Biomedical subjects

Dieter Blaas

Publications and source records attributed to Dieter Blaas.

34 records · Page 2Linked to original sources

PubMed2004

Bioimprinted QCM sensors for virus detection-screening of plant sap.

Surface imprinting techniques on polymer-coated quartz-crystal microbalances (QCM) have been used to detect tobacco mosaic viruses (TMV) in aqueous media. Molecularly imprinted polymers (MIP), tailor-made by self organisation of monomers around a template (TMV), were generated directly on the gold electrodes. Imprinted trenches on the polymer surface mimicking the shape and surface functionality of the virus serve as recognition sites for re-adsorption after washing out of the template. The sensors are applicable to TMV detection ranging from 100 ng mL(-1) to 1 mg mL(-1) within minutes. Furthermore, direct measurements without time-consuming sample preparation are possible in complex matrices such as tobacco plant sap.

Biosensing Techniques↗

PubMed2004

Binding of fluorescent dye to genomic RNA inside intact human rhinovirus after viral capsid penetration investigated by capillary electrophoresis.

RiboGreen is used for concentration measurements of RNA. Upon binding to the RNA, an approximately 1000-fold increase in sensitivity in comparison with the UV absorbance of the free polynucleotide is observed. In the present work, we demonstrate that this dye can penetrate in a time- and temperature-dependent manner the intact viral capsids of human rhinovirus serotypes 2 and 14, where it forms a fluorescent complex with the viral RNA. Capillary electrophoresis with laser-induced fluorescence detection of virus incubated with RiboGreen shows that the electrophoretic mobility of the viruses remained unchanged upon dye-binding. As shown for human rhinovirus serotype 2, its native conformation was conserved, since it still bound a recombinant soluble receptor fragment derived from the very low density lipoprotein receptor. The labeled RNA was released by heat-induced uncoating of the virus, and the RNA-dye complex could be directly detected if degradation was prevented with an RNase inhibitor. This in vitro labeling of viral RNA encased within a protein shell demonstrates the virion's dynamic nature that temporarily allows access of a low-molecular-mass compound to the otherwise protected RNA. It might be of great value for experiments requiring fluorescent viral particles with an unmodified surface, such as investigations of endocytosis and viral uncoating on the single molecule level.

Capsid Proteins↗

PubMed2004

Capillary electrophoresis of biological particles: viruses, bacteria, and eukaryotic cells.

A review about the application of electrophoretic methods in the capillary format for the investigation of large biological assemblies like viruses, bacteria, yeast or entire mammalian cells is given. These entities are of a size ranging between some nanometers and tens of micrometers. They can form colloidal solutions or dispersions and move under the influence of an electric field. They are separated by zone electrophoresis according to their different electrophoretic velocity, and characterized by the electrophoretic mobility, which is easily determinable in free solution in capillaries or in other microdevices. As the charge of these particles, when being amphoteric, is pH-dependent, isoelectric focusing can also be carried out and the capillary format is increasingly being employed for their separation and determination of pI values. Furthermore, interactions with ligands can be assessed by various modes of affinity capillary electrophoresis. Capillary zone electrophoresis has thus become a valuable tool for investigation of large macromolecular assemblies in the field of biochemistry, clinical chemistry, toxicology, and nutrition chemistry amongst many others.

Bacteria↗

PubMed2004

Identification of the human rhinovirus serotype 1A binding site on the murine low-density lipoprotein receptor by using human-mouse receptor chimeras.

Human rhinovirus serotype 1A (HRV1A) binds more strongly to the mouse low-density lipoprotein receptor (LDLR) than to the human homologue (M. Reithmayer, A. Reischl, L. Snyers, and D. Blaas, J. Virol. 76:6957-6965, 2002). Here, we used this fact to determine the binding site of HRV1A by replacing selected ligand binding modules of the human receptor with the corresponding ligand binding modules of the mouse receptor. The chimeric proteins were expressed in mouse fibroblasts deficient in endogenous LDLR and LDLR-related protein, both used by minor group HRVs for cell entry. Binding was assessed by virus overlay blots, by immunofluorescence microscopy, and by measuring cell attachment of radiolabeled virus. Replacement of ligand binding repeat 5 of the human LDLR with the corresponding mouse sequence resulted in a substantial increase in HRV1A binding, whereas substitution of repeats 3 and 4 was without effect. Replacement of human receptor repeats 1 and 2 with the murine homologues also increased virus binding. Finally, murine receptor modules 1, 2, and 5 simultaneously introduced into the human receptor resulted in HRV1A binding indistinguishable from mouse wild-type receptor. Thus, repeats 1 and/or 2 and repeat 5 are involved in HRV1A attachment. Changing CDGGPD in the acidic cluster of module 5 in the human receptor to CDGEAD present in the mouse receptor led to substantially increased binding of HRV1A, indicating an important role of the glutamate residue in HRV1A recognition.

Amino Acid Sequence↗

PubMed2004

Human rhinovirus type 2-antibody complexes enter and infect cells via Fc-gamma receptor IIB1.

HeLa cells were stably transfected with a cDNA clone encoding the B1 isoform of the mouse FcgammaRII receptor (hereafter referred to as HeLa-FcRII cells). The receptor was expressed at high level at the plasma membrane in about 90% of the cells. These cells bound and internalized mouse monoclonal virus-neutralizing antibodies 8F5 and 3B10 of the subtype immunoglobulin G2a (IgG2a) and IgG1, respectively. Binding of the minor-group human rhinovirus type 2 (HRV2) to its natural receptors, members of the low-density lipoprotein receptor family, is dependent on the presence of Ca(2+) ions. Thus, chelating Ca(2+) ions with EDTA prevented HRV2 binding, entry, and infection. However, upon complex formation of (35)S-labeled HRV2 with 8F5 or 3B10, virus was bound, internalized, and degraded in HeLa-FcRII cells. Furthermore, challenge of these cells with HRV2-8F5 or HRV2-3B10 complexes resulted in de novo synthesis of viral proteins, as shown by indirect immunofluorescence microscopy. These data demonstrate that minor-group receptors can be replaced by surrogate receptors to mediate HRV2 cell entry, delivery into endosomal compartments, and productive uncoating. Consequently, the conformational change and uncoating of HRV2 appears to be solely triggered by the low-pH (pH </= 5.6) environment in these compartments.

Animals↗

PubMed2004

Cryoelectron microscopy analysis of the structural changes associated with human rhinovirus type 14 uncoating.

Release of the human rhinovirus (HRV) genome into the cytoplasm of the cell involves a concerted structural modification of the viral capsid. The intracellular adhesion molecule 1 (ICAM-1) cellular receptor of the major-group HRVs and the low-density lipoprotein (LDL) receptor of the minor-group HRVs have different nonoverlapping binding sites. While ICAM-1 binding catalyzes uncoating, LDL receptor binding does not. Uncoating of minor-group HRVs is initiated by the low pH of late endosomes. We have studied the conformational changes concomitant with uncoating in the major-group HRV14 and compared them with previous results for the minor-group HRV2. The structure of empty HRV14 was determined by cryoelectron microscopy, and the atomic structure of native HRV14 was used to examine the conformational changes of the capsid and its constituent viral proteins. For both HRV2 and HRV14, the transformation from full to empty capsid involves an overall 4% expansion and an iris type of movement of viral protein VP1 to open up a 10-A-diameter channel on the fivefold axis to allow exit of the RNA genome. The beta-cylinders formed by the N termini of the VP3 molecules inside the capsid on the fivefold axis all open up in HRV2, but we propose that only one opens up in HRV14. The release of VP4 is less efficient in HRV14 than in HRV2, and the N termini of VP1 may exit at different points. The N-terminal loop of VP2 is modified in both viruses, probably to detach the RNA, but it bends only inwards in HRV2.

Capsid↗

PubMed2004

Monitoring RNA release from human rhinovirus by dynamic force microscopy.

Human rhinoviruses were imaged under physiological conditions by dynamic force microscopy. Topographical images revealed various polygonal areas on the surfaces of the 30-nm viral particles. RNA release was initiated by exposure to a low-pH buffer. The lengths of the RNAs that were released but still connected to the virus capsid varied between 40 and 330 nm, whereas RNA molecules that were completely released from the virus were observed with lengths up to 1 micro m. Fork-like structure elements with 30-nm extensions were sometimes resolved at one end of the RNA molecules. They possibly correspond to the characteristic multi-stem-loop conformation, the internal ribosomal entry site, located at the 5' region of the genome. This study demonstrates that dynamic force microscopy can be used to study viral RNA release in situ under physiological conditions.

Capsid↗

PubMed2003

Virus-ligand interactions: identification and characterization of ligand binding by NMR spectroscopy.

We demonstrate the detection and characterization of ligand binding to viruses via NMR. To illustrate the methodology, the interaction of an antiviral compound with human rhinovirus serotype 2 (HRV2) was investigated. Specific interaction of a capsid-binding inhibitor and native HRV2 was monitored utilizing saturation transfer difference (STD) NMR. STD NMR experiments at atomic resolution allowed those regions of the ligand that are involved in the interaction with the virus to be determined. The approach allows for (i) the fast and robust assessment of binding, (ii) the determination of the ligand binding epitope at atomic resolution without the necessity to crystallize virus-ligand complexes, and (iii) the reuse of the virus in subsequent assays. This methodology enables one to easily identify binding of drugs, peptides, and receptor or antibody fragments to the viral capsid.

Antiviral Agents↗

PubMed2003

Sequence and structure of human rhinoviruses reveal the basis of receptor discrimination.

The sequences of the capsid protein VP1 of all minor receptor group human rhinoviruses were determined. A phylogenetic analysis revealed that minor group HRVs were not more related to each other than to the nine major group HRVs whose sequences are known. Examination of the surface exposed amino acid residues of HRV1A and HRV2, whose X-ray structures are available, and that of three-dimensional models computed for the remaining eight minor group HRVs indicated a pattern of positively charged residues within the region, which, in HRV2, was shown to be the binding site of the very-low-density lipoprotein (VLDL) receptor. A lysine in the HI loop of VP1 (K224 in HRV2) is strictly conserved within the minor group. It lies in the middle of the footprint of a single repeat of the VLDL receptor on HRV2. Major group virus serotypes exhibit mostly negative charges at the corresponding positions and do not bind the negatively charged VLDL receptor, presumably because of charge repulsion.

Amino Acid Sequence↗

PubMed2003

A cellular receptor of human rhinovirus type 2, the very-low-density lipoprotein receptor, binds to two neighboring proteins of the viral capsid.

The very-low-density lipoprotein receptor (VLDL-R) is a receptor for the minor-group human rhinoviruses (HRVs). Only two of the eight binding repeats of the VLDL-R bind to HRV2, and their footprints describe an annulus on the dome at each fivefold axis. By studying the complex formed between a selection of soluble fragments of the VLDL-R and HRV2, we demonstrate that it is the second and third repeats that bind. We also show that artificial concatemers of the same repeat can bind to HRV2 with the same footprint as that for the native receptor. In a 16-A-resolution cryoelectron microscopy map of HRV2 in complex with the VLDL-R, the individual repeats are defined. The third repeat is strongly bound to charged and polar residues of the HI and BC loops of viral protein 1 (VP1), while the second repeat is more weakly bound to the neighboring VP1. The footprint of the strongly bound third repeat extends down the north side of the canyon. Since the receptor molecule can bind to two adjacent copies of VP1, we suggest that the bound receptor "staples" the VP1s together and must be detached before release of the RNA can occur. When the receptor is bound to neighboring sites on HRV2, steric hindrance prevents binding of the second repeat.

Binding Sites↗

PubMed2003

Human rhinovirus type 2 is internalized by clathrin-mediated endocytosis.

Using several approaches, we investigated the importance of clathrin-mediated endocytosis in the uptake of human rhinovirus serotype 2 (HRV2). By means of confocal immunofluorescence microscopy, we show that K(+) depletion strongly reduces HRV2 internalization. Viral uptake was also substantially reduced by extraction of cholesterol from the plasma membrane with methyl-beta-cyclodextrin, which can inhibit clathrin-mediated endocytosis. In accordance with these data, overexpression of dynamin K44A in HeLa cells prevented HRV2 internalization, as judged by confocal immunofluorescence microscopy, and strongly reduced infection. We also demonstrate that HRV2 bound to the surface of HeLa cells is localized in coated pits but not in caveolae. Finally, transient overexpression of the specific dominant-negative inhibitors of clathrin-mediated endocytosis, the SH3 domain of amphiphysin and the C-terminal domain of AP180, potently inhibited internalization of HRV2. Taken together, these results indicate that HRV2 uses clathrin-mediated endocytosis to infect cells.

Animals↗

PubMed2003

Conformational changes, plasma membrane penetration, and infection by human rhinovirus type 2: role of receptors and low pH.

Human rhinovirus type 2 (HRV2) is internalized by members of the low-density lipoprotein (LDL) receptor (LDLR) family. It then progresses into late endosomes, where it undergoes conversion from D- to C-antigenicity at pH < 5.6. Upon uncoating, the viral RNA is transferred into the cytoplasm across the endsosomal membrane. However, C-antigenic particles fail to attach to LDLR; this raised the question of whether the virus remains attached to the receptors and is carried to late compartments or rather falls off at the higher pH in early endosomes. We therefore determined the pH dependence of virus-receptor dissociation and virus conversion to C-antigen under conditions preventing endocytosis. (35)S-HRV2 was attached to HeLa cells at 4 degrees C and incubated in buffers of pH 7.4 to 5.0; levels of native virus and C-antigenic particles remaining cell associated or having been released into the medium were determined by immunoprecipitation. At pH 6.0, HRV2 was readily released from plasma membrane receptors in its native form, whereas at pH < or = 5.4, it was entirely converted to C-antigen, which, however, only dissociated from the surface upon prolonged incubation. The antigenic conversion occurred at the same pH regardless of whether HRV2 was free in solution or bound to its receptors. These data suggest that, in vivo, the virus is no longer bound to its receptors when the antigenic conversion and uncoating occur in more acidic late endosomes. When virus was bound to HeLa cells at 4 degrees C, converted into C-antigen by exposure to pH 5.3, and subsequently warmed to 34 degrees C in the presence of bafilomycin (to prevent endosomal uncoating), viral de novo synthesis was detected. This study demonstrates for the first time that a nonenveloped virus such as HRV2 can infect from the plasma membrane when artificially exposed to low pH. This implies that the viral RNA can gain access to the cytoplasm from the plasma membrane.

Cell Membrane↗

PubMed2002

Kinetics of thermal denaturation of human rhinoviruses in the presence of anti-viral capsid binders analyzed by capillary electrophoresis.

In vivo, the icosahedral capsid of human rhinoviruses undergoes well-defined transitions during the infection pathway. Native virus, sedimenting at 150S, is converted to subviral particles with a sedimentation coefficient of 135S, which have lost the innermost capsid protein VP4. Upon release of the genomic RNA empty 80S capsids remain. Similar structural modifications are observed in vitro upon exposure to low pH and/or elevated temperature. Virions are stabilized against these transitions by various antiviral compounds, which bind to a hydrophobic pocket in the capsid protein VP1. Using capillary electrophoresis the kinetics of viral decay in the presence of such hydrophobic drugs was investigated. Assuming first-order kinetics, the increase of the time constant reflects the extent of stabilization. Exposure of the virions to 55 degrees C after presaturation with the antivirals increased the time constants (as compared to native virus) by a factor of 8-30, from a few minutes to several ten minutes. Denaturation of the stabilized capsid gave rise to heterogeneous material rather than to defined subviral particles. This was confirmed by electron microscopy and indicates that the structural modification of the virus follows a kinetically well-defined pathway which is disturbed by the drugs resulting in disorganized disruption of the virion.

Antiviral Agents↗

PubMed2002

The concerted conformational changes during human rhinovirus 2 uncoating.

Delivery of the rhinovirus genome into the cytoplasm involves a cooperative structural modification of the viral capsid. We have studied this phenomenon for human rhinovirus serotype 2 (HRV2). The structure of the empty capsid has been determined to a resolution of better than 15 A by cryo-electron microscopy, and the atomic structure of native HRV2 was used to examine conformational changes of the capsid. The two proteins around the 5-fold axes make an iris type of movement to open a 10 A diameter channel which allows the RNA genome to exit, and the N terminus of VP1 exits the capsid at the pseudo 3-fold axis. A remarkable modification occurs at the 2-fold axes where the N-terminal loop of VP2 bends inward, probably to detach the RNA.

Capsid↗

PubMed2002

Species-specific receptor recognition by a minor-group human rhinovirus (HRV): HRV serotype 1A distinguishes between the murine and the human low-density lipoprotein receptor.

Human rhinoviruses (HRV) of the minor receptor group use several members of the low-density lipoprotein receptor superfamily for cell entry. These proteins are evolutionarily highly conserved throughout species and are almost ubiquitously expressed. Their common building blocks, cysteine-rich ligand binding repeats about 40 amino acids in length, exhibit considerable sequence similarity. Various numbers of these repeats are present in the different receptors. We here demonstrate that HRV type 1A (HRV1A) replicates in mouse cells without adaptation. Furthermore, although closely related to HRV2, it fails to bind to the human low-density lipoprotein receptor but recognizes the murine protein, whereas HRV2 binds equally well to both homologues. This difference went unnoticed due to the presence of other receptors, such as the low-density lipoprotein receptor-related protein, which allow species-independent attachment. The species specificity of HRV1A reported here will aid in defining amino acid residues establishing the contact between the viral surface and the receptor.

Animals↗

PubMedDate not supplied

Dynamic force microscopy imaging of native membranes.

We employed magnetic ACmode atomic force microscopy (MACmode AFM) as a novel dynamic force microscopy method to image surfaces of biological membranes in their native environments. The lateral resolution achieved under optimized imaging conditions was in the nanometer range, even when the sample was only weakly attached to the support. Purple membranes (PM) from Halobacterium salinarum were used as a test standard for topographical imaging. The hexagonal arrangement of the bacteriorhodopsin trimers on the cytoplasmic side of PM was resolved with 1.5nm lateral accuracy, a resolution similar to images obtained in contact and tapping-mode AFM. Human rhinovirus 2 (HRV2) particles were attached to mica surfaces via nonspecific interactions. The capsid structure and 2nm sized protein loops of HRV2 were routinely obtained without any displacement of the virus. Globular and filamentous structures on living and fixed endothelial cells were observed with a resolution of 5-20nm. These examples show that MACmode AFM is a favorable method in studying the topography of soft and weakly attached biological samples with high resolution under physiological conditions.

Capsid↗