Antibodies induced with recombinant VP1 from human rhinovirus exhibit cross-neutralisation.

Human rhinoviruses (HRVs) are the major cause of the common cold and account for 30-50% of all acute respiratory illnesses. Although HRV infections are usually harmless and invade only the upper respiratory tract, several studies demonstrate that HRV is involved in the exacerbation of asthma. VP1 is one of the surface-exposed proteins of the viral capsid that is important for the binding of rhinoviruses to the corresponding receptors on human cells. Here we investigated its potential usefulness for vaccination against the common cold. We expressed VP1 proteins from two distantly related HRV strains, HRV89 and HRV14, in Escherichia coli. Mice and rabbits were immunised with the purified recombinant proteins. The induced antibodies reacted with natural VP1 and with whole virus particles as shown by immunoblotting and immunogold electron microscopy. They exhibited strong cross-neutralising activity for different HRV strains. Therefore, recombinant VP1 may be considered a candidate HRV vaccine to prevent HRV-induced asthma exacerbations.

Charge-reduced nano electrospray ionization combined with differential mobility analysis of peptides, proteins, glycoproteins, noncovalent protein complexes and viruses.

This study explores the potential of a novel electrospray-based method, termed gas-phase electrophoretic mobility molecular analysis (GEMMA), allowing the molecular mass determination of peptides, proteins and noncovalent biocomplexes up to 2 MDa (dimer of immunglobulin M). The macromolecular ions were formed by nano electrospray ionization (ESI) in the 'cone jet' mode. The multiple charged state of the monodisperse droplets/ions generated was reduced by means of bipolar ionized air (generated by an alpha-particle source) to yield exclusively singly charged positive and negative ions as well as neutrals. These ions are separated subsequently at atmospheric pressure using a nano differential mobility analyzer according to their electrophoretic mobility in air. Finally, the ions are detected using a standard condensation particle counter. Data were expressed as electrophoretic mobility diameters by applying the Millikan equation. The measured electrophoretic mobility diameters, or Millikan diameters, of 32 well-defined proteins were plotted against their molecular weights in the range 3.5 to 1920 kDa and exhibited an excellent squared correlation coefficient (r(2) = 0.999). This finding allowed the exact molecular weight determination of large (glyco)proteins and noncovalent biocomplexes by means of this new technique with a mass accuracy of +/-5.6% up to 2 MDa at the femtomole level. From the molecular masses of the weakly bound, large protein complexes thus obtained, the binding stoichiometry of the intact complex and the complex stability as a function of pH, for example, can be derived. Examples of specific protein complexes, such as the avidin or catalase homo-tetramer, are used to illustrate the potential of the technique for characterization of high-mass biospecific complexes. A discussion of current and future applications of charge-reduced nano ESI GEMMA, such as chemical reaction monitoring (reduction process of immunglobulin G) or size determination of an intact virus, a supramolecular complex, and monitoring of partial dissociation of a human rhinoviruses, is provided.

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.

Complexes between monoclonal antibodies and receptor fragments with a common cold virus: determination of stoichiometry by capillary electrophoresis.

Complex formation between monoclonal antibodies or soluble receptor fragments and a human rhinovirus is quantified by relating the concentration of the antibody or receptor under equilibrium conditions to the initial concentration of the virus. Within a given concentration range of the reactants, the shape of the resulting curve depends only on the value of the dissociation constant of the particular system studied. Using antibodies and receptor fragments, cases for high, low, and intermediate affinity were investigated. For high-affinity systems, the curve approximates a decaying straight line and the binding stoichiometry can be accurately determined from the intercept with the x-axis. For the case of intermediate affinity, the curve can be linearized at low virus concentrations with the receptors present in large excess. Extrapolation of this line allows derivation of the binding stoichiometry from the intercept with the x-axis, although with less accuracy. For intermediate affinities, an estimate of the dissociation constant can be obtained from fitting the curve to the data points measured. Finally, in the case of low affinity none of the binding parameters can be quantified, although a rough estimate of the lower limit of the dissociation constant is possible. The method was applied for two different monoclonal antibodies, a Fab fragment and a receptor fragment, binding to human rhinovirus serotype 2. Thirty copies of the monoclonal antibody 8F5 were found to bind to the virion, which is in agreement with data from electron cryomicroscopy. The complex between monovalent human very-low-density lipoprotein receptor encompassing repeats 2 and 3 and human rhinovirus serotype 2 showed 60 receptor molecules bound per virion.

Viral evolution toward change in receptor usage: adaptation of a major group human rhinovirus to grow in ICAM-1-negative cells.

Major receptor group common cold virus HRV89 was adapted to grow in HEp-2 cells, which are permissive for minor group human rhinoviruses (HRVs) but which only marginally support growth of major-group viruses. After 32 blind passages in these cells, each alternating with boosts of the recovered virus in HeLa cells, HRV89 acquired the capacity to effectively replicate in HEp-2 cells, attaining virus titers comparable to those in HeLa cells although no cytopathic effect was observed. Several clones were isolated and shown to replicate in HeLa cells whose ICAM-1 was blocked with monoclonal antibody R6.5 and in COS-7 cells, which are devoid of ICAM-1. Blocking experiments with recombinant very-low-density lipoprotein receptor fragments and enzyme-linked immunosorbent assays indicated that the mutants bound a receptor different from that used by minor-group viruses. Determination of the genomic RNA sequence encoding the capsid protein region revealed no changes in amino acid residues at positions equivalent to those involved in the interaction of HRV14 or HRV16 with ICAM-1. One mutation was within the footprint of a very-low-density lipoprotein receptor fragment bound to minor-group virus HRV2. Since ICAM-1 not only functions as a vehicle for cell entry but has also a "catalytic" function in uncoating, the use of other receptors must have important consequences for the entry pathway and demonstrates the plasticity of these viruses.

VLDL receptor fragments of different lengths bind to human rhinovirus HRV2 with different stoichiometry. An analysis of virus-receptor complexes by capillary electrophoresis.

The formation of complexes between the minor receptor group human rhinovirus HRV2 and two recombinant soluble receptor fragments derived from the human very low density lipoprotein receptor (VLDLR) and containing ligand-binding repeats 1-3 (MBP.VLDLR(1-3)) or 1-8 (MBP.VLDLR(1-8)) fused to the carboxyl terminus of the maltose-binding protein was analyzed by affinity capillary electrophoresis. At low molar ratios of receptor/virus, the peaks corresponding to substoichiometric complexes were broad indicating heterogeneity. When the receptors were present in molar excess with respect to the virus, the peaks were sharp, suggesting saturation of all binding sites. For the determination of the stoichiometry, constant amounts of receptor were incubated with increasing amounts of virus, and the peak areas corresponding to free receptor were measured and plotted versus total virus concentration. Extrapolation of the linear part of the resulting curve to zero concentration of free receptor enabled quantitation of the molar ratios of the components present in the complex. Using this method, we determined that about 60 molecules of MBP.VLDLR(1-3) but only about 30 molecules of MBP.VLDLR(1-8) were bound per virion.

Inhibition of clathrin-dependent endocytosis has multiple effects on human rhinovirus serotype 2 cell entry.

Minor group human rhinoviruses (exemplified by human rhinovirus serotype 2 (HRV2)) use members of the low density lipoprotein receptor family for cell entry; all these receptors possess clathrin-coated pit localization signals. Viral infection should thus be inhibited under conditions of impaired clathrin-mediated endocytosis. However, Madshus et al. reported an increase in the cytopathic effect of HRV2 infection in HEp-2 cells upon suppression of clathrin-dependent endocytosis by hypotonic shock and potassium depletion (Madshus, I. H., Sandvig, K., Olsnes, S., and van Deurs, B. (1987) J. Cell. Physiol. 131, 14-22.) To resolve this apparent contradiction, we investigated the binding, internalization, conformational changes, and productive uncoating of HRV2 in HeLa cells subjected to hypotonic shock and potassium depletion. This treatment led to an increase in HRV2 binding, with internalization being barely affected. The generation of C-antigenic particles requiring pH

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 A 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.

Expression and folding of human very-low-density lipoprotein receptor fragments: neutralization capacity toward human rhinovirus HRV2.

Minor group human rhinoviruses (HRVs) use members of the low-density lipoprotein receptor family for cell entry. To investigate the utility of receptor fragments as viral inhibitors, various polypeptide segments derived from the ligand binding domain of human very-low-density lipoprotein receptor (VLDLR) were expressed in a soluble form in bacteria. Whereas none of the fragments was active in virus binding immediately after recovery from the cell lysates, constructs encompassing complement type repeats 1-3, 1-6, and 1-8 spontaneously acquired virus binding activity by incubation at 4 degrees C in buffer containing Ca(2+) ions and lacking any redox system. When immobilized receptor-associated protein (RAP), a specific chaperone for VLDLR, was present during the incubation, the yield of protein active in ligand binding was substantially increased. A VLDLR fragment with repeats 4-6 failed to bind virus; however, it bound RAP. Bacterial expression of truncated VLDLR 1-3 at high yield, easy purification, and folding together with high inhibitory activity toward HRV2 makes this protein a promising starting point for the development of an oligopeptide-based antiviral agent. Using sucrose density gradient centrifugation, we demonstrate the formation of virus-receptor complexes. The recombinant receptors can thus be used for structure determination by electron cryo-microscopy.

Affinity capillary electrophoresis for the assessment of complex formation between viruses and monoclonal antibodies.

The formation of complexes of human rhinovirus (serotype HRV2 and HRV14) with nonaggregating neutralizing monoclonal antibodies was investigated by affinity capillary electrophoresis. The method is based on preincubation of virus with antibody, followed by CE analysis. At low antibody-to-virus ratios, peaks corresponding to the complexes were broad, pointing to the presence of a heterogeneous population of virions with various numbers of antibodies bound; at a high molar ratio between virus and antibody, the peak became narrow again, indicating saturation of the 60 equivalent viral epitopes with the antibodies being attached bivalently. As SDS was used as an additive in the background electrolyte to allow for separation, its influence on complex formation was investigated. Once formed, HRV2-antibody complexes were found to be stable in the presence of the detergent but complex formation in buffer containing SDS was severely impaired. HRV14-antibody complexes were rapidly dissociated by SDS. The method proved to be useful for a rapid assessment of complex formation and might allow for an estimation of the binding stoichiometry.

Structure of human rhinovirus serotype 2 (HRV2).

Human rhinoviruses are classified into a major and a minor group based on their binding to ICAM-1 or to members of the LDL-receptor family, respectively. They can also be divided into groups A and B, according to their sensitivity towards a panel of antiviral compounds. The structure of human rhinovirus 2 (HRV2), which uses the LDL receptor for cell attachment and is included in antiviral group B, has been solved and refined at 2.6 A resolution by X-ray crystallography to gain information on the peculiarities of rhinoviruses, in particular from the minor receptor group. The main structural differences between HRV2 and other rhinoviruses, including the minor receptor group serotype HRV1A, are located at the internal protein shell surface and at the external antigenic sites. In the interior, the N termini of VP1 and VP4 form a three-stranded beta-sheet in an arrangement similar to that present in poliovirus, although myristate was not visible at the amino terminus of VP4 in the HRV2 structure. The betaE-betaF loop of VP2, a linear epitope within antigenic site B recognized by monoclonal antibody 8F5, adopts a conformation considerably different from that found in the complex of 8F5 with a synthetic peptide of the same sequence. This either points to considerable structural changes impinged on this loop upon antibody binding, or to the existence of more than one single conformation of the loop when the virus is in solution. The hydrophobic pocket of VP1 was found to be occupied by a pocket factor apparently identical with that present in the major receptor group virus HRV16. Electron density, consistent with the presence of a viral RNA fragment, is seen stacked against a conserved tryptophan residue.

Capillary electrophoresis with postcolumn infectivity assay for the analysis of different serotypes of human rhinovirus (common cold virus).

Differentiation of virus serotypes with capillary zone electrophoresis was demonstrated. For four serotypes of human rhinovirus (HRV2, HRV14, HRV16, HRV49), different electrophoretic mobility was achieved at pH 8.3 (borate/boric acid buffer, 100 mmol/L). Addition of detergent (Triton X-100-R, deoxycholate, and/or SDS) to the background electrolyte was required for reduction of wall adsorption and improvement of peak shape. A major nonviral contaminant, present in all virus samples, was best separated from the viral peaks with 10 mmol/L SDS as additive. The method allowed detecting serotypes HRV16 and HRV49 in crude, partially purified virus preparations. An infectivity assay carried out off-line with fractions collected at the capillary outlet enabled the sensitive and biospecific identification of the peaks of HRV2 and HRV14.

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

Determination of infectious progeny virus and in vivo labelling with [(35)S]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.

Separation and biospecific identification of subviral particles of human rhinovirus serotype 2 by capillary zone electrophoresis.

During infection, human rhinoviruses undergo structural rearrangements of their capsid proteins from D-antigenic native virus (sedimenting at 150S upon sucrose density gradient centrifugation) to C-antigenic A-particles (sedimenting at 135S) and B-particles (sedimenting at 80S); the latter remain after release of the viral genomic RNA into the cytosol. Subviral particles with very similar properties can also be produced in vitro upon exposure to elevated temperatures or to low-pH buffers. This paper reports on the successful separation of native virus and 80S B-particles by capillary zone electrophoresis. Separation was carried out in an untreated fused-silica capillary (50 microns i.d., total length 50.0 cm, effective length 41.5 cm) at 20 degrees C and monitored with UV detection. The separation buffer was 100 mmol/L boric acid/borate (pH 8.3) and contained 0.5% sodium deoxycholate, 0.05% SDS, and 0.5% Triton X100R; the detergents were required to prevent viral aggregation and adsorption to the capillary wall. The analytes were identified from their characteristic spectra as determined by fast spectral scanning. Final confirmation was obtained by comparison of electropherograms from samples prior and after immunodeplition with antibodies specifically precipitating D- or C-antigen. The present method enables one to easily monitor and quantify these structural changes and thus to determine the most favorable conditions for complete conversion of native virus to 80S B-particles.

Crystallization and preliminary X-ray analysis of human rhinovirus serotype 2 (HRV2).

Human rhinoviruses, the major cause of mild recurrent infections of the upper respiratory tract, are small icosahedral particles. Over 100 different serotypes have been identified. The majority (91 serotypes) use intercellular adhesion molecule 1 as the cell-attachment site; ten serotypes (the minor group) bind to members of the low-density lipoprotein receptor. Three different crystal forms of the minor-group human rhinovirus serotype 2 (HRV2) were obtained by the hanging-drop vapour-diffusion technique using ammonium sulfate and sodium/potassium phosphate as precipitants. Monoclinic crystals, space group P2(1), diffracted at least to 2.8 A resolution, and two complete virus particles were located in the crystal asymmetric unit. A second type of crystals had a compact cubic like morphology and diffracted beyond 2.5 A resolution. These crystals belong to a primitive orthorhombic space group, with unit-cell parameters a = 309.3, b = 353.5, c = 759.6 A, and contain one virus particle in the asymmetric unit. A third type of crystals, with a prismatic shape and belonging to space group I222, was also obtained under similar crystallization conditions. These latter crystals, with unit-cell parameters a = 308.7, b = 352.2, c = 380.5 A, diffracted to high resolution (beyond 1.8 A) and contained 15 protomers per asymmetric unit; this requires that three perpendicular crystal twofold axes coincide with three of the viral particle's dyad axes.

Analysis of common cold virus (human rhinovirus serotype 2) by capillary zone electrophoresis: the problem of peak identification.

Different preparations of human rhinovirus serotype 2 (HRV2), a common cold virus, were analyzed by capillary zone electrophoresis (CZE) in untreated fused-silica capillaries using borate buffer (100 mmol/L, pH 8.3) and sodium dodecyl sulfate (10 mmol/L) as additive to prevent wall adsorption. The electropherograms showed one major peak at 205- and 254-nm detection wavelengths. The identity of the peak as originating from native virus was confirmed by several indirect methods. Heating to 56 degrees C is known to lead to release of the genomic RNA from the viral capsid; this treatment resulted in the disappearance of the major peak and the emergence of a new predominant peak that was identified as RNA by enzymatic digestion. As expected, RNase treatment of the unheated sample remained without effect as the viral genome is inaccessible in the native viral shell. A monoclonal, virus-aggregating antibody was used for immunodepletion of native virus; again, the major peak disappeared upon removal of viral aggregates by centrifugation prior to CZE analysis. In combination, these results allowed for the unambiguous identification of the main peak as native HRV2 and of the minor peaks as contaminants present in various amounts in the different viral preparations. It is demonstrated that CZE allows for an extremely easy and rapid assessment of conformational state and purity of virions in a given viral preparation.

Soluble LDL minireceptors. Minimal structure requirements for recognition of minor group human rhinovirus.

Soluble human low density lipoprotein minireceptors with less than seven ligand binding repeats (as are present in the native membrane receptor) were expressed in Sf9 insect cells with a hexa-His tag fused to the C terminus. The recombinant truncated proteins were affinity purified from the tissue culture supernatants by Ni-NTA column chromatography. Minireceptors with more than two repeats bound to rabbit beta very low density lipoprotein and could thus be further purified by affinity chromatography. Binding and cell protection assays indicated that two ligand binding repeats are sufficient for attachment of minor group human rhinoviruses to immobilized receptors, whereas at least three ligand binding repeats are required to protect HeLa cells against viral infection.

Effect of bafilomycin A1 and nocodazole on endocytic transport in HeLa cells: implications for viral uncoating and infection.

Bafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects the transport of endocytosed material from early to late endocytic compartments. To characterize the endocytic route in HeLa cells that are frequently used to study early events in viral infection, we used 35S-labeled human rhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up via receptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of <5.6, resulting in release of the genomic RNA and ultimately in infection (E. Prchla, E. Kuechler, D. Blaas, and R. Fuchs, J. Virol. 68:3713-3723, 1994). As revealed by fluorescence microscopy and subcellular fractionation of microsomes by free-flow electrophoresis (FFE), baf arrests the transport of all markers in early endosomes. In contrast, the microtubule-disrupting agent nocodazole was found to inhibit transport by accumulating marker in endosomal carrier vesicles (ECV), a compartment intermediate between early and late endosomes. Accordingly, lysosomal degradation of HRV2 was suppressed, whereas its conformational change and infectivity remained unaffected by this drug. Analysis of the subcellular distribution of HRV2 and fluid-phase markers in the presence of nocodazole by FFE revealed no difference from the control incubation in the absence of nocodazole. ECV and late endosomes thus have identical electrophoretic mobilities, and intraluminal pHs of <5.6 and allow uncoating of HRV2. As bafilomycin not only dissipates the low endosomal pH but also blocks transport from early to late endosomes in HeLa cells, its inhibitory effect on viral infection could in part also be attributed to trapping of virus in early endosomes which might lack components essential for uncoating. Consequently, inhibition of viral uncoating by bafilomycin cannot be taken to indicate a low pH requirement only.

Very-low-density lipoprotein receptor fragment shed from HeLa cells inhibits human rhinovirus infection.

The large family of human rhinoviruses, the main causative agents of the common cold, is divided into the major and the minor group based on receptor specificity. Major group viruses attach to intercellular adhesion molecule 1 (ICAM-1), a member of the immunoglobulin superfamily, whereas minor group viruses use low-density lipoprotein receptors (LDLR) for cell entry. During early attempts aimed at isolating the minor group receptor, we discovered that a protein with virus binding activity was released from HeLa cells upon incubation with buffer at 37 degreesC (F. Hofer, B. Berger, M. Gruenberger, H. Machat, R. Dernick, U. Tessmer, E. Kuechler, and D. Blaas, J. Gen. Virol. 73:627-632, 1992). In light of the recent discovery of several new members of the LDLR family, we reinvestigated the nature of this protein and present evidence for its being derived from the human very-low density lipoprotein receptor (VLDLR). A soluble VLDLR fragment encompassing the eight complement type repeats and representing the N-terminal part of the receptor was then expressed in the baculovirus system; both the shed protein and the recombinant soluble VLDLR bind minor group viruses and inhibit viral infection of HeLa cells in a concentration-dependent manner.