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PubMed2005

Parvovirus uncoating in vitro reveals a mechanism of DNA release without capsid disassembly and striking differences in encapsidated DNA stability.

The uncoating mechanism of parvoviruses is unknown. Their capsid robustness and increasing experimental data would suggest an uncoating mechanism without capsid disassembly. We have developed an in vitro system to detect and quantify viral DNA externalization and applied the assay on two parvoviruses with important differences in capsid structure, human B19 and minute virus of mice (MVM). Upon briefly treating the capsids to increasing temperatures, the viral genome became accessible in its full-length in a growing proportion of virions. Capsid disassembly started at temperatures above 60 degrees C for B19 and 70 degrees C for MVM. For both viruses, the externalization followed an all-or-nothing mechanism, without transitions exposing only a particular genomic region. However, the heat-induced DNA accessibility was remarkably more pronounced in B19 than in MVM. This difference was also evident under conditions mimicking endosomal acidification (pH 6.5 to 5), which triggered the externalization of B19-DNA but not of MVM-DNA. The externalized ssDNA was a suitable template for the full second-strand synthesis. Immunoprecipitation with antibodies against conformational epitopes and quantitative PCR revealed that the DNA externalized by heat was mostly dissociated from its capsid, however, the low pH-induced DNA externalization of B19 was predominantly capsid-associated. These results provide new insights into parvovirus uncoating suggesting a mechanism by which the full-length viral genome is released without capsid disassembly. The remarkable instability of the encapsidated B19 DNA, which is easily released from its capsid, would also explain the faster heat inactivation of B19 when compared to other parvoviruses.

Animals↗

PubMed2001

The use of scintigraphy to demonstrate the rapid esophageal transit of the oval film-coated placebo risedronate tablet compared to a round uncoated placebo tablet when administered with minimal volumes of water.

As our population ages, and the consumption of pharmaceutical products rises, the incidence of solid oral dosage forms lodging in the esophagus is likely to increase and may be formulation dependent. The aim of this study was to compare the esophageal transit of the commercial film-coated risedronate tablet and a round uncoated tablet resembling the alendronate 10 mg tablet which is reported to cause esophagitis if ingested with little to no water. Water volumes of 30 ml and 50 ml were selected as these volumes can detect formulations prone to esophageal adhesion and a habits and practice study showed that these volumes are within the range preferred by women (7-385 ml). A total of 28 healthy postmenopausal women completed the four-way crossover scintigraphy study. For both volumes of water, the film-coated placebo risedronate tablet had a statistically significant faster esophageal transit time than the uncoated placebo tablet (P=0.002 for 30 ml water and P<0.001 for 50 ml water). Among those taking the round, flat, uncoated tablet, five subjects had esophageal stasis (transit >20 s) and in three subjects the tablet remained in the esophagus at the end of the 10-min imaging period. No stasis was observed for the oval film-coated placebo risedronate tablet. This study demonstrates that tablet size, shape and coating are pharmaceutical parameters which can be controlled to minimize esophageal contact of a dosage form with esophageal tissue.

Calcium Channel Blockers↗

PubMed1997

Endothelial damage caused by uncoated and fluorocarbon-coated poly(methyl methacrylate) intraocular lenses.

PURPOSE: To assess endothelial damage induced by poly(methyl methacrylate) (PMMA) intraocular lenses (IOLs) coated with a fluorocarbon polymer, Teflon AF, to make them highly hydrophobic. SETTING: Department of Ophthalmology. Hôtel-Dieu Hospital, Paris, France. METHODS: Ten Teflon-coated and 10 uncoated PMMA IOLs were used in an in vitro static touch model. The corneal endothelium was placed in direct contact with the IOL for 15 seconds and then stained with trypan blue and alizarin red. The endothelial damage produced by each IOL in the area of contact was assessed semiquantitatively and quantitatively. RESULTS: Teflon-coated IOLs produced significantly less endothelial damage than uncoated PMMA IOLs (P < .0001). Endothelial cells in contact with Teflon-coated IOLs did not usually adhere to the IOL surface. In contrast, the uncoated IOLs produced large areas of endothelial cell loss. CONCLUSION: Teflon-coated PMMA IOLs have an antiadhesive effect that reduced endothelial damage after IOL insertion in an in vitro model.

Animals↗

PubMed2000

Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin.

Coordination between sequential steps in synaptic vesicle endocytosis, including clathrin coat formation, fission, and uncoating, appears to involve proteinprotein interactions. Here, we show that compounds that disrupt interactions of the SH3 domain of endophilin with dynamin and synaptojanin impair synaptic vesicle endocytosis in a living synapse. Two distinct endocytic intermediates accumulated. Free clathrin-coated vesicles were induced by a peptide-blocking endophilin's SH3 domain and by antibodies to the proline-rich domain (PRD) of synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. We suggest that the SH3 domain of endophilin participates in both fission and uncoating and that it may be a key component of a molecular switch that couples the fission reaction to uncoating.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Structure of the functional fragment of auxilin required for catalytic uncoating of clathrin-coated vesicles.

The three-dimensional structure of the C-terminal 20 kDa portion of auxilin, which consists of the clathrin binding region and the C-terminal J-domain, has been determined by NMR. Auxilin is an Hsp40 family protein that catalytically supports the uncoating of clathrin-coated vesicles through recruitment of Hsc70 in an ATP hydrolysis-driven process. This 20 kDa auxilin construct contains the minimal sequential region required to uncoat clathrin-coated vesicles catalytically. The tertiary structure consists of six helices, where the first three are unique to auxilin and believed to be important in the catalytic uncoating of clathrin. The last three helices correspond to the canonical J-domain of Hsp40 proteins. The first helix, helix 1, which contains a conserved FEDLL motif believed to be necessary for clathrin binding, is transient and not packed against the rest of the structure. Helix 1 is joined to helix 2 by a flexible linker. Helix 2 packs loosely against the J-domain surface, whereas helix 3 packs tightly and makes critical contributions to the J-domain core. A long insert loop, also unique to the auxilin J-domain, is seen between helix 4 and helix 5. Comparison with a previously reported structure of auxilin containing only helices 3-6 shows a significant difference in the invariant HPD segment of the J-domain. The region where helix 1 is located corresponds to the expected region of the unstructured G/F-rich domain seen in DnaJ, i.e., the canonical N-terminal J-domain protein. In contrast, the location of helix 1 differs from the substrate binding regions of two other Hsp40 proteins, Escherichia coli Hsc20 and viral large T antigen. The variety of biological functions performed by Hsp40 proteins such as auxilin, as well as the observed differences in the structure and function of their substrate binding regions, supports the notion that Hsp40 proteins act as target-specific adaptors that recruit their more general Hsp70 partners to specific biological roles.

Adenosine Triphosphatases↗

PubMed2004

Structure of an auxilin-bound clathrin coat and its implications for the mechanism of uncoating.

Clathrin-coated pits invaginate from specific membrane compartments and pinch off as coated vesicles. These vesicles then uncoat rapidly once released. The Hsc70 molecular chaperone effects the uncoating reaction, and is guided to appropriate locations on clathrin lattices by the J-domain-containing co-chaperone molecule auxilin. This raises the question of how a local event such as ATP hydrolysis by Hsc70 can catalyse a global disassembly. Here, we have used electron cryomicroscopy to determine 12-A-resolution structures of in-vitro-assembled clathrin coats in association with a carboxy-terminal fragment of auxilin that contains both the clathrin-binding region and the J domain. We have located the auxilin fragment by computing differences between these structures and those lacking auxilin (described in an accompanying paper). Auxilin binds within the clathrin lattice near contacts between an inward-projecting C-terminal helical tripod and the crossing of two 'ankle' segments; it also contacts the terminal domain of yet another clathrin 'leg'. It therefore recruits Hsc70 to the neighbourhood of a set of critical interactions. Auxilin binding produces a local change in heavy-chain contacts, creating a detectable global distortion of the clathrin coat. We propose a mechanism by which local destabilization of the lattice promotes general uncoating.

Animals↗

PubMed2003

Heparin-coated versus uncoated extracorporeal circuit in patients undergoing coronary artery bypass graft surgery.

OBJECTIVE: To assess the effect of heparin-coated circuits on bleeding, transfusion, and platelet count in patients undergoing primary coronary artery bypass grafting with full heparinization. DESIGN: Randomized, double-blind study. SETTING: Tertiary-care academic medical center. PARTICIPANTS: Eighty-eight patients undergoing coronary artery bypass grafting requiring cardiopulmonary bypass (CPB) without previous sternotomy. INTERVENTIONS: Subjects received either a heparin-coated or an uncoated extracorporeal circuit for CPB. Heparin, 300 micro/kg, was administered, and supplemental amounts were administered to maintain an activated coagulation time of greater than 480 seconds. Platelet counts were determined during CPB. Mediastinal chest tube drainage was collected in the intensive care unit for 24 hours. MEASUREMENTS AND MAIN RESULTS: The mean platelet counts were similar between the groups during CPB. There was no significant difference in 24-hour mediastinal chest tube drainage (mean +/- standard deviation; median) between the heparin-coated (n = 44, 1096 +/- 401, 1015 mL) and uncoated group (n = 44, 1150 +/- 548, 1040 mL; p = 0.91). The heparin-coated group received less allogeneic packed red blood cells (0.9 +/- 1.6, 0.0 v 1.5 +/- 1.8, 1.0 U; p = 0.04). CONCLUSIONS: The use of a heparin-coated or uncoated cardiopulmonary bypass circuit and full heparinization marginally reduced only red blood cell transfusion but was not associated with platelet sparing or reduced perioperative bleeding.

Adult↗

PubMed2006

A burst of auxilin recruitment determines the onset of clathrin-coated vesicle uncoating.

Clathrin-coated pits assemble on a membrane and pinch off as coated vesicles. The released vesicles then rapidly lose their clathrin coats in a process mediated by the ATPase Hsc70, recruited by auxilin, a J-domain-containing cofactor. How is the uncoating process regulated? We find that during coat assembly small and variable amounts of auxilin are recruited transiently but that a much larger burst of association occurs after the peak of dynamin signal, during the transition between membrane constriction and vesicle budding. We show that the auxilin burst depends on domains of the protein likely to interact with lipid head groups. We conclude that the timing of auxilin recruitment determines the onset of uncoating. We propose that, when a diffusion barrier is established at the constricting neck of a fully formed coated pit and immediately after vesicle budding, accumulation of a specific lipid can recruit sufficient auxilin molecules to trigger uncoating.

Animals↗

PubMed1970

DNA and gene therapy: uncoating of polyoma pseudovirus in mouse embryo cells.

Pseudovirions (host DNA fragments encapsidated by polyoma virus-coat protein) and polyoma virions are produced after infection of primary mouse embryo cells with polyoma virus. Purified pseudovirions are adsorbed to, and uncoated by, mouse embryo cells. The evidence for this uncoating is based on the conversion by pancreatic DNase of the uncoated pseudovirus DNA from an acid-insoluble to an acid-soluble form. The implications of these experiments to the eventual use of DNA for gene therapy are discussed.

Animals↗

PubMed2004

Impaired nuclear transport and uncoating limit recombinant adeno-associated virus 2 vector-mediated transduction of primary murine hematopoietic cells.

Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second- strand DNA synthesis and transgene expression. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.

Active Transport, Cell Nucleus↗

PubMed2002

Halogen versus high-intensity light-curing of uncoated and pre-coated brackets: a shear bond strength study.

AIM: To evaluate the shear bond strengths of adhesive pre-coated brackets (APC) and conventional uncoated brackets (Victory) cured with two different light-curing units: a conventional halogen light (Visilux 2) and a micro-xenon light (Aurys). SETTING: Ex vivo study MATERIALS AND METHODS: Sixty freshly extracted bovine permanent mandibular incisors were randomly assigned to one of four groups, each group consisting of 15 specimens. Two groups (one for each type of bracket) were exposed to the halogen light for 20 seconds and used as controls. The remaining two groups were cured with the micro-xenon light for 2 seconds. After 24 hours, all samples were tested in a shear mode on an Instron Machine. Analysis was by two-way ANOVA with Scheffé's test for comparisons, Kaplan-Meier survival estimates, and Cox model. The Chi-square (chi(2)) test was used to determine significant differences in the ARI scores. RESULTS: The mean shear bond strength of the uncoated brackets cured with Visilux 2 was significantly higher than those of all the other groups tested. Both groups cured with Visilux 2 produced significantly higher mean shear bond strengths than those of the corresponding groups cured with Aurys. No statistically significant differences were found between the two groups cured with Aurys. CONCLUSIONS: Compared to halogen light-curing, the micro-xenon light enables the clinician to reduce significantly the curing time of both APC and uncoated brackets, and although significantly lower, their shear bond strengths may be clinically acceptable.

Analysis of Variance↗

PubMed1995

Xenotransplantation of porcine and bovine islets without immunosuppression using uncoated alginate microspheres.

Uncoated spherical hydrogel microspheres (calcium alginate, nominal M(r) exclusion of > 600 kD) 800-900 microns in diameter were employed to prevent immune rejection of discordant islet xenografts isolated from pigs and cows. The islets were immobilized in the microspheres and injected into the peritoneum of 14 nonimmunosuppressed streptozotocin (STZ)-induced diabetic C57BL/6J mice. Four recipients received islet grafts from bovine calves, and 10 received islet grafts from pigs. In the control group of 15 diabetic mice implanted with nonencapsulated islets, 6 received i.p. porcine islets and 5 received i.p. bovine islets, whereas remaining 4 received porcine islets under the kidney capsule. Plasma glucose concentrations in recipients of the alginate-encapsulated islets promptly dropped from a preimplantation value of 498 +/- 47 (mean +/- SEM) to 142 +/- 6 (bovine) and 178 +/- 7 mg/dl (porcine) during the first wk. All the animals sustained these levels for at least 1 mo. Two mice implanted with bovine islets subsequently reverted to diabetes (plasma glucose > 250 mg/dl) at 43 days postimplantation. The remaining grafts maintained function for > 10 wk. In contrast, nonencapsulated islets failed to function, or sustained euglycemia for < 4 days. Mice receiving encapsulated islets showed a 23-38% gain in body weight during the first mo after implantation, compared with < 1% (P < 0.002) and 32% (P = 0.84) for the untreated diabetic (n = 6) and normal control (n = 6) groups. Immunohistochemical staining of long-term grafts (> 10 wk) revealed viable islets, with well-granulated alpha, beta, and delta cells; the external surfaces of the microreactors were free of fibrotic overgrowth and exhibited only occasional host cell adherence. Uptake studies with IgG and thyroglobulin (M(r) of 669 kD) suggest that the microreactors were permeable to molecules with a molecular weight of up to > 600 kD (including the various proteins of the complement system, M(r) of 24-570 kD). Spheres implanted in the peritoneum after only 1 wk stained positive for both IgG and for the C3 component of complement. These findings suggest that prolonged survival of discordant xenografts of porcine and bovine islets in the STZ diabetic mouse model can be achieved with uncoated alginate microspheres that are permeable to IgG and complement. The question of whether similar results can be achieved with uncoated alginate microspheres in higher animals remains to be fully determined.

Alginates↗

PubMed2004

Retrievability of uncoated versus paclitaxel-coated Günther-Tulip IVC filters in an animal model.

PURPOSE: To compare in a pilot study, the retrievability and inferior vena cava (IVC) wall reaction elicited by uncoated and paclitaxel-coated Günther-Tulip filters in the animal model. MATERIALS AND METHODS: Three groups with five pigs each underwent infrarenal IVC implantation of Günther-Tulip filters. Paclitaxel-coated filters were used in Groups A and B and uncoated filters were used in Group C. Filters were removed at 14, 19, 22, 26, and 30 days after implantation. A laparotomy was performed to remove filters from animals in group A and filters from animals in groups B and C that could not be retrieved via the right transjugular approach. Filter-induced venous wall changes were evaluated by examination of IVC venography, feasibility of filter removal at different implantation times, and laparotomy and microscopic findings. Feasibility of filter retrieval and venous wall changes were correlated. RESULTS: IVC cavography showed no abnormality. Filters in animals in group B were uneventfully removed by a right jugular approach. Uncoated filter removal was not feasible in three of five animals in group C (19, 22, and 26 days). Microscopically, animals in group A had absent filter-induced IVC wall changes at 14, 19, 22, and 26 days and minimal changes at 30 days post implantation; animals in group B had absent filter-induced IVC wall changes at 14, 19, and 22 days and minimal changes at 26 and 30 days; animals in group C had moderate filter-induced IVC wall changes at 14 days and severe changes at 19, 22, 26, and 30 days. CONCLUSIONS: This pilot study suggests that endothelial reaction to the presence of IVC filters in the porcine model is diminished by addition of paclitaxel coating to these filters. Further studies are necessary to substantiate these results.

Animals↗

PubMed1975

The uncoating of native and reconstituted TMV by dimethylsulphoxide: the polarity of stripping.

The effect of increasing concentrations of dimethylsulphoxide (DMSO) on the stability of native and reconstituted tobacco mosaic virus (TMV) has been investigated by means of electron microscopy and infectivity assay. The removal of protein subunits from TMV by DMSO was found to be a stepwise process. Subunits are first removed from the 3'OH end of the rod but at about 72% DMSO the site at which uncoating occurs shifts to the 5' end. Studies with reconstituted and partially reconstituted TMV confirm the stepwise nature of the uncoating process. Complete uncoating of reconstituted TMV was found to occur at smaller DMSO concentrations than that of native TMV.

Dimethyl Sulfoxide↗

PubMed1989

IgG-neutralized influenza virus undergoes primary, but not secondary uncoating in vivo.

Even when neutralized by saturating amounts of monoclonal IgG directed against the haemagglutinin, influenza virus attaches to cells with kinetics similar to those of infectious virus. It then enters those cells and is uncoated; its RNA becomes localized within the nucleus and its lipid envelope and associated proteins remain in the cytoplasm. In this report we show that despite the apparent normality of these early stages of virus-cell interaction, neutralized virus underwent no detectable primary transcription. In contrast, there was only a slight inhibition of transcription by neutralized virus in vitro which was insufficient to account for the loss in infectivity, despite using mRNA to measure the production of capped oligonucleotides or to prime the elongation step. To test whether the absence of primary transcription in vivo resulted from non-accessibility of the genome rather than an effect on the transcriptase complex itself, we examined the susceptibility to RNase of virion RNA after inoculation of cells with neutralized virus. Data clearly show that, unlike RNA of infectious virus, RNA of neutralized virus did not become sensitive to RNase and we conclude that neutralization of influenza virus by IgG results in failure of virus to undergo a secondary uncoating process which is necessary for the activity of the virion transcriptase complex. Finally we show that by treatment of virions in vitro with detergent it is possible to produce a core structure which is stable and has some of the properties expected of a structure resulting from primary uncoating.

Animals↗

PubMed1991

Complete uncoating of the 5' leader sequence of tobacco mosaic virus RNA occurs rapidly and is required to initiate cotranslational virus disassembly in vitro.

Destabilizing events required for subsequent cotranslational disassembly of tobacco mosaic virus (TMV) particles in vitro were studied. Brief treatment of U-32P-labelled TMV (strain vulgare or U2) with 1% SDS exposed only 2.5% of the RNA (160 5' nucleotides) in a susceptible subpopulation of virions. Limited uncoating occurred almost immediately and appeared to be synchronous because the amount of 5' oligonucleotide marker (omega) recovered remained constant throughout a 15 min period in SDS. Additional RNase T1-sensitive oligonucleotides were exposed only after 1 to 2 min in SDS. Coat protein (CP) subunits released from virions 'destabilized' by ultracentrifugation at between pH 7.2 and 9.2 were quantified using L-[35S]methionine-labelled particles of TMV strain U2. CP recovery and virus particle translation results were consistent with increasing numbers of virions uncoating for approximately 200 nucleotides. In the presence of sparsomycin (SPN), the TMV strain vulgare 5' leader and the first AUG codon can bind two 80S ribosomes. Electron microscopy of pH 7.5-treated TMV particles incubated in SPN-treated wheatgerm extract or rabbit reticulocyte lysate, showed that approximately 10% of virions complexed with one ribosome and approximately 10% with two bound ribosomes, confirming that omega at least had been uncoated. Nucleocapsids in these complexes were shorter than untreated TMV by 9 to 10 nm (i.e. equivalent to 192 to 217 nucleotides exposed). The template activities of virions pretreated at pH 7.2 to 9.2 were destroyed by RNase H when short cDNAs were hybridized to sequences at, or immediately 3' to, the first AUG codon. We propose that the complete 5' leader of TMV RNA interacts weakly with CP subunits and that this micro-instability is due to the absence of G residues and is essential for initiation of cotranslational virus disassembly.

Base Sequence↗

PubMed1999

Serum albumin inhibits echovirus 7 uncoating.

Echoviruses induce a wide spectrum of diseases in man, the most severe being meningitis. In neonates, however, a severe systemic infection can be observed, leading to death. Serum albumin is the most abundant protein in plasma and most interstitial fluids, and its functions include osmoregulation and transport and delivery of hydrophobic molecules such as fatty acids and steroids. The results of cold-synchronized one-step growth analysis of echovirus 7 infection and sucrose-gradient analysis of A-particles suggest that physiological concentrations of albumin block echovirus 7 infection by inhibiting uncoating. The blockage was reversible and was still effective when albumin was added 30 min after virus adsorption. Inhibition of uncoating was confirmed by using rhodanine, a known specific inhibitor of echovirus uncoating. After removal of the albumin blockage, addition of rhodanine perpetuated the inhibition. Serum and interstitial albumin concentrations may limit echovirus infection in vivo and thereby act as an extracellular determinant for echovirus tropism.

Animals↗

PubMed1991

Streptococcal adherence to uncoated and saliva-coated restoratives.

The adherence of Streptococcus sanguis and S. mutans to seven restoratives in the presence and absence of an artificial salivary pellicle has been investigated. The physiochemical surface characteristics of the bacteria and of the restoratives were also measured, together with the effect of salivary coating of restoratives. Zeta potential of uncoated restoratives showed a positive correlation with the number of adherent S. mutans cells and S. sanguis cells. S. sanguis showed a positive correlation with the hydrophobicity of uncoated restoratives. The surface hydrophobicity was decreased upon saliva coating, whereas the zeta potential was slightly increased. The contact angles or zeta potentials of saliva-coated restoratives were positively correlated with those of uncoated restoratives. The number of adherent cells of both S. sanguis and S. mutans to saliva-coated restoratives were decreased after the coating. Zeta potential of saliva-coated restoratives showed a positive correlation with the numbers of adherent S. mutans cells, suggesting that non-specific electrostatic interactions have an important role in the adherence of this species.

Bacterial Adhesion↗