NO173099B - RECEPTOR FROM THE LITTLE RHINOVIRUS RECEPTOR GROUP, PROCEDURE FOR MANUFACTURING AND IN VITRO USING THIS RECEPTOR - Google Patents

Description

The present invention relates to a receptor with binding activity for the smallest group of rhinoviruses for use in vitro, a method for its production, as well as its use.

Human rhinovirus represents a large genus within the Picorna virus family, and includes over 90 different serotypes (6,11). These RNA viruses attack the respiratory tract in humans and cause acute infections, which lead to runny nose, cough, hoarseness, etc., and are usually referred to as the common cold (15). Infections with rhinovirus belong to them

most frequent diseases in humans. Although the course of the disease is usually of a harmless nature, the common cold nevertheless leads to a general weakening of the organism. This can lead to secondary infections with other pathogens.

The large group of human rhinovirus can be divided into two subgroups, when one uses the competition for binding sites on the cell surface of human cells in cell cultures (usually HeLa cells) as a criterion for the assignment. This original assignment of a few representatives of rhinoviruses (10) could be extended by broadly designed experiments on 88 representatives (4, 1). The result of these experiments makes it reasonable to conclude that, despite the large number, there are surprisingly only two different receptors on the cell surface, to which representatives of one or the other rhinovirus group can bind. Until now, 78 serotypes from the large "rhinovirus receptor group" could be assigned to 8 from the small "rhinovirus receptor group" (RVRG). 2 further representatives did not agree, so that no final division was possible (1).

In recent years, a significant increase in rhinovirus infections in densely populated areas has been established. While most other infectious diseases result in a long-term or permanent immunity against the source of the disease in question, infections with rhinovirus can occur again and again. The reason for the lack of permanent immunity is the large number of rhinovirus strains, which in between show no or only little immunological cross-reaction (6, 11) . After the infection has occurred, it is indeed possible to detect antibodies against the virus strain in question, but these provide no protection against other rhinovirus strains. Due to the large number of strains circulating in the population, repeated infections with rhinovirus are possible.

Therefore, the presence of only two receptors offers the promise of multiple attack options for a successful fight against rhinoviral infections.

As receptors are usually very specific, it is possible to achieve a targeted effect on the receptors with suitable substances, e.g. by blocking the receptors. When you insert substances that block the receptor, you can prevent the receptor's specific virus from entering the cell. The same substances that can prevent an infection in this way can also be used in the therapy of a manifest rhinovirus infection. The production of such substances becomes significantly easier, in many cases only then intentionally made possible, when the corresponding receptor has been characterized.

It was therefore a task for the present invention to isolate and to purify the receptor for the small

RVRG.

The only information on rhinoviral receptors that has been available so far concerns the receptors from the large RVRG.

The purification and characterization of the receptors from the large RVRG was carried out using a monoclonal antibody, which was produced by immunization of mice with HeLa cells. This receptor is glycosylated and has a molecular weight of 440 kD in the natural state, denaturation with sodium lauryl sulfate leads to dissociation into subunits of 9 0 kD, which makes it reasonable to conclude that the functional receptor exists as a pentamer (17). The receptor for the small RVRG has so far been neither characterized nor purified. The only data for this receptor refer to its structure from protein, and also show that this - or similar proteins are also present on cells from a number of other species. This distinguishes the receptor from the small RVRG significantly from the receptor from the large group, which could only be found on human and in a few cases on monkey cells. An influence, e.g. blocking the receptor with substances that prevent the penetration of viruses into the cell seems suitable as a possible prevention or also as therapy for an already existing infection.

The receptor according to the invention is characterized by the fact that the molecular weight, determined by gel permeation chromatography, amounts to approximately 450 kD; the sedimentation constant, determined by sugar gradient centrifugation in the presence of surfactants, corresponds to approximately 28.4 S; it is bound by Lens culinaris lectin; that it is not bound by heparin-sepharose; it binds irreversibly to an anion exchanger; its binding activity is not destroyed by neuraminidase; it consists of subunits that are associated by intermolecular disulfide bridges; it shows no binding to rhinovirus in the presence of EDTA; and iodoacetamide only slightly affect the binding activity of rhinovirus.

It was the task of the present invention to provide the prerequisites for the production of substances which enable protection against infections with rhinovirus from the small receptor group. This task is solved with the present invention in that

a. membranes are isolated and purified from cells,

preferably HeLa cells,

b. the receptors in the membranes are solubilized with surfactants, preferably Triton-X100, CHAPS, Zwittergent, octylglucoside or DOC,

particularly preferred is octylglucoside,

c. the insoluble components are removed and

d. the receptors are purified by chromatography, preferably on a column consisting of Concanavalin-A, resin-sepharose or Lens culinaris lectin.

These cells were cultured in suspension in a manner known per se, the cells were blasted, the cell nuclei removed and the membranes cleaned. The receptors that were in the membranes were then solubilized. To optimize the solubilization of active receptors from purified HeLa cells, different surfactants were tested at different concentrations. Decisive for the choice of a particular surfactant was the ability to solubilize as much membrane material as possible with as high a virus binding activity as possible. 1% 1-O-n-octyl-^-D-glucopyranoside proved to be the most suitable (Fig. 0).

The insoluble components were removed, and those receptors that were in solution were further purified. In order to be able to follow the virus binding activity, a sensitive filter binding test was developed, which enabled the binding of <35>S-labeled virus to receptors that had been immobilized on nitrocellulose paper.

The necessary virus for the test was cultured and purified in a manner known per se (13).

As it is known that most membrane proteins are glycosylated, the receptor was purified on a Lens culinaris lectin column. This lectin has specificity for α-D-glucose and α-D-mannose units (16). Bound material was eluted with IM α-D-methylglucoside in phosphate-buffered NaCl solution with 1% octylglucoside.

Aliquots were applied to nitrocellulose in duplicate and incubated with both native and heated virus. Autoradiography of the fractions showed strong binding to natural virus by the material that was eluted, compared to the fractions from the run-through. The heated virus showed weak binding to the flow-through material. This suggests non-specific interactions that are induced by the high proportion of hydrophobic proteins; heated rhinovirus exhibits increased hydrophobicity (9). As it had been determined that purified virus during long-term storage at 4°C is eventually converted into particles exhibiting the same antigenicity as heated virus (C-determinants), these contaminants were separated immediately before carrying out the binding test by immunoprecipitation with specific monoclonal antibodies for the C determinants, for example MAK 2G2. These monoclonal antibodies are obtained in a known manner by immunizing mice or baby rabbits with C-determinants and subsequent cloning according to Kohler and Milstein (18).

In addition to the L. culinaris lectin column, concanavalin A, ricin and heparin sepharose columns were also used to purify the receptor. Flow-through and eluted material were tested as above. Con.A sepharose columns were eluted with IM α-D-methylmannoside, whereby approximately 20% binding activity could be recovered; elution of the castor column with IM galactose led to approximately 100% recoverable binding activity. Unlike the receptor for the Coxsackie B virus group (7), heparin-Sepharose did not retain the binding activity. The eluate from the L. culinaris column was separated on a superose column using FPLC (Pharmacia) (gel permeation chromatography). By comparison with labeled proteins, the molecular weight of the active receptor could be determined to be 450 kD. At the same time, a larger part of the contaminating proteins could be separated (Fig. 1).

The receptor from the small rhinovirus receptor group migrates in a polyacrylamide gel in the presence of SDS with an apparent molecular weight of 120 kD. In some cases, a barely visible band with a somewhat lower molecular weight can also be observed, which possibly represents a modification of the bulk of the receptor protein (Fig. 5, column 1). The molecular weights for both receptor forms are significantly higher than that found for the large rhinovirus receptor (90 kD).

As the receptor proteins in both groups have a molecular weight of approx. 450 kD in its natural form, it is not unlikely that their build-up of individual units takes place in a similar way. The real molecular weight may still differ from that determined by gel permeation chromatography, which is due to the small difference in the retention volume of the proteins in this high molecular weight range. The picornavirus structure shows a deep gorge (canyon) which lies around the fivefold axes of the icosahedral symmetry and which possibly contains the receptor binding sites (19) . It is believed that the receptor from the large rhinovirus receptor group and the receptor for the Coxsackie B virus group bind the viruses across the fivefold axis (20). The question of whether the receptor from the small group is a pentamer remains unanswered, as the molecular weight of the individual units is very high compared to the receptor from the large group.

By means of centrifugation in a sugar gradient, it was possible to determine the sedimentation constant for the receptor. For this purpose, receptor purified on L. culinaris was applied to a sugar gradient and centrifuged. The activity maximum was found in the position of the gradient which corresponds to a sedimentation constant of 28.4 S (Fig. 2).

In the preliminary experiments, it had been determined that the receptor could no longer be eluted from a chromatography column with anion yields. As the receptor is insensitive to neuraminidase, the sialic acid was removed from the glycoprotein to reduce the ion interaction with the column material. The sample was then applied to a mono Q column (Pharmacia), and the receptor eluted with a NaCl gradient. The binding activity could be detected as a broad peak at approximately 250 mM NaCl (Fig. 3).

The purification of the receptors according to the invention is also possible by a combination of the chromatographic purification steps on different chromatography materials.

The chemical properties and the structural requirements of the receptor according to the invention for the viral binding were found with the help of enzymes and chemical reagents (table 1).

Trypsin treatment completely destroys the binding activity. This agrees with already known results from enzymatic treatment of cell surfaces (14), and alludes to the receptor's protein character.

Treatment of the solubilized receptor with neuraminidase reproducibly led to a slight increase in binding activity. This treatment possibly leads to a better accessibility of the region on the receptor molecule which constitutes the target for the virus interaction. It follows from this that the sialic acid is not necessary for virus binding.

Dithiothreitol (DTT) destroys the binding activity, suggesting the participation of disulfide bridges in maintaining the correct folding of the protein. The surprisingly high molecular weight, determined by gel permeation chromatography and gradient centrifugation, suggests an oligomeric structure of the receptor molecule. The sensitivity to DTT could indicate that intermolecular disulphide bridges are necessary for the binding of the hypothetical single units.

Treatment with iodoacetamide reduces the binding activity only slightly, suggesting that free sulfhydryl groups are not necessary for efficient binding.

In the presence of ethylenediaminetetraacetic acid (EDTA) during the incubation of the nitrocellulose filters with <35>S-labeled virus, no binding could be determined. This is consistent with previous investigations that showed the necessity of the presence of divalent cations for the interaction of rhinovirus with the cell surface (12).

Competitive binding studies between pairs of serotypes were used to assign the human rhinoviruses to the two receptor classes (10, 1). In the present invention, HRV2 and HRV49 were therefore used as representatives of the small receptor group and HRV89 as a representative of the large receptor group in competition experiments to find the specificity for the receptors according to the invention. The nitrocellulose filters with immobilized receptors were incubated with labeled HRV2 in the presence of an approximately 20-fold excess of either HRV2 or HRV89. As shown in Table II, binding was massively suppressed in the presence of untagged HRV2, but not affected by HRV89. To confirm these results, experiments were repeated with labeled HRV49 using HRV2 and HRV89 as competitors. Again, it is obvious that HRV2 massively reduces binding, while HRV89 has no influence.

Although HRV2 and HRV89 bind to different receptors, their capsid proteins show surprising similarity (5). A detailed structural comparison between HRV2 and HRV14, based on the X-ray structural analysis of HRV14, has just been performed (2). Both HRV14 and HRV89 bind to the receptor in the large RVRG. It can therefore be expected that a cluster of conserved amino acids will be found at the hypothetical receptor binding site. Previously, however, it has not been possible to discover a single pattern of conservative amino acids within the Canyon region.

Only with the present invention is it possible to produce receptors for the small RVRG in an essentially pure form.

Only at the receptors according to the invention is it possible to investigate the virus/receptor interaction in a targeted manner. Of particular importance here is the localization of the areas on the receptors that are ultimately responsible for the viral effect. By knowing these areas, it should be possible to produce substances that are specifically aimed at these areas, so as to possibly achieve a blocking of the receptors for several different rhinoviruses.

Subject matter of the present invention are also those receptors which can be produced from the natural receptors by treatment with reducing agents, and which can, for example, be purified by electrophoretic methods. For example, these individual units can be used for the production of polyclonal and/or monoclonal antibodies which can be used preparatively, diagnostically and/or therapeutically in a similar way as the corresponding antibodies against the natural receptors. The individual receptor units can also be used in a similar way to the natural receptors.

The natural receptors and/or their individual units that can be modified by targeted enzymatic treatment. As was shown in the present invention, the treatment with trypsin destroys the binding activity of the receptors according to the invention, while neuraminidase caused a slight increase in the activity. It is therefore conceivable and possible for the person skilled in the art to verify in a non-inventive way that certain enzymes and/or chemical reagents lead to receptors that are either improved in their effect and/or are better applicable and usable and/or exhibit improved stability, compared to the natural receptors. These modifications can, for example, lead to parts of the protein chain being separated, excised and/or the protein chain being cut into all or some subunits of the natural receptors.

In addition to these modifications, it is also possible to transfer the natural receptors, for example by controlled reduction either completely or partially in the subunits. These more or less large subunits can also be joined by, for example, targeted oxidation into more or less large units, which are rearranged in relation to the natural receptors.

Reducing agents which are suitable for splitting disulphide bridges are, for example, thiol compounds, such as thiophenol, 4-nitrothiophenol, 1,4-butanedithiol and especially 1,4-dithiothreityl. The reduction is advantageously carried out in an aqueous alkaline medium, for example in the dilute aqueous solution of an alkali metal hydroxide, e.g. sodium hydroxide, of an alkali metal carbonate, e.g. sodium carbonate or of an organic base, especially of a tri-lower alkylamine, e.g. triethylamine, at room temperature.

Oxidizing agents which are suitable for relinking disulfide bonds in the reduced polypeptides are e.g. air-oxygen, which is passed through an aqueous solution of the polypeptide, and to which in this case a catalytic amount of a transition metal salt, e.g. iron (III) sulfate, iron (III) chloride or copper (II) sulfate; iodine, also in the form of the potassium iodide adduct KJ3, which is preferably used in alcoholic, e.g. methanolic, or aqueous-alcoholic, e.g. aqueous methanolic solution; potassium hexacyanoferrate(III) in aqueous solution; 1,2-diiodoethane or azodicarboxylic acid dimethyl ester or -diethyl ester, which is reacted in water or in a mixture consisting of water and a water-miscible alcohol, e.g. methanol. The oxidation is especially carried out at room temperature.

The separation of the reagents, especially of the salts and the oxidising and reducing agents and their by-products from the desired compound, takes place according to methods known per se, for example by

molecular weight filtration, e.g. on Sephadex or Biogel.

All modifications can be used in a similar way to the natural receptors according to the invention.

The receptor according to the invention is soluble, so that it can be easily handled and used.

However, it is also possible to bind the receptors to a solid carrier, and use them in this form for diagnostic and preparative purposes. As carriers, all common solid carriers such as polystyrene, glass, dextran, but also biological membranes and lipid vesicles come into question.

It is also possible to bind common markers to the receptors, and use them diagnostically in this form.

If the receptors according to the invention are bound to a carrier, they can be used both diagnostically and also preparatively for binding the viral protein, for example by means of so-called affinity chromatography. Diagnostically, a viral protein can be picked up in the usual way by a receptor that is bound to a carrier, e.g. using antibodies or labeled antibodies. Examples of labeling include radioactive labeling, an enzyme or a fluorescent group.

Of great importance is the use of the receptors according to the invention in vitro for the production of polyclonal and/or monoclonal antibodies which act specifically against the receptors located in the cell membranes. Such antibodies can in return be used primarily diagnostically for the production and determination of the receptors on cells or cell biological material.

They thus open completely new paths and possibilities.

Explanation of the figures

Fig. 0 Solubilization of the receptors with different surfactants (A: natural virus, B:

denatured virus).

Fig. 1 Gel permeation chromatography of the solubilized receptor on a Superose 6 HR 10/30 column. Membranes equivalent to 10<8> cells were solubilized in OG (15 mM sodium phosphate pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2 (PBS) with 1% octyl glucoside applied to a 1 ml L. culinaris column, and the adsorbed material eluted with 1 M α-D-methylglucoside in OG. The eluate was concentrated in a small Centricon tube to 0.5 ml and applied to the Superose column. The column was developed at 0.2 ml/min OG, and fractions of 0 .5 ml was collected The binding activity for the individual

the fractions, the positions of the labeled proteins (determined in a special experiment) and the extinction at 280 nm are shown. Fig. 2 Sugar gradient centrifugation of the solubilized receptor. The material eluted from a L. culinaris column was concentrated (see legend to Fig. 1) and partitioned on a 10-4 0% sugar gradient in OG. The binding activity of the fractions (0.4 ml), the position of the labeled proteins catalase (Cat) and aldolase (Aid) as well as the extinction at 280 nm are shown. Fig. 3 Mono Q anion exchange chromatography. Fractions 14-16

from the gel permeation chromatography (Fig. 1) was treated with neuraminidase and the material separated by means of FPLC on mono Q. The binding activity of the individual fractions (0.5 ml), the extinction at 280 nm and the course of the gradient are shown.

Fig. 4 Detection of the receptor on Western Blots. The virus binding activity of the mono P anion exchange column was applied to a Superose 6 HR 10/30 column. Fractions of 1.2 ml were collected. The proteins were traced as A280, the positions of the labeled proteins (thyroglobulin, 670k; apoferritin 440, ; β-amylase, 200k) are also shown (a). The proteins in the fractions from the Superose column were concentrated and applied to three 6% SDS-polyacrylamide gels. One gel was stained with Coomassie blue (b), the proteins from the second and third gel were transferred onto nitrocellulose membranes (c, d). The blot was incubated with <35>S-labeled HRV2 in the absence (c) or in the presence of an excess of unlabeled HRV2 (d); the positions of the labeled proteins (2-macroglobulin, 180k; /3-galactosidase, 116k; fructose-6-phosphate kinase, 84k) and of the receptor bands are marked with arrows.

Fig. 5 Autoradiography of Western Blots obtained from the pooled fractions with virus binding activity from the Superose column (see Fig. 4). These blots were incubated with <35>S-labelled HRV2. Before loading onto the gel, the samples were incubated with SDS at room temperature, lane 1; the sample was boiled in SDS, lane 2; the sample was incubated with 10 mM dithiothreitol, lane 3; a blot identical to lane 1 was incubated with <35>S-labeled HRV2 in the presence of 10 mm EDTA, lane 4; or was incubated with <35>S-labeled HRV2, which had been heated to 56 °C for 10 min, lane 5.

Materials

l-O-n-octyl-/?-p-glucopyranosides, Tween 40 and 3-(3-chiolamidopropyl)-dimethylammonium-l-propanesulfonate (CHAPS) were from Sigma, N-tetradecyl-N,N-dimethylammonium-3- propane sulphonate (Zwittergen 3-14) from Serva. The other surfactants were from Merck, trypsin from Miles, <35>S-methionine (1350 Ci/mmol) from Amersham.

Example 1:

Production of viruses

HRV2, HRV49, and HRV89 were grown and purified essentially as described in HeLa cell suspension (13). As an example, the cultivation, isolation and purification of

HRV2.

HeLa cells (strain HeLa-Ohio, 03-147, Flow Laboratories, England) were cultured in suspension at 37°C. The suspension medium (Thomas, D.S., Conant, R.M. and Hamparian, V.U., 1970, Proe. Soc. Exp. Biol. Med. 133, 62-65; Stott, E.J. and Heath, G.F., 1970, J. Gen. Virol, 6, 15-24) consisted of a Joklik modification of MEM for Suspension (Gibco 072-1300) and 7% horse serum (Seromed 0135). The inoculation density was 5-10 x 10<*> cells/ml, the volume 500 ml. The suspension was centrifuged at a cell density of 1 x 10<6> cells/ml under sterile conditions at 300 g for 10 min. The supernatant was suctioned off and the cells resuspended in 100 ml of infection medium (Joklik modification of MEM for suspension culture with 2% horse serum and 2 mM MgCl2) • By careful suction several times in a 20 ml pipette, the cells were distributed homogeneously in the infection medium. It was then filled up to 500 ml. The cell suspension was then brought to 34°C and infected with HRV2 (twice Plaque purified) at a multiplicity of 0.1 virus/cell. The HRV2 strain was provided by the American Type Culture Collection (ATCC VR-482 and VR-1112). The strain used was neutralized with antiserum against HRV2 (American Type Culture Collection, Cat.No. ATCC VR-1112

AS/GP).

An antiserum against HRV7 (Cat.No. ATCC VR-1117 AS/GP), which showed no neutralization, served as a control serum. After 60 hours at 34°C, the virus was harvested. Viruses were recovered both from the cells, respectively the cell fragments, and from the medium.

For this purpose, the medium was separated and aspirated from the infected cells and cell fragments by centrifugation for 10 minutes at 1500 g. The precipitate was frozen at -70°C.

The cell pellets from 12 l suspension culture were combined, resuspended in 40 ml TM buffer (20 mM Tris/HCl, pH 7.5, 2 mM MgCl2), placed on ice for 15 minutes, then broken up in a Dounce homogenizer and the mixture centrifuged in 30 minutes at 6000 g. The precipitate was then washed once more in 10 ml of TM buffer. Both supernatants were pooled and centrifuged for 3 hours at 110,000 g to pellet the virus. The virus pellet was then taken up in 10 ml KTMP buffer (50 mM KCl, 50 mM Tris/HCl, pH 7.5, 5 mM MgCl2, 2 mM mercaptoethanol, 1 mM puromycin, 0.5 mM GTP) and after addition of 150 yig DNase I (Sigma, ribonuclease free) incubated for 1 hour on ice.

From the infection medium, the virus was precipitated under agitation at 4°C with polyethylene glycol 6000 (PEG 6000; Merck) at a concentration of 7% and 450 mM*NaCl (Korant, B.D., Lonberg-Holm, K., Noble, J. and Stasny, J. T. 1972, Virology 48, 71-86). After 4 h at low temperature, the virus was centrifuged for 30 min at 1500 g, the pellet resuspended in 10 ml of KTMP buffer, which contained 75 iq of DNase I, the mixture incubated for 1 h on ice and then frozen at -70°C.

The virus suspensions recovered from the cells and from the medium were combined, incubated 5 min at 37°C, cooled by the addition of 60 ml cold TE buffer (10 mM Tris/HCl, pH 7.4, 1 mM EDTA) and then sonicated for 5 min in an ice bath.

It was then centrifuged for 30 min at 6000 g. 920 ml TE buffer containing 7% PEG 6000 and 450 mM NaCl was added to the supernatant, gently stirred at 4°C for 4 h, and the resulting precipitate pelleted for 30 min at 6000 g. The precipitate was taken up again in 100 ml of TM buffer, the virus precipitated as above by adding PEG 6000 and NaCl and pelleted. The precipitate was resuspended in 40 ml of TM buffer, the suspension centrifuged for 30 min at 6000 g, and the virus was pelleted for 3 hours at 110,000 g. The precipitate was dissolved in 1 ml of TM buffer, after the addition of 50 µg of DNase I incubated in 1 hour at 4°C and then added 1 ml of TE buffer. For further purification, the virus suspension was centrifuged on sucrose gradients (10-30% w/w in TE buffer) for 4 hours at 4°C at 110,000 g. Using extinction at 260 nm, the fractions containing the virus were taken out and diluted with TM buffer, so that the final concentration of sucrose was 10%. Then centrifuged for 8 hours at 85,000 g. The virus pellet was taken up in 1 ml of TM buffer and stored in wood

-70°C. To test the purity of the virus preparation, an electrophoresis was carried out on a 12.5% polyacrylamide gel in the presence of 0.1% sodium dodecyl sulfate

(Laemmli, U.K., 1970 Nature (London) 277, 680-685) and the protein bands stained with Coomassie brilliant blue.

Example 2:

Preparation of 35 S- methionine-labelled human rhinovirus serotype 2

(HRV2)

2 HeLa cell monolayers in 165 cm<2> petri dishes were infected at an MOI (Multiplicity of Infection) of 40 with HRV2 for 1 hour at 34°C in methionine-free MEM medium (Gibco) with 2% dialyzed fetal calf serum (Flow). The cells were washed twice with PBS and the incubation continued at 34°C in fresh medium. After 3 hours, 1 mCi <35>S-methionine (1350 Ci/mmol, Amersham) was added to each monolayer and further incubated for a total of 24 hours. The medium of infected cells and cell fragments was separated and aspirated by centrifugation for 10 min at 1500 g. The pellet was frozen at -70°C in 5 ml of 10 mM Tris, 10 mM EDTA, pH 7.5 (Tris/EDTA ) and thawed again. Supernatant and frozen/thawed pellet were pooled and centrifuged for 30 min at 45,000 g. The supernatant from this centrifugation was centrifuged for 2 h at 140,000 g. The virus pellet was resuspended in 300 µl Tris/EDTA, and the virus was purified as above over a 10 -30% sucrose gradient. The individual fractions from the gradient were analyzed using SDS electrophoresis in 12% polyacrylamide gels. The pure virus fractions were pooled and stored in the presence of 1% BSA (bovine serum albumin) at 4°C for a maximum of 4 weeks.

In order to remove viruses with altered capsid structure from the preparations from example 1 and example 2, 20 μl of immunoadsorbant, which contained monoclonal antibodies against the C-determinants, e.g. mAK 2G2, was incubated with the purified virus for 30 min and pelleted before the viral samples was removed.

Preparation of the immunoadsorbance

Sta<p>hvlococcus aureus (BRL) cells were suspended to a 10% w/w suspension in water. The cells were washed twice with PBS, added 1/5 vol. baby rabbit anti-mouse IgG serum (Behring); the suspension was incubated for 1 hour at room temperature. The cells were washed twice with PBS and re-incubated for 1 hour with 1/5 vol. mouse ascites fluid that contained monoclonal antibodies against the C determinants (eg mAK 2G2). After washing twice, the pellet was pelleted, the pellet resuspended in PBS (10% W/W) and added to the preparations with the radioactive virus.

Example 3:

Solubilization of the receptor from HeLa cells

HeLe cells (strain HeLa-Ohio, 03-147, Flow Laboratories, England) were cultured in suspension at 37°C. The suspension medium (Thomas, D.C., Conant, R.M. and Hamparian, V.U., 1970, Proe. Soc. Exp. Biol. Med. 133, 62-65; Stott, E.J. and Heath, G.F., 1970, J. Gen. Virol, 6, 15-24) consisted of a Joklik modification of MEM for suspension (Gibco 072-1300) and 7% horse serum (Seromed 0135). The inoculation density was 5-10 x 10<*> cells/ml, the volume 500 ml. The suspension was centrifuged at a cell density of 1 x 10<6> cells/ml under sterile conditions at 300 g for 10 min. The supernatant was aspirated off and the cells were washed twice with phosphate-buffered saline (PBS). 10<9> cells were suspended in 20 ml of isotonic buffer (10 mM HEPES-KOH, pH 7.9, 140 mM KCl, 1.5 mM MgCl2, 0.5 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride) and lysed at low temperature with 200 shocks in a 50 ml Dounce homogenizer. Cell nuclei were removed by centrifugation for 3 min at 1000 g. The membranes were then further purified using the two-phase method (3). Membranes corresponding to 2 x 10<8> cells/ml were taken up in PBS and stored in liquid nitrogen. For solubilization, 2 x 10<8> cell equivalents were suspended in 1 ml of 1% octyl glucoside in PBS (OG) and insoluble material separated by centrifugation at 80,000 g/h. The supernatant was used for the column chromatography.

Example 4:

Filter binding test

The fractions from the column chromatography that were to be examined for activity were applied in a Dot-blot apparatus (Bio-Rad) to a nitrocellulose membrane (BA85, Schleicher and Schull). The samples were allowed to soak in at room temperature. The liquid residues were then sucked off with a weak water jet vacuum and non-specific protein binding sites desaturated with 2% bovine serum albumin (BSA) in PBS at 4°C overnight. The filters were then incubated for 1 h with 10<5> cpm <35>S-methionine-labeled HRV2 in 1% Tween 40, 0.5% sodium deoxycholate, and 10 mM (3-(3-cholamidopropyl)-dimethylammonium 1-propanesulfonate) in PBS. The membranes were washed twice with 2% BSA in PBS, dried and the round areas corresponding to the samples punched out; the radioactivity was measured in a liquid scintillation counter.

As a specificity control, HRV2 was heated for 10 min at 56°C before the incubation of the nitrocellulose filters (8). After this treatment, no binding to one of the samples could be determined anymore (Fig. 1B). From this it was deduced that the binding of natural HRV2 to immobilized material must in reality be traced back to a specific interaction between the virus and the receptor.

Example 5;

Affinity chromatography on columns of Lens culinaris lectin

10<8> cell equivalents were solubilized as described and applied to a L. culinaris column (1 ml) equilibrated with OG. The column was washed with 5 ml of OG and bound material eluted with 2 ml of 1 M α-D-methylglucoside in OG. The binding test showed that almost 100% of the binding activity could be recovered, while on the other hand around 90% of the total protein had been removed.

Example 6:

Gel permeation chromatography

The eluate from the L. culinaris column was concentrated with a small Centricon tube (30 kD exception) to 0.5 ml, and separated on a Superose 6 HR 10/30 column (equilibrated with OG buffer) using FPLC (Pharmacia) . By comparison with labeled proteins, the molecular weight of the active receptor could be determined to be 450 kD. At the same time, a larger part of the contaminating proteins could be separated (fig. 1) .

Example 7:

Centr i f uer ing on sugar gradient

Receptor purified on L. culinaris (as above) was applied to a sugar gradient (10-40% in OG) and centrifuged for 8 hours at 38 rpm at 4°C. The activity maximum was found in the gradient position corresponding to the sedimentation constant of 28.4 S. The positions of the labeled proteins were determined in another gradient (Fig. 2). Due to the presence of surfactants, the markers sedimented at calculated sedimentation coefficients of 15.0 S and 21.9 S (7.3 S and 1.3 S in the absence of surfactants).

Example 8;

Anion yield chromatography

In preliminary experiments, it had been established that the receptor could no longer be eluted from mono Q HR 5/5 columns (Pharmacia). Therefore, receptor prepurified over L. culinaris and gel permeation was treated with 1 U neuraminidase/mg protein for 60 min at 37°C to remove the strongly acidic neuraminic acid groups. The sample was then diluted two-fold with 10 mM sodium phosphate buffer pH 7, 1% octylglucoside and applied to a mono Q column. The column was developed with a gradient of 0-1 M NaCl in the same buffer. The binding activity could be detected as a broad peak at approximately 250 mM NaCl (Fig. 3).

Example 9:

Isolation and purification of the receptor

Plasma membranes from 2 x 10<9> HeLa cells were solubilized for 10 min at room temperature in 5 ml PBS, containing 1% w/v l-O-n-octyl-£-D-glucopyranoside and 0.01% w/v of each of L-α-p-tosyl-L-lysine chloromethyl ketone (TLCK), L-l-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) and phenylmethylsulfonyl fluoride (PMSF)

(all from Sigma). Insoluble material was centrifuged off during 30 min at 30 krpm in a Beckmann 65 fixed angle rotor.

The supernatant was applied to a 25 ml L. culinaris lectin column equilibrated with OG (PBS, 1% w/v octylglucoside), and the bound material eluted with 5 ml OG, containing 1 M α-methylglucoside. The eluate was saturated to 50% with an equal volume of saturated ammonium sulfate solution. The precipitated material was dissolved in 2 ml of buffer A (10 mM TRIS-HCl (pH 7.5), 5 mM EDTA, 1% w/v octylglucoside), and injected into a mono P anion exchange column connected to an FPLC- system (Pharmacia).

The proteins were separated using a gradient of 0-100% buffer B (like buffer A, but containing 1.5 M NaCl). Those fractions containing virus binding activity were pooled, concentrated with a small Centricon tube to 0.5 ml and partitioned on a Superose 6 column equilibrated with OG containing 5 mM EDTA. The protein concentration was followed as extinction at 280 nm (Fig. 4a).

The fractions from this column were concentrated to 50

/µl, brought up to 0.1% w/v SDS, and aliquots separated on three 6% polyacrylamide gels containing 5 mM EDTA (21). The resolved proteins in the gel were then either stained with Coomassie blue (Fig. 4b) or electrophoretically transferred onto a nitrocellulose membrane (22) which had been incubated with 4 x 10<5> cpm<35>S-labeled HRV2 (see above). The nitrocellulose was dried and autoradiographed (Fig. 4c). As a control for specific binding, an identically prepared nitrocellulose replica was incubated in the presence of a 20-fold excess of unlabeled HRV2 (Fig. 4d). It was determined that fractions 5 and 7 from the Superose column contained material capable of binding HRV2 when transferred onto nitrocellulose.

The autoradiography showed some bands with an apparent molecular weight greater than 300 kD in addition to a position corresponding to about 120 kD, compared to labeled proteins developed on the same gel (Fig. 4c). Only these 120 kD bands containing excess untagged virus disappear in the control (Fig. 4d) and thus demonstrate specific interaction between this protein and HRV2. The polyacrylamide gel containing identical samples and stained with Coomassie blue showed a very faint band in a position corresponding to the radioactive bands on the Wester blot. These bands are found exclusively in the samples from fractions 6 and 7 that showed virus binding activity (fig. 4b).

Example 10:

Binding tests

The regeneration of an active receptor depends on mild conditions; boiling in SDS destroys e.g. the activity irreversibly (compare Fig. 5, columns 1 and 2). If the receptor preparation was incubated with 10 mM dithiothreitol before application to the gel, no binding could be observed (Fig. 5, column 3). As the specific interactions of rhinoviruses with their receptors depend on the presence of divalent cations (12), the blot from a sample identical to that applied to lane 1 (Fig. 5) was incubated with virus in the presence of EDTA . Under these conditions, no binding could be observed (Fig. 5, column 4).

As a further test, the incubation of the nitrocellulose membrane was carried out with HRV2 heated to 56°C. This treatment leads to structural changes in the viral capsid that make it impossible for the virus to attach to the HeLa cell surface (23). As can be seen in fig. 5, column 5, no binding occurs under these conditions.

The filters were incubated as described with a 20-fold excess of unlabeled virus.

Bibliography

1. Abraham, G., and Colonno, R.J. (1984). Many rhinovirus serotypes share the same cellular receptor. J. Virol. 51, 340-345. 2. Blaas, D., Kuechler, E., Vriend, G., Arnold, E., Griffith, J.P., Luo, M., Rossmann, M.G. (1987). Comparison of three-dimensional structure of two human rhinoviruses (HRV2 and HRV14). Proteins (in press). 3. Brunette, D.M., and Till, J.E., (1971). A rapid method for the isolation of L-cell surface membranes using an aqueous two-phase polymer system. J. Membr. Biol. 5, 215-224. 4. Colonne, R.J., Callahan, P.L., and Long, W.J. (1986), Isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses. J. Virol. 57, 7-12. 5. Duechler, M., Skern, T., Sommergruber, W., Neubauer, Ch., Gruendler, P., Fogy, I., Blaas, D. and Kuechler, E.

(1987). Evolutionary relationships within the human

rhinovirus genus; comparison of serotypes 89, 2 and 14.

Pro. Nati. Acad. Pollock. U.S.A. (in press).

6. Fox, J.P. (1976). Is a rhinovirus vaccine possible?

American J. Epidemiol. 103. 345-354.

7. Krah, D.L., and Crowell, R.L. (1985). Properties of the desoxycholate-solubilized HeLa cell plasma membrane receptor for binding group B coxsackieviruses. J. Virol. 53: 867-870. 8. Lonberg-Holm, K., and Yin, F.H. (1973). Antigenic determinants of infective and inactivated human rhinovirus type 2. J. Virol. 12, 114-123. 9. Lonberg-Holm, K., and Whiteley, N.M. (1976. Physical and metabolic requirements for early interaction of poliovirus and human rhinovirus with HeLa cells. J. Virol. 19, 857-870. 10. Lonberg-Holm, K., Crowell, R.L., and Philipson, L. (1976) . Unrelated animal viruses share receptors. Nature 259, 679-681. 11. Melnick, J.L. (1980). Taxonomy of Viruses. Proe. Med. Virol. 26, 214-232. 12. Noble-Harvey, J., and Lonberg -Holm, K. (1974). Sequential steps in attachment of human rhinovirus type 2 to HeLa

cells. J.Gen. Virol..25, 83-91.

13. Skern, T., Sommergruber, W., Blaas, D., Pieler, Ch., and Kuechler, E. (1984). The sequence of the polymerase gene of

human rhinovirus type 2. Virology 136. 125-132.

14. Stott, E.J., and Heath, G.F. (1970). Factors affecting the growth of rhinovirus 2 in suspension cultures of L132 cells.

J. gen. Virol. 6, 15-24.

15. Stott, E.J., Killington, R.A. (1972). Rhinoviruses. Ann.

Fox. Microbiol. 26, 503-525.

16. Young, N.M., Leon, M.A., Takahashi, T., Howard, I.K., and

Sage, H.J. (1971). Studies on a phytohemagglutinin from the lentil. III. Reaction of Lens culinaris hemagglutinin with polysaccharides, glycoproteins, and lymphocytes, J. Biol. Chem. 271, 1596-1601. 17. Tomassini, J.E. and Colonno, R.J. (1986). Isolation of a receptor protein involved in attachment of human rhinoviruses. J. Virol. 58, 290-295. 18. Kohler, G. and Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity.

Nature (London) 256, 495-497.

19. Rossmann, M.G., Arnold, E., Erickson, J.W., Frankenberger, E.A., Griffith, J.P., Hecht, H-J., Johnson, J.E., Kamer, G., Luo, M., Mosser, A.M., Rueckert, R.R., Sherry, B. A., & Vriend, G. (1985). Structure of a common cold virus, human

rhinovirus 14 (HRV14), Nature (London) 317, 145-154.

20. Mapoles, J.E., Krah, D.L. & Crowell, R.L. (1985). Purification of a HeLa cell receptor protein for group B coxsackieviruses. Journal of Virology 55, 560-566. 21. Laemmli, U.K. (1979). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 277, 680-685. 22. Burnette, W.N. (1981). Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate - poly-acrylamide gels to unmodified nitrocellulose and radio-graphic detection with antibody and radioiodinated protein

A. Analytical Biochemistry 112, 195-203.

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Claims (11)

1. Receptor with binding activity for the smallest group of rhinoviruses for use in vitro characterized in that the molecular weight, determined by gel permeation chromatography, amounts to approximately 450 kD; the sedimentation constant, determined by sugar gradient centrifugation in the presence of surfactants, corresponds to approximately 28.4 S; it is bound by Lens culinaris lectin; that it is not bound by heparin-sepharose; it binds irreversibly to an anion exchanger; its binding activity is not destroyed by neuraminidase; it consists of subunits that are associated by intermolecular disulfide bridges; it shows no binding to rhinovirus in the presence of EDTA; and iodoacetamide only slightly affect the binding activity of rhinovirus. 2. Receptor subunit according to claim 1, characterized in that it can be produced by complete reduction from the receptor according to claim 1. 3. Receptor according to claim 1, characterized in that it can be produced by targeted reduction of the receptor according to claim 1. 4. Receptor according to claim 1, characterized in that it can be produced by targeted oxidation of at least two of the receptor subunits according to claim 2. 5. Process for producing receptors according to claim 1, with binding activity for rhinovirus belonging to the smallest group, characterized in that a. membranes are isolated and purified from cells, preferably HeLa cells, b. the receptors in the membranes are solubilized with surfactants, preferably Triton-X100, CHAPS, Zwittergent, octylglucoside or DOC, octylglucoside is particularly preferred, c. the insoluble the components are removed and d. the receptors are purified by chromatography, preferably on a column consisting of Concanavalin-A, resin-sepharose or Lens culinaris lectin. 6. Method according to claim 5, characterized in that the receptors after treatment with neuraminidase are chromatographed over an anion exchange column, preferably over a Mono Q column. 7. Method according to one of claims 5 or 6, characterized in that, in addition to the first chromatographic purification, a second chromatographic purification is carried out on a Superose column. 8. Method for producing the receptor subunits according to claim 2, characterized in that the receptor according to claim 1 is purposefully reduced. 9. Method for producing the receptor according to claim 1, characterized in that at least two of the receptor subunits according to claim 2 are purposefully oxidized. 10. Use in vitro of the receptors according to one of claims 1-4, - for qualitative and/or quantitative detection of rhinovirus, whereby the receptors can preferably be bound to a solid carrier; - for analytical characterization or purification of rhinovirus; - for the production of polyclonal and/or monoclonal antibodies. 11. Test kit for the determination of rhinovirus, characterized in that it contains receptors according to one of claims 1-4.