Proteolytic processing of tomato ringspot nepovirus 3C-like protease precursors: definition of the domains for the VPg, protease and putative RNA-dependent RNA polymerase.

Wang, A; Carrier, Karma; Chisholm, Joan; Wieczorek, Andrew; Huguenot, C.; Sanfa√ßon, H

doi:10.1099/0022-1317-80-3-799

Cited by 30 publications

(49 citation statements)

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“…In vitro, the ToRSV NTB-VPg cleavage site is a predominant cis-cleavage site, and accumulation of NTB-VPg is not detected. Rather, the mature NTB and the VPg-Pro intermediate were shown to accumulate (54,55). The accumulation of NTB-VPg in infected plants suggests that cellular factors (such as the membrane environment) modulate the recognition of the NTB-VPg cleavage site by the ToRSV proteinase.…”

Section: Discussionmentioning

confidence: 99%

“…1A) (43). The proteinase processes P1 at five cleavage sites in vitro, and the precise locations of the NTB-VPg, VPg-Pro, and Pro-Pol cleavage sites have been determined experimentally (54,55). The NTB protein has sequence elements similar to those found in known RNA helicases (24) and has homology with the picornavirus 2C and 3A proteins.…”

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confidence: 98%

“…The membranes were blocked with PBS containing 5% skim milk powder and incubated with the primary antibodies (diluted in PBS buffer) for 1 h at room temperature. The anti-NTB, anti-VPg, anti-movement protein (anti-MP), and anti-coat protein (anti-CP) antibodies were described previously (45,54,56). The anti-Bip and anti-␤-xylosyl antibodies were gifts from M. Chrispeels (University of California, San Diego) and A. Sturm (Friedrich Miescher Institute), respectively.…”

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confidence: 99%

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

Han

Sanfaçon²

2003

J Virol

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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.Replication of the genomes of all characterized positivestrand RNA viruses occurs in large complexes which are associated with intracellular membranes (5). Many positive-strand RNA viruses induce extensive proliferation and modification of intracellular membranes in their hosts, which often results in the accumulation of numerous membranous vesicles. Doublestranded RNA (dsRNA) replication intermediates and viral replication factors are associated with the membranous vesicles, which are thought to be the site of the replication (6,7,18,25,48,49,52). The requirement for intact membranes for successful virus replication has also been demonstrated using cell-free replication systems (2, 35, 59). Different viruses associate with and modify different types of intracellular membranes (5). Although the importance of the association of viral replication factors with intracellular membranes is well documented, the mechanisms by which replication complexes are fixed on specific membrane sites remain poorly understood.Picornaviruses and several plant viruses with similar genomic organization have been shown to replicate in association with membranes derived from the endoplasmic reticulum (ER) (6,42,47,51). One or several viral proteins are thought to act as membrane an...

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Section: Discussionmentioning

confidence: 99%

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confidence: 98%

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confidence: 99%

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

Han

Sanfaçon²

2003

J Virol

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“…It has been observed earlier that in some viruses, VPg influences the activity of the protease. For instance, in Tomato ringspot nepovirus (TomRSV), VPg-Pro precursor was shown to be more active in cleavage at one of the sites (4). In cowpea mosaic virus, expression of the protease along with the N-terminal extension corresponding to the VPg sequence enhanced its proteolytic activity (5).…”

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confidence: 99%

“Natively Unfolded” VPg Is Essential for Sesbania Mosaic Virus Serine Protease Activity

Satheshkumar

Gayathri

Prasad

et al. 2005

Journal of Biological Chemistry

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Polyprotein processing is a major strategy used by many plant and animal viruses to maximize the number of protein products obtainable from a single open reading frame. In Sesbania mosaic virus, open reading frame-2 codes for a polyprotein that is cleaved into different functional proteins in cis by the N-terminal serine protease domain. The soluble protease domain lacking 70-amino-acid residues from the N terminus (⌬N70Pro, where Pro is protease) was not active in trans. Interestingly, the protease domain exhibited trans-catalytic activity when VPg (viral protein genome-linked) was present at the C terminus. Bioinformatic analysis of VPg primary structure suggested that it could be a disordered protein. Biophysical studies validated this observation, and VPg resembled "natively unfolded" proteins. CD spectral analysis showed that the ⌬N70Pro-VPg fusion protein had a characteristic secondary structure with a 230 nm positive CD peak. Mutation of Trp-43 in the VPg domain to phenylalanine abrogated the positive peak with concomitant loss in cis-and trans-proteolytic activity of the ⌬N70Pro domain. Further, deletion of VPg domain from the polyprotein completely abolished proteolytic processing. The results suggested a novel mechanism of activation of the protease, wherein the interaction between the natively unfolded VPg and the protease domains via aromatic amino acid residues alters the conformation of the individual domains and the active site of the protease. Thus, VPg is an activator of protease in Sesbania mosaic virus, and probably by this mechanism, the polyprotein processing could be regulated in planta.

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“…The genome of Tomato ringspot nepovirus (ToRSV) consists of two molecules of RNA (45). RNA1 encodes a polyprotein (P1) containing the domains for the replication proteins, including the RNA-dependent RNA polymerase, a 3C-like proteinase, the genome-linked protein (VPg), a putative nucleoside triphosphate-binding protein (NTB), and two additional proteins (X1 and X2) of unknown function (42,59,61). The 3C-like proteinase is responsible for cleavage of P1 and of the RNA2-encoded polyprotein (13).…”

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confidence: 99%

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

Zhang¹,

Zhang

Yang³

et al. 2005

J Virol

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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...

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Proteolytic processing of tomato ringspot nepovirus 3C-like protease precursors: definition of the domains for the VPg, protease and putative RNA-dependent RNA polymerase.

Cited by 30 publications

References 50 publications

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

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

“Natively Unfolded” VPg Is Essential for Sesbania Mosaic Virus Serine Protease Activity

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

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