scholarly journals Microtubule-Mediated and Microtubule-Independent Transport of Adenovirus Type 5 in HEK293 Cells

2007 ◽  
Vol 81 (13) ◽  
pp. 6899-6908 ◽  
Author(s):  
Carmen Yea ◽  
Joanna Dembowy ◽  
Laura Pacione ◽  
Martha Brown
Keyword(s):  
Viral Genome ◽  
Fluorescent Protein ◽  
Cluster Formation ◽  
Adenovirus Type ◽  
Spindle Pole ◽  
Hek293 Cells ◽  
Productive Infection ◽  
Microtubule Organizing Center ◽  
293 Cells ◽  
Green Fluorescent

ABSTRACT Adenovirus serotypes 2 and 5 are taken into cells by receptor-mediated endocytosis, and following release from endosomes, destabilized virions travel along microtubules to accumulate around the nucleus. The entry process culminates in delivery of the viral genome through nuclear pores. This model is based on studies with conventional cell lines, such as HeLa and HEp-2, but in HEK293 cells, which are routinely used in this laboratory because they are permissive for replication of multiple adenovirus serotypes, a different trafficking pattern has been observed. Nuclei of 293 cells have an irregular shape, with an indented region, and virions directly labeled with carboxyfluorescein accumulate in a cluster within that indented region. The clusters, which form in close proximity to the microtubule organizing center (MTOC) and to the Golgi apparatus, are remarkably stable; a fluorescent signal can be seen in the MTOC region up to 16 h postinfection. Furthermore, if cells are infected and then undergo mitosis after the cluster is formed, the signal is found at each spindle pole. Despite the sequestration of virions near the MTOC, 293 cells are no less sensitive than other cells to productive infection with adenovirus. Even though cluster formation depends on intact microtubules, infectivity is not compromised by disruption of microtubules with either nocodazole or colchicine, as determined by expression of an enhanced green fluorescent protein reporter gene inserted in the viral genome. These results indicate that virion clusters do not represent the infectious pathway and suggest an alternative route to the nucleus that does not depend on nocodazole-sensitive microtubules.

scholarly journals Protease-Dependent Uncoating of a Complex Retrovirus

2005 ◽  
Vol 79 (14) ◽  
pp. 9244-9253 ◽  
Author(s):  
Jacqueline Lehmann-Che ◽  
Marie-Lou Giron ◽  
Olivier Delelis ◽  
Martin Löchelt ◽  
Patricia Bittoun ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Viral Genome ◽  
Productive Infection ◽  
Wild Type Virus ◽  
Target Cells ◽  
Wild Type ◽  
Microtubule Organizing Center ◽  
Viral Protease ◽  
Virus Life Cycle ◽  
Organizing Center

ABSTRACT Although retrovirus egress and budding have been partly unraveled, little is known about early stages of the replication cycle. In particular, retroviral uncoating, a process during which incoming retroviral cores are altered to allow the integration of the viral genome into host chromosomes, is poorly understood. To get insights into these early events of the retroviral cycle, we have used foamy complex retroviruses as a model. In this report, we show that a protease-defective foamy retrovirus is noninfectious, although it is still able to bud and enter target cells efficiently. Similarly, a retrovirus mutated in an essential viral protease-dependent cleavage site in the central part of Gag is noninfectious. Following entry, wild-type and mutant retroviruses are able to traffic along microtubules towards the microtubule-organizing center (MTOC). However, whereas nuclear import of Gag and of the viral genome was observed for the wild-type virus as early as 8 hours postinfection, incoming capsids and genome from mutant viruses remained at the MTOC. Interestingly, a specific viral protease-dependent Gag cleavage product was detected only for the wild-type retrovirus early after infection, demonstrating that cleavage of Gag by the viral protease at this stage of the virus life cycle is absolutely required for productive infection, an unprecedented observation among retroviruses.

scholarly journals N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site

Toxins ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 802
Author(s):  
Oksana V. Nekrasova ◽  
Alexandra L. Primak ◽  
Anastasia A. Ignatova ◽  
Valery N. Novoseletsky ◽  
Olga V. Geras’kina ◽  
...  
Keyword(s):  
Binding Site ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Hek293 Cells ◽  
Kv1.3 Channel ◽  
C Terminus ◽  
Peptide Toxins ◽  
Fluorescent Ligands ◽  
Voltage Gated Channels ◽  
Green Fluorescent

Recently developed fluorescent protein-scorpion toxin chimeras (FP-Tx) show blocking activities for potassium voltage-gated channels of Kv1 family and retain almost fully pharmacological profiles of the parental peptide toxins (Kuzmenkov et al., Sci Rep. 2016, 6, 33314). Here we report on N-terminally green fluorescent protein (GFP)-tagged agitoxin 2 (GFP-L2-AgTx2) with high affinity and selectivity for the binding site of Kv1.3 channel involved in the pathogenesis of various (primarily of autoimmune origin) diseases. The basis for this selectivity relates to N-terminal location of GFP, since transposition of GFP to the C-terminus of AgTx2 recovered specific interactions with the Kv1.1 and Kv1.6 binding sites. Competitive binding experiments revealed that the binding site of GFP-L2-AgTx2 overlaps that of charybdotoxin, kaliotoxin 1, and agitoxin 2, the known Kv1.3-channel pore blockers. GFP-L2-AgTx2 was demonstrated to be applicable as a fluorescent probe to search for Kv1.3 pore blockers among individual compounds and in complex mixtures, to measure blocker affinities, and to visualize Kv1.3 distribution at the plasma membrane of Kv1.3-expressing HEK293 cells. Our studies show that definite combinations of fluorescent proteins and peptide blockers can result in considerable modulation of the natural blocker-channel binding profile yielding selective fluorescent ligands of certain channels.

scholarly journals Formation of Spindle Poles by Dynein/Dynactin-Dependent Transport of Numa

2000 ◽  
Vol 149 (4) ◽  
pp. 851-862 ◽  
Author(s):  
Andreas Merdes ◽  
Rebecca Heald ◽  
Kumiko Samejima ◽  
William C. Earnshaw ◽  
Don W. Cleveland
Keyword(s):  
Fluorescent Protein ◽  
Gel Filtration ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Nuclear Envelope Breakdown ◽  
Spindle Poles ◽  
Mitotic Spindle Assembly ◽  
Green Fluorescent ◽  
Dependent Transport ◽  
Dynactin Complex

NuMA is a large nuclear protein whose relocation to the spindle poles is required for bipolar mitotic spindle assembly. We show here that this process depends on directed NuMA transport toward microtubule minus ends powered by cytoplasmic dynein and its activator dynactin. Upon nuclear envelope breakdown, large cytoplasmic aggregates of green fluorescent protein (GFP)-tagged NuMA stream poleward along spindle fibers in association with the actin-related protein 1 (Arp1) protein of the dynactin complex and cytoplasmic dynein. Immunoprecipitations and gel filtration demonstrate the assembly of a reversible, mitosis-spe-cific complex of NuMA with dynein and dynactin. NuMA transport is required for spindle pole assembly and maintenance, since disruption of the dynactin complex (by increasing the amount of the dynamitin subunit) or dynein function (with an antibody) strongly inhibits NuMA translocation and accumulation and disrupts spindle pole assembly.

scholarly journals Analysis of a Spindle Pole Body Mutant Reveals a Defect in Biorientation and Illuminates Spindle Forces

2005 ◽  
Vol 16 (1) ◽  
pp. 141-152 ◽  
Author(s):  
Tennessee J. Yoder ◽  
Mark A. McElwain ◽  
Susan E. Francis ◽  
Joy Bagley ◽  
Eric G.D. Muller ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Spindle Pole ◽  
Microtubule Organizing Center ◽  
Bipolar Spindle ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
Mutant Cells

The spindle pole body (SPB) is the microtubule organizing center in Saccharomyces cerevisiae. An essential task of the SPB is to ensure assembly of the bipolar spindle, which requires a proper balancing of forces on the microtubules and chromosomes. The SPB component Spc110p connects the ends of the spindle microtubules to the core of the SPB. We previously reported the isolation of a mutant allele spc110-226 that causes broken spindles and SPB disintegration 30 min after spindle formation. By live cell imaging of mutant cells with green fluorescent protein (GFP)-Tub1p or Spc97p-GFP, we show that spc110-226 mutant cells have early defects in spindle assembly. Short spindles form but do not advance to the 1.5-μm stage and frequently collapse. Kinetochores are not arranged properly in the mutant cells. In 70% of the cells, no stable biorientation occurs and all kinetochores are associated with only one SPB. Examination of the SPB remnants by electron microscopy tomography and fluorescence microscopy revealed that the Spc110-226p/calmodulin complex is stripped off of the central plaque of the SPB and coalesces to from a nucleating structure in the nucleoplasm. The central plaque components Spc42p and Spc29p remain behind in the nuclear envelope. The delamination is likely due to a perturbed interaction between Spc42p and Spc110-226p as detected by fluorescence resonance energy transfer analysis. We suggest that the force exerted on the SPB by biorientation of the chromosomes pulls the Spc110-226p out of the SPB; removal of force exerted by coherence of the sister chromatids reduced fragmentation fourfold. Removal of the forces exerted by the cytoplasmic microtubules had no effect on fragmentation. Our results provide insights into the relative contributions of the kinetochore and cytoplasmic microtubules to the forces involved in formation of a bipolar spindle.

scholarly journals Fluorescently tagged canine adenovirus via modification with protein IX–enhanced green fluorescent protein

2005 ◽  
Vol 86 (12) ◽  
pp. 3201-3208 ◽  
Author(s):  
Long P. Le ◽  
Jing Li ◽  
Vladimir V. Ternovoi ◽  
Gene P. Siegal ◽  
David T. Curiel
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Neutralizing Antibodies ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Adenovirus Type ◽  
Canine Model ◽  
Heterologous Proteins ◽  
Green Fluorescent ◽  
Protein Ix

Canine adenovirus type 2 (CAV2) has become an attractive vector for gene therapy because of its non-pathogenicity and the lack of pre-existing neutralizing antibodies against this virus in the human population. Additionally, this vector has been proposed as a conditionally replicative adenovirus agent under the control of an osteocalcin promoter for evaluation in a syngeneic, immunocompetent canine model with spontaneous osteosarcoma. In this study, a CAV2 vector labelled with the fluorescent capsid fusion protein IX–enhanced green fluorescent protein (pIX–EGFP) was developed. Expression of the fluorescent fusion-protein label in infected cells with proper nuclear localization, and incorporation into virions, could be detected. The labelled virions could be visualized by fluorescence microscopy; this was applicable to the tracking of CAV2 infection, as well as localizing the distribution of the vector in tissues. Expression of pIX–EGFP could be exploited to detect the replication and spread of CAV2. These results indicate that pIX can serve as a platform for incorporation of heterologous proteins in the context of a canine adenovirus xenotype. It is believed that capsid-labelled CAV2 has utility for vector-development studies and for monitoring CAV2-based oncolytic adenovirus replication.

scholarly journals Alteration of intracellular histamine H2 receptor cycling precedes antagonist-induced upregulation

2005 ◽  
Vol 289 (5) ◽  
pp. G880-G889 ◽  
Author(s):  
Satoshi Osawa ◽  
Masayoshi Kajimura ◽  
Seiji Yamamoto ◽  
Mutsuhiro Ikuma ◽  
Chihiro Mochizuki ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Inverse Agonist ◽  
Recycling Endosome ◽  
Histamine H2 Receptor ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Gfp Fluorescence ◽  
Term Administration ◽  
Green Fluorescent

Long-term administration of a histamine H2 receptor (H2R) antagonist (inverse agonist) induces upregulation of H2R in parietal cells, which may be relevant to the rebound hypersecretion of gastric acid that occurs after withdrawal of treatment. The mechanisms underlying this effect are unknown. We hypothesized that the H2R upregulation could be related to receptor trafficking and used H2R-green fluorescent protein (H2R-GFP) to test the hypothesis. Human H2R-GFP was generated and functionally expressed in HEK-293 cells. Binding of the H2R antagonist [3H]tiotidine was performed to quantify H2R expression, and H2R-GFP was imaged in living cells by confocal and evanescent wave microscopy. The binding affinity of [3H]tiotidine was not significantly different between H2R-GFP- and wild-type H2R-expressing HEK-293 cells, both of which had constitutive activity of adenylate cyclase. Visualization of H2R-GFP revealed that the agonist-induced H2R internalization and the antagonist-induced recycling of the internalized H2R from the recycling endosome within 2 h. Long exposure to the antagonist increased GFP fluorescence in the plasma membrane and also induced upregulation of H2R-GFP estimated by the binding assay, whereas long exposure to the agonist enhanced degradative trafficking of H2R-GFP. We examined whether the upregulation reflected an increase in receptor synthesis. Treatment with antagonist did not augment H2R mRNA, and subsequent inhibition of protein synthesis by cycloheximide had no effect on H2R upregulation. These findings suggested that upon exposure to an antagonist (inverse agonist), the equilibrium between receptor endocytosis and recycling is altered before H2R upregulation, probably via suppressing H2R degradation.

scholarly journals Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor

2002 ◽  
Vol 157 (7) ◽  
pp. 1211-1222 ◽  
Author(s):  
László Hunyady ◽  
Albert J. Baukal ◽  
Zsuzsanna Gáborik ◽  
Jesus A. Olivares-Reyes ◽  
Márta Bor ◽  
...  
Keyword(s):  
Cell Surface ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Slow Component ◽  
Ang Ii ◽  
Hek 293 ◽  
Recycling Endosomes ◽  
293 Cells ◽  
Early Endosomes ◽  
Green Fluorescent

Agonist-induced endocytosis and processing of the G protein–coupled AT1 angiotensin II (Ang II) receptor (AT1R) was studied in HEK 293 cells expressing green fluorescent protein (GFP)– or hemagglutinin epitope–tagged forms of the receptor. After stimulation with Ang II, the receptor and its ligand colocalized with Rab5–GFP and Rab4–GFP in early endosomes, and subsequently with Rab11–GFP in pericentriolar recycling endosomes. Inhibition of phosphatidylinositol (PI) 3-kinase by wortmannin (WT) or LY294002 caused the formation of large endosomal vesicles of heterogeneous Rab composition, containing the ligand–receptor complex in their limiting membranes and in small associated vesicular structures. In contrast to Alexa®–transferrin, which was mainly found in small vesicles associated with the outside of large vesicles in WT-treated cells, rhodamine–Ang II was also segregated into small internal vesicles. In cells labeled with 125I-Ang II, WT treatment did not impair the rate of receptor endocytosis, but significantly reduced the initial phase of receptor recycling without affecting its slow component. Similarly, WT inhibited the early, but not the slow, component of the recovery of AT1R at the cell surface after termination of Ang II stimulation. These data indicate that internalized AT1 receptors are processed via vesicles that resemble multivesicular bodies, and recycle to the cell surface by a rapid PI 3-kinase–dependent recycling route, as well as by a slower pathway that is less sensitive to PI 3-kinase inhibitors.

scholarly journals Visualization of distinct patterns of subcellular redistribution of the thyrotropin-releasing hormone receptor-1 and Gqα /G11α induced by agonist stimulation

1999 ◽  
Vol 340 (2) ◽  
pp. 529-538 ◽  
Author(s):  
Tomas DRMOTA ◽  
Jiri NOVOTNY ◽  
Gwyn W. GOULD ◽  
Petr SVOBODA ◽  
Graeme MILLIGAN
Keyword(s):  
Plasma Membrane ◽  
G Proteins ◽  
Hormone Receptor ◽  
Fluorescent Protein ◽  
Thyrotropin Releasing Hormone ◽  
Hek293 Cells ◽  
Low Density ◽  
Rapid Separation ◽  
Releasing Hormone ◽  
Green Fluorescent

The rat thyrotropin-releasing hormone receptor-1 (TRHR-1) was modified by the addition of green fluorescent protein (GFP) and expressed stably in HEK293 cells. Extensive overlap of plasma membrane distribution of autofluorescent TRHR-1-GFP with that of the phosphoinositidase C-linked G-proteins Gqα/G11α, identified by indirect immunofluorescence, was monitored concurrently. Addition of thyrotropin-releasing hormone resulted in rapid separation of TRHR-1-GFP and Gqα/G11α signals as the receptor was internalized. This situation persisted for more than an hour. At longer time periods a fraction of the cellular Gqα/G11α was also internalized, although much of the Gqα/G11α immunoreactivity remained associated with the plasma membrane. Parallel experiments, in which the cellular distribution of TRHR-1-GFP and Gqα/G11α immunoreactivity were monitored in sucrose-gradient fractions following cell disruption, also demonstrated a rapid, agonist-induced movement of TRHR-1-GFP away from the plasma membrane to low-density vesicular fractions. At later time points, a fraction of the cellular Gqα/G11α immunoreactivity was also redistributed to overlapping, but non-identical, low-density-vesicle-containing fractions. Pretreatment of the cells with cytochalasin D or nocodazole prevented agonist-induced redistribution of G-protein but not TRHR-1-GFP, further indicating resolution of the mechanics of these two processes. The combination of a GFP-modified receptor and immunostaining of the G-proteins activated by that receptor allows, for the first time, concurrent analysis of the varying dynamics and bases of internalization and redistribution of two elements of the same signal-transduction cascade.

scholarly journals Intracellular Targeting of Gag Proteins of the Drosophila Telomeric Retrotransposons

2003 ◽  
Vol 77 (11) ◽  
pp. 6376-6384 ◽  
Author(s):  
S. Rashkova ◽  
A. Athanasiadis ◽  
M.-L. Pardue
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Cluster Formation ◽  
Intracellular Localization ◽  
Ltr Retrotransposons ◽  
Protein Protein Interactions ◽  
Gag Proteins ◽  
Intracellular Targeting ◽  
Identify Amino Acid ◽  
Green Fluorescent

ABSTRACT Drosophila has two non-long-terminal-repeat (non-LTR) retrotransposons that are unique because they have a defined role in chromosome maintenance. These elements, HeT-A and TART, extend chromosome ends by successive transpositions, producing long arrays of head-to-tail repeat sequences. These arrays appear to be analogous to the arrays produced by telomerase on chromosomes of other organisms. While other non-LTR retrotransposons transpose to many chromosomal sites, HeT-A and TART transpose only to chromosome ends. Although HeT-A and TART belong to different subfamilies of non-LTR retrotransposons, they encode very similar Gag proteins, which suggests that Gag proteins are involved in their unique transposition targeting. We have recently shown that both Gags localize efficiently to nuclei where HeT-A Gag forms structures associated with telomeres. TART Gag does not associate with telomeres unless HeT-A Gag is present, suggesting a symbiotic relationship in which HeT-A Gag provides telomeric targeting. We now report studies to identify amino acid regions responsible for different aspects of the intracellular targeting of these proteins. Green fluorescent protein-tagged deletion derivatives were expressed in cultured Drosophila cells. The intracellular localization of these proteins shows the following. (i) Several regions that direct subcellular localizations or cluster formation are found in both Gags and are located in equivalent regions of the two proteins. (ii) Regions important for telomere association are present only in HeT-A Gag. These are present at several places in the protein, are not redundant, and cannot be complemented in trans. (iii) Regions containing zinc knuckle and major homology region motifs, characteristic of retroviral Gags, are involved in protein-protein interactions of the telomeric Gags, as they are in retroviral Gags.

scholarly journals BRET-based self-cleaving biosensors for SARS-CoV-2 3CLpro Inhibitor Discovery

2021 ◽  
Author(s):  
Ningke Hou ◽  
Chen Peng ◽  
Lijing Zhang ◽  
Yuyao Zhu ◽  
Qi Hu
Keyword(s):  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Hiv Protease ◽  
Activation Mechanism ◽  
Hek293 Cells ◽  
Renilla Luciferase ◽  
Limiting Factor ◽  
Inhibitor Discovery ◽  
Green Fluorescent

The 3C-like protease (3CLpro) of SARS-CoV-2 is an attractive drug target for developing antivirals against SARS-CoV-2. A few small molecule inhibitors of 3CLpro are in clinical trials for COVID-19 treatments and more inhibitors are being developed. One limiting factor for 3CLpro inhibitors development is that the cellular activities of such inhibitors have to be evaluated in a Biosafety Level 3 (BSL-3) or BSL-4 laboratory. Here, we design genetically encoded biosensors that can be used in BSL-2 laboratories to set up cell-based assays for 3CLpro inhibitor discovery. The biosensors were constructed by linking a green fluorescent protein (GFP2) to the N-terminus and a Renilla luciferase (RLuc8) to the C-terminus of SARS-CoV-2 3CLpro, with the linkers derived from the cleavage sequences of 3CLpro. After over-expression of the biosensors in HEK293 cells, 3CLpro can be released from GFP2 and RLuc by self-cleavage, resulting in a decrease of the bioluminescence resonance energy transfer (BRET) signal. Using one of these biosensors, pBRET-10, we evaluated the cellular activities of several 3CLpro inhibitors. These inhibitors restored the BRET signal by blocking the proteolysis of pBRET-10, and their relative activities measured using pBRET-10 were consistent with their anti-SARS-CoV-2 activities reported previously. We conclude that the biosensor pBRET-10 is a useful tool for SARS-CoV-2 3CLpro inhibitor discovery. Furthermore, our strategy can be used to design biosensors for other viral proteases that share the same activation mechanism as 3CLpro, such as HIV protease PR and HCV protease NS3.

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