59 end (VPg), is polyadenylated at its 39 end and encodes one large polyprotein. The RNA1-encoded polyprotein (P1) 4Present address: The Hospital for Sick Children, Cancer Research Program, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8. 3Present address: Southern Crop Protection and Food Research Centre, 1391 Sandford Street, London, Ontario, Canada, N5V 4T3. 0001-9612 G 2004 Canadian Government Printed in Great Britain 535 Journal of General Virology (2004), 85, 535–545 DOI 10.1099/vir.0.19612-0