scholarly journals Dual-colour imaging of membrane protein targeting directed by poa semilatent virus movement protein TGBp3 in plant and mammalian cells

2002 ◽  
Vol 83 (3) ◽  
pp. 651-662 ◽  
Author(s):  
A. A. Zamyatnin ◽  
A. G. Solovyev ◽  
A. A. Sablina ◽  
A. A. Agranovsky ◽  
L. Katul ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Protein Trafficking ◽  
Mammalian Cells ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Triple Gene Block ◽  
Gene Block ◽  
Movement Function ◽  
Dual Colour

The movement function of poa semilatent hordeivirus (PSLV) is mediated by the triple gene block (TGB) proteins, of which two, TGBp2 and TGBp3, are membrane proteins. TGBp3 is localized to peripheral bodies in the vicinity of the plasma membrane and is able to re-direct TGBp2 from the endoplasmic reticulum (ER) to the peripheral bodies. For imaging of TGBp3-mediated protein targeting, PSLV TGBp3 tagged with a red fluorescent protein (DsRed) was used. Coexpression of DsRed-TGBp3 with GFP targeted to the ER lumen (ER-GFP) demonstrated that ER-GFP was contained in typical ER structures and peripheral bodies formed by TGBp3 protein, suggesting an ER origin for these bodies. In transient coexpression with viral membrane proteins tagged with GFP, DsRed-TGBp3 directed to the peripheral bodies the homologous TGBp2 protein and two unrelated membrane proteins, the 6 kDa movement protein of beet yellows closterovirus and the putative movement protein encoded by the genome component 4 of faba bean necrotic yellows nanovirus. However, coexpression of TGBp3 with GFP derivatives targeted to the ER membranes by artificial hydrophobic tail sequences suggested that targeting to the ER membranes per se was not sufficient for TGBp3-directed protein trafficking to peripheral bodies. TGBp3-induced targeting of TGBp2 also occurred in mammalian cells, indicating the universal nature of the protein trafficking signals and the cotargeting mechanism.

scholarly journals Viral protein targeting to the cortical endoplasmic reticulum is required for cell–cell spreading in plants

2011 ◽  
Vol 193 (3) ◽  
pp. 521-535 ◽  
Author(s):  
Chih-Hang Wu ◽  
Shu-Chuan Lee ◽  
Chao-Wen Wang
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Protein Targeting ◽  
Triple Gene Block ◽  
Intercellular Junctions ◽  
Cell Spreading ◽  
Gene Block ◽  
Sorting Signal ◽  
Viral Spread ◽  
Cortical Endoplasmic Reticulum

Many plant RNA viruses use their nonstructural proteins to target and move through the cortical endoplasmic reticulum (ER) tubules within the plant intercellular junction for cell-to-cell spreading. Most of these proteins, including the triple-gene-block 3 protein (TGBp3) of Potexvirus, are ER membrane proteins. We previously showed that TGBp3 of the Bamboo mosaic potexvirus partitions into tubular subdomains of the ER in both yeast and plants, but the mechanism and physiological significance of this localization is unclear. Here, we demonstrate that a sorting signal present in TGBp3 is necessary and sufficient for its oligomerization and for targeting integral membrane proteins into puncta within curved ER tubules. Mutations in the TGBp3 sorting signal impair viral spread, and plants infected with viruses harboring these mutants were either asymptomatic or had reduced symptoms. Thus, we propose that Potexvirus use the sorting signal in TGBp3 to target infectious viral derivatives to cortical ER tubules for transmission through the intercellular junctions in plants.

scholarly journals P42 Movement Protein of Beet necrotic yellow vein virus Is Targeted by the Movement Proteins P13 and P15 to Punctate Bodies Associated with Plasmodesmata

2000 ◽  
Vol 13 (5) ◽  
pp. 520-528 ◽  
Author(s):  
M. Erhardt ◽  
M. Morant ◽  
C. Ritzenthaler ◽  
C. Stussi-Garaud ◽  
H. Guilley ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Triple Gene Block ◽  
Point Mutations ◽  
Cell Movement ◽  
Leading Edge ◽  
Yellow Vein ◽  
Body Formation ◽  
Movement Proteins ◽  
Gene Block

Cell-to-cell movement of Beet necrotic yellow vein virus (BNYVV) is driven by a set of three movement proteins—P42, P13, and P15—organized into a triple gene block (TGB) on viral RNA 2. The first TGB protein, P42, has been fused to the green fluorescent protein (GFP) and fusion proteins between P42 and GFP were expressed from a BNYVV RNA 3-based replicon during virus infection. GFP-P42, in which the GFP was fused to the P42 N terminus, could drive viral cell-to-cell movement when the copy of the P42 gene on RNA 2 was disabled but the C-terminal fusion P42-GFP could not. Confocal microscopy of epidermal cells of Chenopodium quinoa near the leading edge of the infection revealed that GFP-P42 localized to punctate bodies apposed to the cell wall whereas free GFP, expressed from the replicon, was distributed uniformly throughout the cytoplasm. The punctate bodies sometimes appeared to traverse the cell wall or to form pairs of disconnected bodies on each side. The punctate bodies co-localized with callose, indicating that they are associated with plasmodesmata-rich regions such as pit fields. Point mutations in P42 that inhibited its ability to drive cell-to-cell movement also inhibited GFP-P42 punctate body formation. GFP-P42 punctate body formation was dependent on expression of P13 and P15 during the infection, indicating that these proteins act together or sequentially to localize P42 to the plasmodesmata.

scholarly journals Transient Coexpression of Individual Genes Encoded by the Triple Gene Block of Potato mop-top virus Reveals Requirements for TGBp1 Trafficking

2004 ◽  
Vol 17 (8) ◽  
pp. 921-930 ◽  
Author(s):  
Andrey A. Zamyatnin ◽  
Andrey G. Solovyev ◽  
Eugene I. Savenkov ◽  
Anna Germundsson ◽  
Maria Sandgren ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Triple Gene Block ◽  
Terminal Region ◽  
Virus Movement ◽  
Cell Periphery ◽  
Movement Proteins ◽  
Gene Block ◽  
Potato Mop Top Virus ◽  
Green Fluorescent

TGBp1, TGBp2, and TGBp3, three plant virus movement proteins encoded by the “triple gene block” (TGB), may act in concert to facilitate cell-to-cell transport of viral RNA genomes. Transient expression of Potato mop-top virus (genus Pomovirus) movement proteins was used as a model to reconstruct interactions between TGB proteins. In bombarded epidermal cells of Nicotiana benthamiana, green fluorescent protein (GFP)-TGBp1 was distributed uniformly. However, in the presence of TGBp2 and TGBp3, GFP-TGBp1 was directed to intermediate bodies at the cell periphery, and to cell wall-embedded punctate bodies. Moreover, GFP-TGBp1 migrated into cells immediately adjacent to the bombarded cell. These data suggest that TGBp2 and TGBp3 mediate transport of GFP-TGBp1 to and through plasmodesmata. Mutagenesis of TGBp1 suggested that the NTPase and helicase activities of TGBp1 were not required for its transport to intermediate bodies directed by TGBp2 and TGBp3, but these activities were essential for the protein association with cell wall-embedded punctate bodies and translocation of TGBp1 to neighboring cells. The C-terminal region of TGBp1 was critical for trafficking mediated by TGBp2 and TGBp3. Mutation analysis also suggested an involvement of the TGBp2 C-terminal region in interactions with TGBp1.

scholarly journals Cell-to-Cell Movement of Potato virus X: The Role of p12 and p8 Encoded by the Second and Third Open Reading Frames of the Triple Gene Block

2001 ◽  
Vol 14 (10) ◽  
pp. 1158-1167 ◽  
Author(s):  
Atsushi Tamai ◽  
Tetsuo Meshi
Keyword(s):  
Potato Virus ◽  
Fluorescent Protein ◽  
Microprojectile Bombardment ◽  
Triple Gene Block ◽  
Cell Movement ◽  
Potato Virus X ◽  
Open Reading Frames ◽  
Gene Block ◽  
Infected Cells ◽  
Green Fluorescent

Potato virus X (PVX) requires three proteins, p25, p12, and p8, encoded by the triple gene block plus the coat protein (CP) for cell-to-cell movement. When each of these proteins was co-expressed with a cytosolic green fluorescent protein (GFP) in the epidermal cells of Nicotiana benthamiana by the microprojectile bombardment-mediated gene delivery method, only p12 enhanced diffusion of co-expressed GFP, indicating an ability to alter plasmodesmal permeability. p25, p12, and CP, expressed transiently in the initially infected cells, transcomplemented the corresponding movement-defective mutants to spread through two or more cell boundaries. Thus, these proteins probably move from cell to cell with the genomic RNA. In contrast, p8 only functioned intracellularly and was not absolutely required for cell-to-cell movement. Since overexpression of p12 overcame the p8 deficiency, p8 appears to facilitate the functioning of p12, presumably by mediating its intracellular trafficking. Considering the likelihood that p12 and p8 are membrane proteins, it is suggested that intercellular as well as intracellular movement of PVX involves a membrane-mediated process.

scholarly journals Interactions of the TGB1 Protein during Cell-to-Cell Movement of Barley Stripe Mosaic Virus

2001 ◽  
Vol 75 (18) ◽  
pp. 8712-8723 ◽  
Author(s):  
Diane M. Lawrence ◽  
A. O. Jackson
Keyword(s):  
Subcellular Localization ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Triple Gene Block ◽  
Cell Movement ◽  
Barley Stripe Mosaic Virus ◽  
Helicase Domain ◽  
Chenopodium Amaranticolor ◽  
Gene Block ◽  
Green Fluorescent

ABSTRACT We have recently used a green fluorescent protein (GFP) fusion to the γb protein of Barley stripe mosaic virus (BSMV) to monitor cell-to-cell and systemic virus movement. The γb protein is involved in expression of the triple gene block (TGB) proteins encoded by RNAβ but is not essential for cell-to-cell movement. The GFP fusion appears not to compromise replication or movement substantially, and mutagenesis experiments demonstrated that the three most abundant TGB-encoded proteins, βb (TGB1), βc (TGB3), and βd (TGB2), are each required for cell-to-cell movement (D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65–75, 2001). We have now extended these analyses by engineering a fusion of GFP to TGB1 to examine the expression and interactions of this protein during infection. BSMV derivatives containing the TGB1 fusion were able to move from cell to cell and establish local lesions in Chenopodium amaranticolor and systemic infections of Nicotiana benthamiana and barley. In these hosts, the GFP-TGB1 fusion protein exhibited a temporal pattern of expression along the advancing edge of the infection front. Microscopic examination of the subcellular localization of the GFP-TGB1 protein indicated an association with the endoplasmic reticulum and with plasmodesmata. The subcellular localization of the TGB1 protein was altered in infections in which site-specific mutations were introduced into the six conserved regions of the helicase domain and in mutants unable to express the TGB2 and/or TGB3 proteins. These results are compatible with a model suggesting that movement requires associations of the TGB1 protein with cytoplasmic membranes that are facilitated by the TGB2 and TGB3 proteins.

scholarly journals Down-Regulation of the Trypanosomatid Signal Recognition Particle Affects the Biogenesis of Polytopic Membrane Proteins but Not of Signal Peptide-Containing Proteins

2007 ◽  
Vol 6 (10) ◽  
pp. 1865-1875 ◽  
Author(s):  
Yaniv Lustig ◽  
Yaron Vagima ◽  
Hanoch Goldshmidt ◽  
Avigail Erlanger ◽  
Vered Ozeri ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Signal Peptide ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Dominant Negative ◽  
Signal Recognition ◽  
Rnai Silencing ◽  
7Sl Rna

ABSTRACT Protein translocation across the endoplasmic reticulum is mediated by the signal recognition particle (SRP). In this study, the SRP pathway in trypanosomatids was down-regulated by two approaches: RNA interference (RNAi) silencing of genes encoding SRP proteins in Trypanosoma brucei and overexpression of dominant-negative mutants of 7SL RNA in Leptomonas collosoma. The biogenesis of both signal peptide-containing proteins and polytopic membrane proteins was examined using endogenous and green fluorescent protein-fused proteins. RNAi silencing of SRP54 or SRP68 in T. brucei resulted in reduced levels of polytopic membrane proteins, but no effect on the level of signal peptide-containing proteins was observed. When SRP deficiency was achieved in L. collosoma by overexpression of dominant-negative mutated 7SL RNA, a major effect was observed on polytopic membrane proteins but not on signal peptide-containing proteins. This study included two trypanosomatid species, tested various protein substrates, and induced depletion of the SRP pathway by affecting either the levels of SRP binding proteins or that of SRP RNA. Our results demonstrate that, as in bacteria but in contrast to mammalian cells, the trypanosome SRP is mostly essential for the biogenesis of membrane proteins.

scholarly journals Evidence that the Linker between the Methyltransferase and Helicase Domains of Potato Virus X Replicase Is Involved in Homologous RNA Recombination

2009 ◽  
Vol 83 (15) ◽  
pp. 7761-7769 ◽  
Author(s):  
Heidrun-Katharina Draghici ◽  
Mark Varrelmann
Keyword(s):  
Coat Protein ◽  
Potato Virus ◽  
Fluorescent Protein ◽  
Triple Gene Block ◽  
Leaf Tissue ◽  
Potato Virus X ◽  
Wild Type Virus ◽  
Rna Recombination ◽  
Gene Block

ABSTRACT Recombination in RNA viruses, one of the main factors contributing to their genetic variability and evolution, is a widespread phenomenon. In this study, an in vivo assay to characterize RNA recombination in potato virus X (PVX), under high selection pressure, was established. Agrobacterium tumefaciens was used to express in Nicotiana benthamiana leaf tissue both a PVX isolate labeled with green fluorescent protein (GFP) containing a coat protein deletion mutation (ΔCP) and a transcript encoding a functional coat protein +3′-ntr. Coexpression of the constructs led to virus movement and systemic infection; reconstituted recombinants were observed in 92% of inoculated plants. Similar results were obtained using particle bombardment, demonstrating that recombination mediated by A. tumefaciens was not responsible for the occurrence of PXC recombinants. The speed of recombination could be estimated by agroinfection of two PVX mutants lacking the 3′ and 5′ halves of the genome, respectively, with an overlap in the triple gene block 1 gene, allowing GFP expression only in the case of recombination. Ten different pentapeptide insertion scanning replicase mutants with replication abilities comparable to wild-type virus were applied in the different recombination assays. Two neighboring mutants affecting the linker between the methyltransferase and helicase domains were shown to be strongly debilitated in their ability to recombine. The possible functional separation of replication and recombination in the replicase molecule supports the model that RNA recombination represents a distinct function of this protein, although the underlying mechanism still needs to be investigated.

scholarly journals Phloem Unloading of Potato virus X Movement Proteins Is Regulated by Virus and Host Factors

2008 ◽  
Vol 21 (8) ◽  
pp. 1106-1117 ◽  
Author(s):  
Tefera Mekuria ◽  
Devinka Bamunusinghe ◽  
Mark Payton ◽  
Jeanmarie Verchot-Lubicz
Keyword(s):  
Transgenic Plants ◽  
Potato Virus ◽  
Fluorescent Protein ◽  
Triple Gene Block ◽  
Proteasome Inhibitors ◽  
Potato Virus X ◽  
Viral Proteins ◽  
Phloem Unloading ◽  
Mesophyll Cells ◽  
Gene Block

To determine the requirements for viral proteins exiting the phloem, transgenic plants expressing green fluorescent protein (GFP) fused to the Potato virus X (PVX) triple gene block (TGB)p1 and coat protein (CP) genes were prepared. The fused genes were transgenically expressed from the companion cell (CC)-specific Commelina yellow mottle virus (CoYMV) promoter. Transgenic plants were selected for evidence of GFP fluorescence in CC and sieve elements (SE) and proteins were determined to be phloem mobile based on their ability to translocate across a graft union into nontransgenic scions. Petioles and leaves were analyzed to determine the requirements for phloem unloading of the fluorescence proteins. In petioles, fluorescence spread throughout the photosynthetic vascular cells (chlorenchyma) but did not move into the cortex, indicating a specific barrier to proteins exiting the vasculature. In leaves, fluorescence was mainly restricted to the veins. However, in virus-infected plants or leaves treated with a cocktail of proteasome inhibitors, fluorescence spread into leaf mesophyll cells. These data indicate that PVX contributes factors which enable specific unloading of cognate viral proteins and that proteolysis may play a role in limiting proteins in the phloem and surrounding chlorenchyma.

scholarly journals Movement protein-derived resistance to triple gene block-containing plant viruses.

1997 ◽  
Vol 78 (6) ◽  
pp. 1241-1246 ◽  
Author(s):  
P Sepp√§nen ◽  
O Fedorkin ◽  
Morozov SYu ◽  
R Puska ◽  
J G Atabekov ◽  
...  
Keyword(s):  
Movement Protein ◽  
Triple Gene Block ◽  
Plant Viruses ◽  
Gene Block

scholarly journals Targeting of Rough Endoplasmic Reticulum Membrane Proteins and Ribosomes in Invertebrate Neurons

2002 ◽  
Vol 13 (5) ◽  
pp. 1778-1791 ◽  
Author(s):  
Melissa M. Rolls ◽  
David H. Hall ◽  
Martin Victor ◽  
Ernst H. K. Stelzer ◽  
Tom A. Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Caenorhabditis Elegans ◽  
Membrane Proteins ◽  
Cell Body ◽  
Rough Endoplasmic Reticulum ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Cell Types ◽  
Endoplasmic Reticulum Membrane ◽  
Unknown Mechanism

The endoplasmic reticulum (ER) is divided into rough and smooth domains (RER and SER). The two domains share most proteins, but RER is enriched in some membrane proteins by an unknown mechanism. We studied RER protein targeting by expressing fluorescent protein fusions to ER membrane proteins in Caenorhabditis elegans. In several cell types RER and general ER proteins colocalized, but in neurons RER proteins were concentrated in the cell body, whereas general ER proteins were also found in neurites. Surprisingly RER membrane proteins diffused rapidly within the cell body, indicating they are not localized by immobilization. Ribosomes were also concentrated in the cell body, suggesting they may be in part responsible for targeting RER membrane proteins.

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