Evidence that Insertion of Tomato Ringspot Nepovirus NTB-VPg Protein in Endoplasmic Reticulum Membranes Is Directed by Two Domains: a C-Terminal Transmembrane Helix and an N-Terminal Amphipathic Helix

Abstract

The NTB-VPg protein of Tomato ringspot nepovirus is an integral membrane protein found in association with endoplasmic reticulum (ER)-derived membranes active in virus replication. A transmembrane helix present in a hydrophobic region at the C terminus of the NTB domain was previously shown to traverse the membranes, resulting in the translocation of the VPg domain in the lumen. We have now conducted an in planta analysis of membrane-targeting domains within NTB-VPg using in-frame fusions to the green fluorescent protein (GFP). As expected, the entire NTB-VPg protein directed the GFP fluorescence to ER membranes. GFP fusion proteins containing the C-terminal 86 amino acids of NTB-VPg also associated with ER membranes, resulting in ER-specific glycosylation at a naturally occurring glycosylation site in the VPg domain. Deletion of the hydrophobic region prevented the membrane association. The N-terminal 80 amino acids of NTB were also sufficient to direct the GFP fluorescence to intracellular membranes. A putative amphipathic helix in this region was necessary and sufficient to promote membrane association of the fusion proteins. Using in vitro membrane association assays and glycosylation site mapping, we show that the N terminus of NTB can be translocated in the lumen at least in vitro. This translocation was dependent on the presence of the putative amphipathic helix, suggesting that oligomeric forms of this helix traverse the membrane. Taken together, our results suggest that at least two distinct elements play a key role in the insertion of NTB-VPg in the membranes: a C-terminal transmembrane helix and an N-terminal amphipathic helix. An updated model of the topology of the protein in the membrane is presented.Infection by positive-strand RNA viruses results in massive proliferation and modification of the structure of the host intracellular membranes. Viral replication occurs in association with intracellular membranes and is associated with various modified membrane structures, including membranous vesicles and membranous webs (8,38,44,48). The specific nature of the membranes affected varies from one virus to another (e.g., endoplasmic reticulum [ER], tonoplast, mitochondrial membranes). Animal picornaviruses induce the formation of replication-competent membranous vesicles which are derived from the ER probably using mechanisms similar to those of the secretory pathway (17,28,43,47,51). Replication proteins and replication intermediates from plant viruses related to picornaviruses (e.g., potyviruses, comoviruses, and nepoviruses) are also found in association with ER-derived membranous vesicles (9,10,40,46). It is thought that these vesicles constitute mini-virus factories which are physically separated from the cytoplasmic content of the cell and offer protective environments for viral RNA replication (44).The initial targeting of viral replication proteins to the ER is a key step in the formation of replication-competent vesicles. One or several viral or host proteins act as membrane anchors for the re...

The Poly(A) Binding Protein Is Internalized in Virus-Induced Vesicles or Redistributed to the Nucleolus during Turnip Mosaic Virus Infection

“…Membrane flotation assays were conducted essentially as previously described (65). Briefly, P30 fraction was resuspended in 400 l of NTE buffer (10 mM Tris-HCl [pH 7.4], 100 mM NaCl, 1 mM EDTA), and 300 l was mixed with 1.6 ml of 85% (wt/vol) sucrose in NTE buffer and overlaid with 7 ml of 65% sucrose in NTE buffer and then overlaid with 3.1 ml of 10% sucrose in NTE buffer.…”

“…The presence of PABP2 in P30 fractions of TuMV-infected leaves may result from a true membrane association or simply protein aggregation. To distinguish between these two possibilities, a membrane flotation assay was used (65). The membrane-enriched fraction (P30) was overlaid with a sucrose step gradient and subjected to centrifugation.…”

Characterization of Membrane Association Domains within the Tomato Ringspot Nepovirus X2 Protein, an Endoplasmic Reticulum-Targeted Polytopic MembraneProtein

“…When expressed independently of other viral proteins, the NTB-VPg protein localizes to ER membranes (52). ER binding of the protein is mediated by two regions present in the NTB domain, i.e., a C-terminal transmembrane helix and an N-terminal amphipathic helix (50,52). These results led us to suggest that NTB and/or a polyprotein containing the NTB domain acts as a membrane anchor for the replication complexes.…”

“…Similarly the 3AB, 2BC, and 2C proteins of poliovirus and other picornaviruses, the 6-kDa protein of potyviruses, and the 60-kDa and 32-kDa proteins of Cowpea mosaic comovirus (CPMV) target to the ER membranes in the absence of other viral proteins and induce modifications of intracellular membranes similar to these found in viral infection (7,12,13,40). Membrane-binding domains have been identified in some viral membrane proteins, and these include transmembrane hydrophobic helices, amphipathic helices, and other less-well-defined sequences (16,17,46,47,52). However, the mechanisms of membrane binding and ER targeting are still poorly understood.…”

“…These proteins are tightly associated with ER membranes and cofractionate with ER-associated VRCs (19). When expressed independently of other viral proteins, the NTB-VPg protein localizes to ER membranes (52). ER binding of the protein is mediated by two regions present in the NTB domain, i.e., a C-terminal transmembrane helix and an N-terminal amphipathic helix (50,52).…”