Dieter Blaas

The cellular receptor to human rhinovirus 2 binds around the 5-fold axis and not in the canyon: a structural view

Human rhinovirus serotype 2 (HRV2) belongs to the minor group of HRVs that bind to members of the LDL‐receptor family including the very low density lipoprotein (VLDL)‐receptor (VLDL‐R). We have determined the structures of the complex between HRV2 and soluble fragments of the VLDL‐R to 15 Å resolution by cryo‐electron microscopy. The receptor fragments, which include the first three ligand‐binding repeats of the VLDL‐R (V1–3), bind to the small star‐shaped dome on the icosahedral 5‐fold axis. This is in sharp contrast to the major group of HRVs where the receptor site for ICAM‐1 is located at the base of a depression around each 5‐fold axis. Homology models of the three domains of V1–3 were used to explore the virus–receptor interaction. The footprint of VLDL‐R on the viral surface covers the BC‐ and HI‐loops on VP1.

Capillary zone electrophoresis with a linear, non-cross-linked polyacrylamide gel: separation of proteins according to molecular mass

Abstract The separation of proteins according to their molecular masses using capillary zone electrophoresis was demonstrated in a buffer containing a linear, non-cross-linked polyacrylamide gel as a sieving medium. The four test proteins, covering a molecular mass range of between 17 800 and 77 000, were applied as sodium dodecyl sulphate (SDS) complexes. The separation was carried out at pH 5.5 (phosphate buffer, 0.5% SDS, 10% liquid, linear polyacrylamide gel). The retention times of the proteins correlated with the logarithm of their molecular masses.

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.

Polypeptide 2A of human rhinovirus type 2: identification as a protease and characterization by mutational analysis

Evidence is presented that the protein 2A of human rhinovirus serotype 2 (HRV2) is a protease. On expression of the VP1-2A region of HRV2 in bacteria, protein 2A was capable of acting on its own N-terminus; derived extracts specifically cleaved a 16 amino acid oligopeptide corresponding to the sequence at the cleavage site. Cleavage of the oligopeptide substrate provides a convenient in vitro assay system. Deletion experiments showed that removal of 10 amino acids from the carboxy terminus inactivated the enzyme. Site-directed mutagenesis identified an essential arginine close to the C-terminus and showed that the enzyme was sensitive to changes in the putative active site. This analysis supports the hypothesis that 2A belongs to the group of sulfhydryl proteases, although sequence comparisons indicate that the putative active site of HRV2 2A is closely related to that of the serine proteases.

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.

Cap-recognizing protein of influenza virus

Abstract Globin mRNA-primed in vitro transcription of influenza virus is inhibited by m 7 GpppGm, m 7 GpppG, and m 7 GTP indicating that the cap analogs block the specific binding site involved in the cap transfer reaction. γ-[4-(Benzoylphenyl)methylamido]-7-methylguanosine-5′-triphosphate, a photoreactive derivative of m 7 GTP, was used as an affinity label for the cap-binding protein. Evidence for the specificity of the labeling reaction is provided by competition with various cap analogs. At low concentrations inhibition of affinity labeling was observed only with guanosine derivatives which also exert a strong inhibitory effect on in vitro transcription. The cap-binding protein was tentatively identified as P2 by electrophoresis on polyacrylamide gels.

Human rhinovirus HRV14 uncoats from early endosomes in the presence of bafilomycin

Determination of infectious progeny virus and in vivo labelling with [35S]methionine followed by immunoprecipitation demonstrates that the major receptor group human rhinovirus HRV14 is able to infect HeLa cells in the presence of the V‐ATPase inhibitor bafilomycin A1. However, host cell shut off is delayed and viral yield is decreased in the presence of the drug. Uncoating can thus take place under conditions that prevent endosomal acidification indicating that it is catalysed by the viral receptor alone. Since transport is arrested in early endosomes upon inhibition of vesicle acidification, the data also suggest that productive uncoating takes place from early endocytic compartments.

Elevated Endosomal pH in HeLa Cells Overexpressing Mutant Dynamin Can Affect Infection by pH-Sensitive Viruses

Many viruses gain access to the cell via the endosomal route and require low endosomal pH for infectivity. The GTPase dynamin is essential for clathrin‐dependent endocytosis, and in HeLa cells overexpressing the nonfunctional dynaminK44A mutant the formation of clathrin‐coated vesicles is halted. HRV2, a human minor group rhinovirus, is internalized by members of the low‐density lipoprotein receptor family in a clathrin‐independent manner. The low endosomal pH then leads to conversion of the capsid to C‐antigen, which is required for release (uncoating) and transfer of the viral RNA into the cytosol and de novo synthesis of infectious virus. We here demonstrate that overexpression of dynaminK44A reduces this antigenic conversion and results in diminished viral synthesis. In contrast, lysosomal degradation is unaffected. The kinetics of the formation of C‐antigen in vitro and in vivo suggest that the pH in endosomes is elevated by about 0.4 units upon overexpression of dynaminK44A. As a consequence, HRV2 uncoating is diminished early after internalization but attains control levels upon prolonged internalization. Thus, overexpression of dynaminK44A, in addition to trafficking defects, results in an elevated endosomal pH and thereby affects virus infection and most likely endosomal sorting and processing.

A monoclonal antibody against the alkaline extracellular β-glucosidase from Trichoderma reesei: reactivity with other Trichoderma β-glucosidases

Monoclonal antibodies were produced against the major extracellular β-glucosidase (76 kDa, isoelectric point 8.4) from Trichoderma reesei, which were able to detect as little as 10 ng of β-glucosidase on Western blots. They recognized a single protein of 76 kDa within the extracellular proteins secreted by T. reesei grown on either cellulose or lactose upon SDS-polyacrylamide gel electrophoresis (PAGE)/Western blotting, which usually was accompanied by one or two closely migrating antigenic proteins, thus indicating microheterogeneity. The antibodies did not react with β-glucosidase from commercial preparations of other Trichoderma sp., nor with those from Aspergillus niger or Penicillium funiculosum. The antibody did, however, recognize the partially purified β-glucosidase solubilized from T. reesei cell membranes and that solubilized from cell walls. It did not recognize the partially purified intracellular β-glucosidase. Separation of crude culture filtrates from T. reesei by FPLC always exhibited one major (IP 8.4) and two minor (IP 4.2 and 4.5) peaks of β-glucosidase activity, which, however, appeared similar in SDS-PAGE/Western blotting and immunostaining. Rechromatofocusing of the ‘acid’ peaks always produced peaks at IP 8.4. Culture filtrates from fermentations run at pH < 4 or from old cultures (older than 7 days) exhibited additional β-glucosidase peaks (IP 5.7–6), which, however, did not react with the antibody. Partial proteolysis of β-glucosidase by trypsin led to rapid appearance of antigenicity in a small peptide fragment of about 20 kDa, concomitantly with disappearance of the 76 kDa band. SDS-PAGE of the degraded β-glucosidase showed that newly formed proteins of 50 and 37 kDa had been formed. Upon chromatofocusing of partially proteolyzed β-glucosidase, two peaks of activity appeared at pH 5.7 and 6.1. From these results it is concluded that the multiplicity of β-glucosidase from T. reesei is due to anomalous chromatographic behaviour and partial proteolysis of a single enzyme species.

Substrate requirements of a human rhinoviral 2A proteinase

The genetic information contained within the RNA genome of picornaviruses is expressed as a single large open reading frame; processing of the primary translation product begins while translation is still in progress. In rhinoviruses and enteroviruses, two picornavirus genera, the virally encoded proteinase 2A begins the processing cascade, cleaving between the C-terminus of VP1 and its own N-terminus. The natural variation in the amino acid sequences amongst rhinoviruses and enteroviruses at the cleavage site of the viral proteinase 2A served as the basis for a mutational analysis of the substrate specificity of the 2A proteinase of human rhinovirus 2. This enzyme was shown to have an unusual preference at the P1 site; out of eight amino acid substitutions made, only the branched amino acids Val and Ile were not readily accepted. The HRV2 2A was shown to process poorly the HRV89 2A cleavage site and to be unable to cleave at sites which included the P region of poliovirus or HRV14. Furthermore, the 2A of HRV89 preferred the cleavage site of HRV2 to its own.