Release of a 22-kDa protein derived from the amino-terminal domain of the 49-kDa NIa of turnip mosaic potyvirus in Escherichia coli

Laliberté, Jean‐François; Nicolas, Olivier; Chatel, Henriette; Lazure, Claude; Morosoli, Rolf

doi:10.1016/0042-6822(92)91244-o

Cited by 28 publications

(13 citation statements)

References 26 publications

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“…Our results indicate that the anti-His antibodies recognized VPg but failed to recognize unprocessed polyprotein which further processed VPg and NIa-Pro. Our results were corresponding to the previous results that the VPg alone without NIa-Pro was detected when the entire coding region of NIa protein of TuMV was expressed in E. coli [36] . The NIa-Pro of TEV was also expressed in E. coli as a recombinant protein with His-tag, but the expression of unprocessed polyprotein was unsuccessful [37] .…”

Section: Resultssupporting

confidence: 80%

Molecular Characterization of Soybean Mosaic Virus NIa Protein and its Processing Event in Bacterial Expression

Choi¹,

Park²,

Ahn³

et al. 2006

American J. of Biochemistry and Biotechnology

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Soybean mosaic virus (SMV)-CN18 is anRsv resistance-breaking (RB) isolate to overcome soybean resistance genes Rsv1, Rsv3 and Rsv4. The aim of this study was to characterize nuclear inclusion protein a (NIa protein) of RB isolate at the molecular level and demonstrate its processing into genome-linked protein (VPg) and NIa-Pro domains in Esherichia coli containing a bacterial expression pET vector inserted with NIa gene. The full-length of NIa gene was synthesized by reverse transcription-polymerase chain reaction (RT-PCR) and its 1298 nucleotides (nt) and 432 amino acids (aa) were deduced. The nt and aa sequences of NIa gene of SMV-CN18 shared high identities with the corresponding sequences of the NIa gene of the known SMV isolates, suggesting that the NIa is a highly conserved protein. The NIa-Pro domain contains a highly conserved structural motif for proteolysis, while the VPg domain contains a nuclear localization signal (NLS), a putative NTPbinding site and cellular factor-binding sites. The phylogenetic tree revealed that less divergence of NIa protein exists among twelve SMV isolates, which can be supported by a low bootstrap value between clades. In addition, the full-length of NIa gene, amplified by RT-PCR, was ligated into pET28b E. coli expression vector with an N-terminal His 6 -tag. Optimal conditions for expression were at 1mM treatment of IPTG at 25°C for 5 hr. The released protein from bacterial lysates remained soluble and proved the processing form of the NIa polyprotein. E. coli expression system shows the processed product of 29 kDa VPg in SDS-PAGE confirmed by western blot analysis in both crude extracts and purified elution products, using Ni 2+ -NTA resin. The present study indicates that the N-terminal region of NIa which is processed and expressed in bacteria.

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Section: Resultssupporting

confidence: 80%

Molecular Characterization of Soybean Mosaic Virus NIa Protein and its Processing Event in Bacterial Expression

Choi¹,

Park²,

Ahn³

et al. 2006

American J. of Biochemistry and Biotechnology

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“…In order to prevent proteolysis by Pro at the 6K-VPg and VPg-Pro junctions, the glutamic acid residue preceding the cleavage bond was modified to histidine. These modifications were shown to prevent processing at the cleavage sites (28). Transient expression of VPg precursors was performed by agroinfiltration in N. benthamiana, which is a host for TuMV.…”

Section: Resultsmentioning

confidence: 99%

“…The resulting plasmids were identified as pGreen/eIF(iso)4E-GFP. 6K-VPg-Pro and VPg-Pro were amplified with the 6K-F/Pro-R and VPg-F/Pro-R primers (Table 1), respectively, from the pET/ 6K2-NIa Quebec isolate of TuMV, which has been modified at the junction of 6K2 and VPg and at the active site of Pro to prevent Pro-mediated proteolysis (28). 6K-VPg-Pro and VPg-Pro cDNA fragments were inserted into HindIII/ BamHI sites of the pGreenDsRed (for VPg-Pro only) and pGreenGFP vectors.…”

Section: Methodsmentioning

confidence: 99%

Visualization of the Interaction between the Precursors of VPg, the Viral Protein Linked to the Genome ofTurnip Mosaic Virus, and the Translation Eukaryotic Initiation Factor iso 4E In Planta

Beauchemin

Boutet

Laliberté

2007

J Virol

149

116

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The RNA genome of Turnip mosaic virus is covalently linked at its 5 end to a viral protein known as VPg. This protein binds to the translation eukaryotic initiation factor iso 4E [eIF(iso)4E]. This interaction has been shown to be important for virus infection, although its exact biological function(s) has not been elucidated. In this study, we investigated the subcellular site of the VPg-eIF(iso)4E interaction using bimolecular fluorescence complementation (BiFC). As a first step, eIF(iso)4E, 6K-VPg-Pro, and VPg-Pro were expressed as full-length green fluorescent protein (GFP) fusions in Nicotiana benthamiana, and their subcellular localizations were visualized by confocal microscopy. eIF(iso)4E was predominantly associated with the endoplasmic reticulum (ER), and VPg-Pro was observed in the nucleus and possibly the nucleolus, while 6K-VPg-Pro-GFP induced the formation of cytoplasmic vesicles budding from the ER. In BiFC experiments, reconstituted green fluorescence was observed throughout the nucleus, with a preferential accumulation in subnuclear structures when the GFP split fragments were fused to VPg-Pro and eIF(iso)4E. On the other hand, the interaction of 6K-VPg-Pro with eIF(iso)4E was observed in cytoplasmic vesicles embedded in the ER. These data suggest that the association of VPg with the translation factor might be needed for two different functions, depending of the VPg precursor involved in the interaction. VPg-Pro interaction with eIF(iso)4E may be involved in perturbing normal cellular functions, while 6K-VPg-Pro interaction with the translation factor may be needed for viral RNA translation and/or replication.One of the initial steps in protein synthesis is the recruitment of mRNAs by the translation eukaryotic initiation factor 4F (eIF4F) complex (17). In plants, this complex is composed of two proteins, eIF4E and eIF4G (4). eIF4E is the cap-binding protein, while eIF4G is a large polypeptide containing binding sites for eIF4E, eIF4A, eIF3, and the poly(A) binding protein. eIF4F simultaneously interacts with the cap structure of mRNA (through eIF4E) and the ribosome-associated eIF3 (through eIF4G). Thus, eIF4F executes the pivotal function of bridging mRNAs with ribosomes. Another cap-binding complex, eIF(iso)4F, has been identified in plants (5) and is composed of a smaller eIF(iso)4E subunit and a larger eIF(iso)4G subunit relative to the eIF4F subunits. Those two complexes perform essentially the same task in translation but have different affinities for certain classes of mRNA substrates and may be involved in different cellular events (14).Potyviruses have a positive-strand RNA genome of approximately 10 kb (53). The genome codes for a single long polyprotein that is processed by viral proteinases into 10 mature proteins. The viral RNA is polyadenylated at its 3Ј end and has a covalently linked virus-encoded protein (VPg) instead of a cap structure at the 5Ј end. Being derived from a polyprotein, VPg exists in various forms (i.e., VPg, VPg-Pro, 6K-VPg-Pro) that fulfill specific functions....

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“…Similarly, Margis et al (1994) reported that maturation of the VPg-Pro intermediate precursor of grapevine fanleaf virus (GFLV) occurred at very low rates in vitro. Also, the potyvirus NIa protein was very inefficiently cleaved at the VPg-Pro site in vitro and in E. coli (Dougherty & Parks, 1991 ;Laliberte! et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

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

Carrier

Chisholm³

et al. 1999

Journal of General Virology

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Release of a 22-kDa protein derived from the amino-terminal domain of the 49-kDa NIa of turnip mosaic potyvirus in Escherichia coli

Cited by 28 publications

References 26 publications

Molecular Characterization of Soybean Mosaic Virus NIa Protein and its Processing Event in Bacterial Expression

Molecular Characterization of Soybean Mosaic Virus NIa Protein and its Processing Event in Bacterial Expression

Visualization of the Interaction between the Precursors of VPg, the Viral Protein Linked to the Genome ofTurnip Mosaic Virus, and the Translation Eukaryotic Initiation Factor iso 4E In Planta

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

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