scholarly journals A Cysteine-Rich Plant Protein Potentiates Potyvirus Movement through an Interaction with the Virus Genome-Linked Protein VPg

2004 ◽  
Vol 78 (5) ◽  
pp. 2301-2309 ◽  
Author(s):  
P. Dunoyer ◽  
C. Thomas ◽  
S. Harrison ◽  
F. Revers ◽  
A. Maule
Keyword(s):  
Transcriptional Control ◽  
Fluorescent Protein ◽  
Virus Genome ◽  
Specific Protein ◽  
Interacting Protein ◽  
Diverse Range ◽  
Phd Finger ◽  
Systemic Movement ◽  
Systemic Symptoms ◽  
Green Fluorescent

ABSTRACT We have identified a cellular factor that interacts with the virus genome-linked proteins (VPgs) of a diverse range of potyviruses. The factor, called Potyvirus VPg-interacting protein (PVIP), is a plant-specific protein with homologues in all the species examined, i.e., pea, Arabidopsis thaliana, and Nicotiana benthamiana. The sequence of PVIP does not identify a specific function, although the existence of a “PHD finger” domain may implicate the protein in transcriptional control through chromatin remodeling. Deletion analysis using the yeast two-hybrid system showed that the determinants of the interaction lay close to the N terminus of VPg; indeed, the N-terminal 16 amino acids were shown to be both necessary and sufficient for the interaction with at least one PVIP protein. From a sequence comparison of different potyvirus VPg proteins, a specific amino acid at position 12 was directly implicated in the interaction. This part of VPg is distinct from regions associated with other functional roles of VPg. Through mutation of Turnip mosaic virus (TuMV) at VPg position 12, we showed that the interaction with PVIP affected systemic symptoms in infected plants. This resulted from reduced cell-to-cell and systemic movement more than reduced virus replication, as visualized by comparing green fluorescent protein-tagged wild-type and mutant viruses. Furthermore, by using RNA interference of PVIP in Arabidopsis, we showed that reduced expression of PVIP genes reduced susceptibility to TuMV infection. We conclude that PVIP functions as an ancillary factor to support potyvirus movement in plants.

Alix (AIP1) is a vasopressin receptor (V2R)-interacting protein that increases lysosomal degradation of the V2R

2007 ◽  
Vol 292 (5) ◽  
pp. F1303-F1313 ◽  
Author(s):  
Xianhua Yi ◽  
Richard Bouley ◽  
Herbert Y. Lin ◽  
Shaliha Bechoua ◽  
Tian-xiao Sun ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Human Kidney ◽  
Lysosomal Degradation ◽  
Interacting Protein ◽  
Parathyroid Hormone Receptor ◽  
Gfp Fluorescence ◽  
Green Fluorescent ◽  
G Protein Coupled ◽  
Regulatory Event

The vasopressin type 2 receptor (V2R) is a G protein-coupled receptor that plays a central role in renal water reabsorption. Termination of ligand (vasopressin) stimulation is an important physiological regulatory event, but few proteins that interact with the V2R during downregulation after vasopressin (VP) binding have been identified. Using yeast two-hybrid screening of a human kidney cDNA library, we show that a 100-kDa protein called ALG-2-interacting protein X (Alix) interacts with the last 29 amino acids of the V2R COOH terminus. This was confirmed by pull-down assays using a GST-V2R-COOH-tail fusion protein. Alix was immunolocalized in principal cells of the kidney, which also express the V2R. The function of the Alix-V2R interaction was studied by transfecting Alix into LLC-PK1 epithelial cells expressing V2R-green fluorescent protein (GFP). Under basal conditions, V2R-GFP localized mainly at the plasma membrane. On VP treatment, V2R-GFP was internalized into perinuclear vesicles in the nontransfected cells. In contrast, V2R-GFP fluorescence was virtually undetectable 2 h after exposure to VP in cells that coexpressed Alix. Western blotting using an anti-GFP antibody showed marked degradation of the V2R after 2 h in the presence of VP and Alix, a time point at which little or no degradation was detected in the absence of Alix. In contrast, little or no degradation of the parathyroid hormone receptor was detectable in the presence or absence of Alix and/or the PTH ligand. The VP-induced disappearance of V2R-GFP was abolished by chloroquine, a lysosomal degradation inhibitor, but not by MG132, a proteosome inhibitor. These data suggest that Alix increases the rate of lysosomal degradation of V2R and may play an important regulatory role in the VP response by modulating V2R downregulation.

scholarly journals Development of Sindbis Viruses Encoding nsP2/GFP Chimeric Proteins and Their Application for Studying nsP2 Functioning

2007 ◽  
Vol 81 (10) ◽  
pp. 5046-5057 ◽  
Author(s):  
Svetlana Atasheva ◽  
Rodion Gorchakov ◽  
Robert English ◽  
Ilya Frolov ◽  
Elena Frolova
Keyword(s):  
Virus Replication ◽  
Sindbis Virus ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Chimeric Protein ◽  
Specific Protein ◽  
Heterologous Proteins ◽  
Peptide Tags ◽  
Infected Cells ◽  
Green Fluorescent

ABSTRACT Sindbis virus (SINV) is one of almost 30 currently known alphaviruses. In infected cells, it produces only a few proteins that function in virus replication and interfere with the development of the antiviral response. One of the viral nonstructural proteins, nsP2, not only exhibits protease and RNA helicase activities that are directly involved in viral RNA replication but also plays critical roles in the development of transcriptional and translational shutoffs in the SINV-infected cells. These multiple activities of nsP2 complicate investigations of this protein's functions and further understanding of its structure. Using a transposon-based approach, we generated a cDNA library of SINV genomes with a green fluorescent protein (GFP) gene randomly inserted into nsP2 and identified a number of sites that can be used for GFP cloning without a strong effect on virus replication. Recombinant SIN viruses encoding nsP2/GFP chimeric protein were capable of growth in tissue culture and interfering with cellular functions. SINV, expressing GFP in the nsP2, was used to isolate nsP2-specific protein complexes formed in the cytoplasm of the infected cells. These complexes contained viral nsPs, all of the cellular proteins that we previously coisolated with SINV nsP3, and some additional protein factors that were not found before in detectable concentrations. The random insertion library-based approach, followed by the selection of the viable variants expressing heterologous proteins, can be applied for mapping the domain structure of the viral nonstructural and structural proteins, cloning of peptide tags for isolation of the protein-specific complexes, and studying their formation by using live-cell imaging. This approach may also be applicable to presentation of additional antigens and retargeting of viruses to new receptors.

scholarly journals The Use of Green Fluorescent Protein-Tagged Recombinant Viruses to Test Lettuce mosaic virus Resistance in Lettuce

2002 ◽  
Vol 92 (2) ◽  
pp. 169-176 ◽  
Author(s):  
T. Candresse ◽  
O. Le Gall ◽  
B. Maisonneuve ◽  
S. German-Retana ◽  
E. Redondo
Keyword(s):  
Green Fluorescent Protein ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Lettuce Mosaic Virus ◽  
Recombinant Viruses ◽  
Systemic Movement ◽  
Resistance Breaking ◽  
Recessive Genes ◽  
Lettuce Breeding ◽  
Green Fluorescent

Seed certification and the use of cultivars containing one of two, probably allelic, recessive genes, mo11 and mo12, are the principal control methods for Lettuce mosaic virus (LMV) in lettuce. Although for a few LMV isolates, mo12 confers resistance with most isolates, the genes mo11 or mo12 confer a tolerance, and virus accumulation is readily detected in mo1-carrying plants. This phenotype complicates evaluation of the resistance status, in particular for mo11, for which there are no viral strains against which a true resistance is expressed. Two green fluorescent protein (GFP)-tagged viruses were constructed, derived from a non-resistance breaking isolate (LMV-0) and from a resistance-breaking isolate (LMV-E). An evaluation of 101 cultivars of known status was carried out with these recombinant viruses. Using the LMV-0-derived recombinant, identification of mo1-carrying cultivars was simple because, contrary to its wild-type parent, systemic movement of LMV-0-GFP was abolished in resistant plants. This assay detected four cases of misidentification of resistance status. In all these cases, further tests confirmed that the prior resistance status information was incorrect, so that a 100% correlation was observed between LMV-0-GFP behavior and the mo1 resistance status. Similarly, the LMV-E-derived recombinant allowed the identification of mo12 lettuce lines because its systemic movement was restricted in mo12 lines but not in susceptible or in mo11 lines. The tagged viruses were able to systemically invade another host, pea, irrespective of its resistance status against another member of the genus Potyvirus, Pea seed-borne mosaic virus. The use of these recombinant viruses could therefore greatly facilitate LMV resistance evaluation and speed up lettuce breeding programs.

scholarly journals In vivo analysis of mouse gastrin gene regulation in enhanced GFP-BAC transgenic mice

2011 ◽  
Vol 300 (2) ◽  
pp. G334-G344 ◽  
Author(s):  
Shigeo Takaishi ◽  
Wataru Shibata ◽  
Hiroyuki Tomita ◽  
Guangchun Jin ◽  
Xiangdong Yang ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Fluorescent Protein ◽  
Artificial Chromosome ◽  
Transitional Zone ◽  
Anterior Lobe ◽  
Gastric Antrum ◽  
Neuroendocrine Cells ◽  
Adult Mice ◽  
Green Fluorescent

Gastrin is secreted from a subset of neuroendocrine cells residing in the gastric antrum known as G cells, but low levels are also expressed in fetal pancreas and intestine and in many solid malignancies. Although past studies have suggested that antral gastrin is transcriptionally regulated by inflammation, gastric pH, somatostatin, and neoplastic transformation, the transcriptional regulation of gastrin has not previously been demonstrated in vivo. Here, we describe the creation of an enhanced green fluorescent protein reporter (mGAS-EGFP) mouse using a bacterial artificial chromosome that contains the entire mouse gastrin gene. Three founder lines expressed GFP signals in the gastric antrum and the transitional zone to the corpus. In addition, GFP(+) cells could be detected in the fetal pancreatic islets and small intestinal villi, but not in these organs of the adult mice. The administration of acid-suppressive reagents such as proton pump inhibitor omeprazole and gastrin/CCK-2 receptor antagonist YF476 significantly increased GFP signal intensity and GFP(+) cell numbers in the antrum, whereas these parameters were decreased by overnight fasting, octreotide (long-lasting somatostatin ortholog) infusion, and Helicobacter felis infection. GFP(+) cells were also detected in the anterior lobe of the pituitary gland and importantly in the colonic tumor cells induced by administration with azoxymethane and dextran sulfate sodium salt. This transgenic mouse provides a useful tool to study the regulation of mouse gastrin gene in vivo, thus contributing to our understanding of the mechanisms involved in transcriptional control of the gastrin gene.

scholarly journals Stress-induced Nuclear Bodies Are Sites of Accumulation of Pre-mRNA Processing Factors

2001 ◽  
Vol 12 (11) ◽  
pp. 3502-3514 ◽  
Author(s):  
Marco Denegri ◽  
Ilaria Chiodi ◽  
Margherita Corioni ◽  
Fabio Cobianchi ◽  
Silvano Riva ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Nuclear Bodies ◽  
Splicing Factors ◽  
Hnrnp A1 ◽  
Interacting Protein ◽  
Multifunctional Protein ◽  
Adenovirus E1a ◽  
Green Fluorescent ◽  
Nuclear Ribonucleoprotein ◽  
Processing Factors

Heterogeneous nuclear ribonucleoprotein (hnRNP) HAP (hnRNP A1 interacting protein) is a multifunctional protein with roles in RNA metabolism, transcription, and nuclear structure. After stress treatments, HAP is recruited to a small number of nuclear bodies, usually adjacent to the nucleoli, which consist of clusters of perichromatin granules and are depots of transcripts synthesized before stress. In this article we show that HAP bodies are sites of accumulation for a subset of RNA processing factors and are related to Sam68 nuclear bodies (SNBs) detectable in unstressed cells. Indeed, HAP and Sam68 are both present in SNBs and in HAP bodies, that we rename “stress-induced SNBs.” The determinants required for the redistribution of HAP lie between residue 580 and 788. Different portions of this region direct the recruitment of the green fluorescent protein to stress-induced SNBs, suggesting an interaction of HAP with different components of the bodies. With the use of the 580–725 region as bait in a two-hybrid screening, we have selected SRp30c and 9G8, two members of the SR family of splicing factors. Splicing factors are differentially affected by heat shock: SRp30c and SF2/ASF are efficiently recruited to stress-induced SNBs, whereas the distribution of SC35 is not perturbed. We propose that the differential sequestration of splicing factors could affect processing of specific transcripts. Accordingly, the formation of stress-induced SNBs is accompanied by a change in the splicing pattern of the adenovirus E1A transcripts.

scholarly journals Subcellular Localization of TatAd of Bacillus subtilis Depends on the Presence of TatCd or TatCy

2009 ◽  
Vol 191 (13) ◽  
pp. 4410-4418 ◽  
Author(s):  
Anja N. J. A. Ridder ◽  
Esther J. de Jong ◽  
Jan D. H. Jongbloed ◽  
Oscar P. Kuipers
Keyword(s):  
Bacillus Subtilis ◽  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Inducible Promoter ◽  
Specific Protein ◽  
Positive Bacterium ◽  
Protein Substrates ◽  
Gfp Fusion Protein ◽  
Dual Localization ◽  
Green Fluorescent

ABSTRACT The gram-positive bacterium Bacillus subtilis contains two minimal Tat translocases, TatAdCd and TatAyCy, which are each involved in the secretion of one or more specific protein substrates. We have investigated the subcellular localization of the TatA components by employing C-terminal green fluorescent protein (GFP) fusions and fluorescence microscopy. When expressed from a xylose-inducible promoter, the TatA-GFP fusion proteins displayed a dual localization pattern, being localized peripherally and showing bright foci which are predominantly located at the division sites and/or poles of the cells. Importantly, the localization of TatAd-GFP was similar when the protein was expressed from its own promoter under phosphate starvation conditions, indicating that these foci are not the result of artificial overexpression. Moreover, the TatAd-GFP fusion protein was shown to be functional in the translocation of its substrate PhoD, provided that TatCd is also present. Furthermore, we demonstrate that the localization of TatAd-GFP in foci depends on the presence of the TatCd component. Remarkably, however, the TatAd-GFP foci can also be observed in the presence of TatCy, indicating that TatAd can interact not only with TatCd but also with TatCy. These results suggest that the formation of TatAd complexes in B. subtilis is controlled by TatC.

scholarly journals Development of a Novel Avian Vaccine Vector Derived From the Emerging Fowl Adenovirus 4

2021 ◽  
Vol 12 ◽  
Author(s):  
Qing Pan ◽  
Yu Zhang ◽  
Aijing Liu ◽  
Hongyu Cui ◽  
Yulong Gao ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Virus Genome ◽  
Open Reading Frames ◽  
Vaccine Vector ◽  
Economic Losses ◽  
Foreign Gene ◽  
The Novel ◽  
Hydropericardium Syndrome ◽  
Fowl Adenovirus ◽  
Green Fluorescent

Severe hepatitis-hydropericardium syndrome (HHS) associated with a novel viral genotype, fowl adenovirus 4 (FAdV-4), has emerged and widely spread in China since 2015, causing severe economic losses to the poultry industry. We previously reported that the hexon gene is responsible for pathogenicity and obtained a non-pathogenic hexon-replacement rHN20 strain; however, the lack of information about the non-essential regions for virus replication limits the development of a FAdV-4 vector. This study first established an enhanced green fluorescent protein (EGFP)-indicator virus based on the FAdV-4 reverse genetic technique, effective for batch operations in the virus genome. Based on this, 10 open reading frames (ORFs) at the left end and 13 ORFs at the right end of the novel FAdV-4 genome were deleted separately and identified as non-essential genes for viral replication, providing preliminary insertion sites for foreign genes. To further improve its feasibility as a vaccine vector, seven combinations of ORFs were successfully replaced with EGFP without affecting the immunogenicity of the vector backbone. Finally, a recombinant rHN20-vvIBDV-VP2 strain, expressing the VP2 protein of very virulent infectious bursa disease virus (vvIBDV), was rescued and showed complete protection against FAdV-4 and vvIBDV. Thus, the novel FAdV-4 vector could provide sufficient protection for HHS and efficient exogenous gene delivery. Overall, our findings systemically identified 23 non-essential ORFs for FAdV-4 replication and seven foreign gene insertion regions, providing valuable information for an in-depth understanding of the novel FAdV-4 pathogenesis and development of multivalent vaccines.

Microvesicle Information Transfer from Lung to Marrow Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 437-437 ◽  
Author(s):  
Jason Aliotta ◽  
Sam Faradyan ◽  
Mark Dooner ◽  
Gerri Dooner ◽  
Peter J. Quesenberry
Keyword(s):  
Information Transfer ◽  
Fluorescent Protein ◽  
Specific Protein ◽  
Conditioned Media ◽  
Lung Epithelial Cells ◽  
Clara Cell ◽  
Normal Lung ◽  
Cell Phenotype ◽  
Green Fluorescent ◽  
Marrow Cells

Abstract A body of work in the literature has shown that after marrow transplantation into irradiated mice, there appear cells in the lung with an epithelial lung cell phenotype, but with markers of the donor marrow cells. After transplantation of green-fluorescent protein positive marrow cells into lethally irradiated mice, we have previously shown that there are from 3–5% non-hematopoietic cells bearing the donor marrow markers in the lung, most of these being cytokeratin positive (Aliotta et al., Exp Hematol., 34(2):230–41, 2006). We have utilized a cell impermeable double culture chamber to co-culture irradiated and non-irradiated lung across from marrow cells. With 2 or 7 days of co-culture, the marrow cells express the lung-specific mRNAs surfactant proteins B and C and Clara Cell specific protein at high levels. This phenomenon is most pronounced when the lung has been exposed to 500 cGy 5 days prior to initiating co-culture, although it is also seen with non-irradiated lung. Conditioned media from lung exerts the same effects and RNase treatment of the conditioned media markedly decreased this effect. Ultracentrifugation of the conditioned media pellets the converting activity, which appears to reside in microvesicles. These microvesicles were demonstrated to enter marrow stem cells and to enhance their capacity to convert to lung epithelial cells after transplantation. Most recently, we have also shown that the capacity to enter marrow cells varies both with the cell cycle status of lineage negative Sca-1+ murine marrow cells and with the source of the microvesicles, either from irradiated or normal lung. Altogether, these data indicate that microvesicle transfer from damaged tissue may be the basis of some forms of marrow plasticity, which have been previously reported.

scholarly journals Nonmyofilament-associated troponin T fragments induce apoptosis

2009 ◽  
Vol 297 (1) ◽  
pp. H283-H292 ◽  
Author(s):  
Euy-Myong Jeong ◽  
Xin Wang ◽  
Kun Xu ◽  
M. Moazzem Hossain ◽  
J.-P. Jin
Keyword(s):  
Fluorescent Protein ◽  
Striated Muscle ◽  
Troponin T ◽  
Variable Region ◽  
Nemaline Myopathy ◽  
Specific Protein ◽  
Cellular Protection ◽  
Cellular Environment ◽  
Nonmuscle Cells ◽  
Green Fluorescent

Troponin T (TnT) is a striated muscle-specific protein and an abundant component of the myofilaments. Nonmyofilament-associated TnT is rapidly degraded in myocytes, implying an importance in the maintenance of the cellular environment. However, if the level of nonmyofilament-associated TnT or TnT fragments exceeds the degradation capacity, it may cause cytotoxicity. To investigate this hypothesis, we constructed bicistronic vectors to express different portions of TnT polypeptide chain, together with nonfusion green fluorescent protein as a tracer for the transfection. Cytotoxicity of the TnT fragments was studied through forced expression in C2C12 myoblasts and human embryonic kidney-293 nonmuscle cells and examination of the viability of the transfected cells. The results demonstrated that, in the absence of myofilaments, the conserved COOH-terminal and middle fragments of TnT were highly effective on inducing cell death via apoptosis, whereas the NH2-terminal variable region was not. As combined effects, nonmyofilament-associated intact cardiac TnT and a COOH-terminal truncated slow TnT fragment found in Amish nemaline myopathy exhibited intermediate cytotoxicity. A particular significance of this finding is that peak releases of TnT or TnT fragments from decomposition of a large number of myofibrils in acute myocardial infarction may breach the cellular protection of proteolytic degradation and result in apoptosis as a potential cause for the loss of cardiomyocytes.

scholarly journals LZTS2 Is a Novel β-Catenin-Interacting Protein and Regulates the Nuclear Export of β-Catenin

2006 ◽  
Vol 26 (23) ◽  
pp. 8857-8867 ◽  
Author(s):  
Gregory Thyssen ◽  
Tzu-Huey Li ◽  
Lynn Lehmann ◽  
Ming Zhuo ◽  
Manju Sharma ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Glycogen Synthase ◽  
Nuclear Export Signal ◽  
Point Mutations ◽  
Cellular Level ◽  
Interacting Protein ◽  
Nuclear Exclusion ◽  
Green Fluorescent ◽  
Wnt Signal

ABSTRACT β-Catenin plays multiple roles in cell-cell adhesion and Wnt signal transduction. Through the Wnt signal, the cellular level of β-catenin is constitutively regulated by the multicomponent destruction complex containing glycogen synthase kinase 3β, axin, and adenomatous polyposis coli. Here, we present multiple lines of evidence to demonstrate that LZTS2 (lucine zipper tumor suppressor 2) interacts with β-catenin, represses the transactivation of β-catenin, and affects the subcellular localization of β-catenin. The LZTS2 gene is located at 10q24.3, which is frequently lost in a variety of human tumors. A functional nuclear export signal (NES) was identified in the C terminus of the protein (amino acids 631 to 641). Appending this motif to green fluorescent protein (GFP) induced nuclear exclusion of the GFP fusion protein. However, introducing point mutations in either one or two leucine residues of this NES sequence abolished the nuclear exclusion of the LZTS2 protein. The nuclear export of LZTS2 can be blocked by leptomycin B (LMB), an inhibitor of the CRM1/exportin-alpha pathway. Intriguingly, β-catenin colocalizes with LZTS2 in the cytoplasm of cells in the absence of LMB but in the nuclei of cells in the presence of LMB. Increasing the LZTS2 protein in cells reduces the level of nuclear β-catenin in SW480 cells. Taken together, these data demonstrate that LZTS2 is a β-catenin-interacting protein that can modulate β-catenin signaling and localization.

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