Assay for Evaluation of Rotavirus-Cell Interactions - Journal of Virology

0 downloads 0 Views 2MB Size Report
Oct 7, 1993 - Identification of an Enterocyte Ganglioside Fraction That. Mediates Group A Porcine Rotavirus Recognition. MARK D. ROLSMA, HOWARD B.
Vol. 68, No. 1

JOURNAL OF VIROLOGY, Jan. 1994, p. 258-268

0022-538X/94/$04.00+0 Copyright ©D 1994, American Society for Microbiology

Assay for Evaluation of Rotavirus-Cell Interactions: Identification of an Enterocyte Ganglioside Fraction That Mediates Group A Porcine Rotavirus Recognition MARK D. ROLSMA, HOWARD B. GELBERG, AND MARK S. KUHLENSCHMIDT* Department of Pathobiology, College of Veterinary Medicine, Veterinary Medicine Basic Science Building, University of Illinois, Urbana, Illinois 61801

Received 5 March 1993/Accepted 7 October 1993 A virus-host cell-binding assay was developed and used to investigate specific binding between group A porcine rotavirus and MA-104 cells or porcine enterocytes. A variety of glycoconjugates and cellular components were screened for their ability to block rotavirus binding to cells. During these experiments a crude ganglioside mixture was observed to specifically block rotavirus binding. On the basis of these results, enterocytes were harvested from susceptible piglets and a polar lipid fraction was isolated by solvent extraction and partitioning. Throughout subsequent purification of this fraction by Sephadex partition, ion-exchange, silicic acid, and thin-layer chromatography, blocking activity behaved as a monosialoganglioside (GMX) that displayed a thin-layer chromatographic mobility between those of GM2 and GM3. The blocking activity of GMX was inhibited by treatment with neuraminidase and ceramide glycanase but not by treatment with protease or heat (100°C). Further purification of GMX by high-pressure liquid chromatography resulted in the resolution of two monosialogangliosides, GMX and a band which comigrated with GM1 on thin-layer chromatography. These data suggest that a cell surface monosialoganglioside or family of monosialogangliosides may function as an in vivo relevant receptor for group A porcine rotavirus and that sialic acid is a required epitope for virus-binding activity.

52, 60) provide strong evidence to support the existence of specific rotavirus receptors. In fact, all of these studies implicate glycoconjugates as the putative receptors. It is uncertain, however, which glycoconjugate functions as the actual receptor in vivo for a given rotavirus serotype. Some of this confusion may be due to the use of a host animal model or cell type that is not homologous to the rotavirus serotype being studied. The type of assay used to measure the virus-host cell interaction may also influence the type of receptor detected. We have developed a whole-cell, virus-binding assay to directly test enterocyte extracts for their ability to compete for specific rotavirus binding to enterocytes or MA-104 cells in suspension. We describe here the use of this assay to identify and partially purify a porcine enterocyte ganglioside that may function as an in vivo relevant cell surface receptor for the homologous group A porcine rotavirus serotype. (The work by M. D. Rolsma was conducted in partial fulfillment of the requirements for a Ph.D. from the University of Illinois, Urbana.)

Group A rotaviruses are among the most important agents associated with severe diarrhea in the young of humans and other animals (6, 8, 9, 18, 24, 54). They are a major cause of morbidity in infants and young children in developed countries and of morbidity and mortality in those in developing countries. It is estimated that more than one million children die each year of human rotavirus infections (3, 5). Rotavirus infections are also of prime agricultural importance since they frequently cause serious neonatal diarrheal diseases of many animal species, most importantly neonatal and postweaning pigs and calves. Morbidity due to rotavirus infections in these species often reaches 80%, and mortality can be as high as 60% (6). Despite considerable research efforts in many laboratories, rotavirus diarrhea continues to be a serious and costly disease in both animals and humans and has recently become important in causing significant illness in immunocompromised people (38, 62) and debilitated neonates (35). Despite extensive efforts including the use of reassortants, attenuated live strains, and vector expression of VP4 and/or VP7, a vaccine which is protective across all rotavirus serotypes is not available. To establish a firm scientific basis for the ultimate prevention and control of rotavirus disease, a greater understanding of the molecular mechanisms of rotavirus host cell interaction is required. The earliest requisite step for productive viral infection is recognition and binding to villous tip enterocytes. The tissue and cell type specific tropisms displayed by rotaviruses is consistent with the hypothesis that a specific host cell surface receptor(s) mediates recognition. Recent reports (2, 21, 45, 47,

MATERIALS AND METHODS Materials. Unless stated otherwise, all reagents and chemicals were obtained from Sigma Chemical Co. and were of the highest purity available. Neisseria meningitidis group C capsular polysaccharide and colominic acid were gifts from Eric Vimr, University of Illinois. Neoglycoproteins were generously donated by Y. C. Lee, The Johns Hopkins University. Glycosaminoglycans were provided by Edward Conrad, Glycomed, Inc., Alameda, Calif., and Theresa Kuhlenschmidt, University of Illinois. Anti-VP7 monoclonal antibodies were kindly donated by David A. Benfield, South Dakota State University. All solvents were Burdick & Jackson high-pressure liquid chromatography (HPLC) grade (Baxter Healthcare Corp.). Thin-layer chromatography (TLC) and high-performance TLC (HPTLC)

* Corresponding author. Mailing address: Department of Pathobiology, College of Veterinary Medicine, Room 2843 VMBSB, University of Illinois, 2001 S. Lincoln Ave., Urbana, IL 61801. Phone: (217) 333-9039. Fax: (217) 333-4628.

258

VOL. 68, 1994

plates were E. Merck Kieselgel 60 plates without a fluorescent indicator (VWR Scientific). Virus propagation and purification. Roller bottles (Corning) containing a nearly confluent monolayer of MA-104 (embryonic African green monkey kidney) cells (M. A. Bioproducts) grown in Eagle's minimal essential medium (MEM; GIBCO) with 10% added calf serum (GIBCO; pH 7.3) were rinsed with MEM (without added serum; pH 7.3) and incubated for 3 h at 37°C in the same serum-free medium. Group A porcine rotavirus (OSU strain) was pretreated for 30 min at 37°C with 10 ,ug of crystallized trypsin per ml. The cells were incubated with rotavirus at a multiplicity of infection of 2 to 5 PFU per cell for 90 min at 37°C. The viral inoculum was aspirated, and the cells were rinsed once with MEM. The roller bottles were then incubated with MEM (15 ml per roller bottle, without added serum) containing 10 jLg of soybean trypsin inhibitor per ml at 37°C until more than 75% of the cell monolayer was destroyed (generally less than 24 h). Roller bottles were freeze-thawed after addition of 100 ,ug of aprotinin (21). Tissue culture fluid was extracted with equal volumes of trichlorotrifluoroethane in a Dounce tissue grinder and centrifuged in 50-ml polypropylene centrifuge tubes (Corning) at 1,750 x g for 10 min. The solvent was reextracted twice with equal volumes of MEM. The aqueous phases were combined and clarified by centrifugation in an SW28 rotor (Beckman) at 9,000 rpm for 30 min. Virus was then pelleted through a 20% sucrose cushion (with 100 mM CaCl2) in an SW28 rotor at 26,000 rpm for 2 h. The viral pellet was resuspended in virus buffer (50 mM Tris, 20 mM NaCl, 100 mM CaCl2 [pH 7.5]) containing sufficient CsCI2 (GIBCO-BRL) to achieve a homogeneous density of 1.37 g/cm3 and centrifuged for 30 h at 35,000 rpm in an SW55 rotor (Beckman). Light-scattering bands located at densities of 1.36 and 1.38 g/cm3, which corresponded to double-shelled (ds) and single-shelled (ss) virus particles, respectively, were removed and rebanded on individual CsCl2 gradients as described above. The individual virus bands were collected and dialyzed against virus buffer by using a continuous-flow microdialysis chamber (Bethesda Research Laboratories). The purity of virus bands was estimated by using negative-staining (ammonium molybdate) electron microscopy by direct count of the number of ds and ss virus particles present in a total of 100 particles. Electron-microscopic determination of the particle concentration was performed by comparing particle numbers with dilutions of 102-nm polystyrene beads (Seradyn) at known concentrations (27). Preparation of radiolabeled rotavirus. Borosilicate culture tubes (12 by 75 mm) were cleaned with ethanol, acetone, and chloroform and thoroughly dried in preparation for coating with 30 p.g of lodogen (Pierce) dissolved in 200 p.l of chloroform. The chloroform was evaporated with a gentle stream of nitrogen. The tubes were prepared immediately prior to use. Tris buffer (10 ,ul; 0.5 M [pH 7.5]) and 0.5 mCi of 1251 (Amersham) were mixed in an lodogen-coated tube. Purified ss or ds rotavirus (10 p.g) was added to the tube and incubated for 3 min at room temperature. The reaction mixture was removed and neutralized by addition of potassium iodide (200 ,ul of a 10-mg/ml solution) followed by desalting on a GF-5 Excellulose column (Pierce) pretreated with 1 ml of a 10mg/ml bovine serum albumin (BSA, gamma globulin free; Sigma) solution and equilibrated with virus buffer. The radioactivity of each 0.6-ml fraction was determined by using a gamma counter (Packard). The final specific radioactivity was typically 9 x 103 cpm/ng. Virus-containing fractions were pooled and adjusted to 5 p.g of virus protein per ml, and BSA

ROTAVIRUS-CELL INTERACTIONS

259

was added to a final concentration of 10 mg/ml. Radiolabeled virus was stored on melting ice. Virus-binding assay. A single-cell suspension of MA-104 cells was obtained by harvesting 150-cm- flasks of confluent MA-104 cells with 0.05% trypsin in 0.53 mM EDTA (GIBCO). Individual enterocytes were harvested from 4-week-old piglets by modifications of a previously described method (1). Briefly, after excised intestinal segments were rinsed with phosphatebuffered saline (PBS), the lumens were filled with digestion medium and incubated at 37°C for 30 min. Rafts of superficial epithelium were collected, and incubation was continued at 37°C with continuous stirring until enterocytes were individualized. MA-104 cells and enterocytes were chilled to 4°C, washed three times with MEM (without serum) by centrifugation at 200 x g, and adjusted to a concentration of 2 x 106 cells per ml. Cell numbers and viability were determined by hemacytometer counts and trypan blue exclusion. Subsequent steps of this assay were performed in a 4°C cold room. Cells in 2 ml of MEM (assay buffer) were mixed with 30 p.l (150 ng) of 125I-labeled ds or -ss rotavirus in corked borosilicate culture tubes (12 by 75 mm) and rotated end over end at 5 rpm. Aliquots (200 p.l) were removed at appropriate time intervals (1, 3, 5, 8, 12, 20, 30, and 45 min), immediately overlaid onto 0.5 ml of a silicone-mineral oil mixture (3:1 ratio of Dow Corning 550 and Sigma light mineral oil) in 1.5-ml microcentrifuge tubes, and centrifuged at 15,000 rpm (15,600 x g) for 45 s in an Eppendorf 5414 centrifuge. The microcentrifuge tubes were inverted, and the bottom of the tube (containing the cell pellet) was cut off. The radioactivity in the pellets and supernatants was enumerated in a gamma counter, and percent virus binding was defined as 100 x the pellet cpm divided by the sum of the supernatant and pellet cpm. Blocking assay. Additives evaluated for their ability to block virus binding were dissolved in 100 p.l of virus buffer and incubated with 10 p.l (50 ng) of '25I-labeled ds rotavirus in an ice bath for 30 min prior to the addition of 1 ml of MA-104 cells (2 x 106' cells per ml). The tubes containing the cell-virus mixture were rotated end over end at 5 rpm in a 4°C cold room for 30 min. Duplicate 200-p.l aliquots of the cell-virus mixture were processed as described above for the binding assay. Inhibitory activity was expressed as a percentage of shamtreated 125I-labeled ds rotavirus (100% binding) in control

incubations. In preliminary experiments designed to validate the blocking assay, 10-p.l (50-ng) aliquots of 125I-labeled ds rotavirus were treated with potential inhibitors or noninhibitors of

binding.

Aliquots of '5JI-labeled ds rotavirus were incubated in 100 p.l of 10 mM EDTA at 37°C for 30 min prior to the addition of MA-104 cells. Additional aliquots of '25I-labeled ds rotavirus

pL. of a 1:200 dilution of anti VP7 monoclonal antibody or 100 pL. of 1-mg/ml BSA for 30 min prior to the addition of MA-104 cells. In all experiments described below, we compared the inhibitory activity of various cell fractions or additives from data collected when all tested fractions were present in the same experiment. Extraction and purification of porcine enterocyte gangliosides. The small intestine from 4- to 7-week-old specificpathogen-free crossbred piglets was excised and thoroughly rinsed with ice-cold PBS. The intestine was opened, and the mucosa was gently scraped with a glass microscope slide. The mucosal scrapings were washed three times with PBS and stored at - 80°C pending extraction. Extraction of mucosal tissue, solvent partitioning of the extract, and purification of a crude ganglioside preparation were performed as previously described (51). The crude ganglioside preparation was initially purified by column partition chromatography by the method of

were incubated on ice with 100

260

J. VIROL.

ROLSMA ET AL.

Rouser as described by Kates (20). Briefly, the crude lipid fraction was loaded onto a Sephadex G-25 column and sequentially eluted with solvent 1 (chloroform, methanol, water [19:1:sat]), solvent 2 (chloroform, methanol, acetic acid, water [19:1:4:sat]), solvent 3 (chloroform, methanol, acetic acid, water [9:1:2.9:sat]), and solvent 4 (methanol, water [1:1]). All four fractions were tested for inhibitory activity. The active ganglioside-enriched fraction (fraction 2) was further purified by anion-exchange chromatography, using a previously described procedure (51) with the following modifications. DEAE-Sepharose was substituted for DEAE-Sepharosil, and gangliosides were batch eluted with pure methanol (DEAE fraction I) followed by solutions of 2, 10, 25, 50, and 100 mM KCl in methanol (DEAE fractions II to VI). The fraction possessing inhibitory activity (DEAE fraction II) was further separated from contaminating substances by silicic acid chromatography. A glass column (2.2 by 100 cm) was packed with 100 g of latrobeads (latron Biochemicals) and equilibrated with chloroform-methanol (95:5). Fraction II was loaded onto the column in chloroform-methanol (8:1) and subjected to batch elution with chloroform-methanol mixtures of 4:1, 3:1, 2:1, and 1:1. The ganglioside containing 3:1, 2:1, and 1:1 fractions were pooled, evaporated under a stream of nitrogen, and stored at 20°C until assayed. TLC, HPTLC, and preparative TLC. Samples were dissolved in chloroform-methanol-0.25% (wt/vol) CaCl2 in water (55: 45:10 for TLC [Merck no. 5626] and 62.5:30:6 for HPTLC [Merck no. 5641]). Appropriate volumes (generally 5 to 20 per lane) of samples were spotted in 0.5-cm streaks (separated by 1.0-cm blank lanes) onto either plate; this was followed by development in the appropriate solvent in a short-bed continuous-development chamber (Regis). Ganglioside bands were visualized by resorcinol spray (28). Preparative TLC was performed under the same conditions as for HPTLC except that the sample (a total of 75 to 100 plA per plate) was spotted in 0.5-cm lanes (separated by 0.5-cm blank lanes) across the entire width of the plate. After development, the first and last lanes of the plate, which were separated from the adjacent lanes by 1.5 cm to facilitate scoring, were cut and sprayed with resorcinol. These lanes were used as guides for dividing the unstained lanes into horizontal bands that extended across the entire width of the plate. Unstained bands were scraped from the plate, and the silica was exhaustively eluted with chloroform-methanol-water (30:60:20). Silica fines were removed by centrifugation and filtration. Following evaporation and resuspension in a volume of solvent equivalent to the original applied volume, appropriate aliquots were tested for inhibitory activity in the blocking assay and were rechromatographed on HPTLC plates. HPLC of gangliosides. HPLC of gangliosides was performed by a modification of a previously described method (14). A Perkin-Elmer Series 3 dual-pump HPLC apparatus was used to equilibrate a Shandon Hypersil column (5-,im-diameter silica; 4.6 by 250 mm) with chloroform-methanol-water (65:25: 3). Pooled latrobead fractions were dissolved in equilibrating solvent. After injection of 100-,ul aliquots, the flow rate was continued at 1 ml/min for 30 min. A linear gradient of chloroform-methanol-water which varied from 65:25:3 to 60: 35:8 was then run at a rate of 1 ml/min for 60 min. Fractions (4 ml) were collected, evaporated by a nitrogen stream, and redissolved in 100 plA of chloroform-methanol-water (55:45:10). Aliquots (20 pA) of each fraction were tested for inhibitory activity in the blocking assay and spotted onto a TLC plate. Following development, ganglioside-containing fractions were identified by resorcinol spray. Treatment of gangliosides. Aliquots of commercial or en-

terocyte gangliosides were treated with neuraminidase, periodate, ceramide glycanase, trypsin, or heat. (i) Neuraminidase. Gangliosides were dissolved in 100 ,u of buffer (100 mM phosphate-citrate [pH 4.4]) and treated with 100 mU of Clostridium perfringens neuraminidase (type X). After incubation at 37°C for 24 h the samples were neutralized to pH 7.5 with NaOH and boiled for 5 min. Sham-treated (no-neuraminidase) controls were processed simultaneously. Both were evaluated for activity in the blocking assay. (ii) Periodate. Gangliosides were dissolved in 100 ,lI of periodate reagent (50 mM sodium acetate, 10 mM sodium metaperiodate [pH 4.5]) and incubated on ice for 24 h in the dark. The periodate was inactivated by the addition of 20 [L of 1 M glucose in 50 mM sodium acetate. Control samples were incubated in periodate reagent under the same conditions; however, the glucose was added at the beginning of the incubation period. Treated and control samples were dialyzed against distilled water and evaluated for activity in the blocking assay. (iii) Ceramide glycanase. Gangliosides were dissolved in 100 RI of buffer (50 mM sodium acetate, 1.75 mM sodium cholate [pH 5.0]). After addition of 0.5 U of ceramide glycanase (V-Labs), the treated samples and untreated controls were incubated at 37°C for 24 h. The samples were neutralized with NaOH, boiled for 5 min, and tested for activity in the blocking assay. (iv) Trypsin. Gangliosides were dissolved in 100 RI of virus buffer containing 10 ,ug of crystallized trypsin and incubated for 24 h at 37°C. Control samples were identically incubated without trypsin. After incubation, all samples were boiled for 5 min and evaluated for activity in the blocking assay. (v) Heat. Gangliosides were dissolved in 100 [lI of virus buffer, boiled for 15 min, and tested for activity in the blocking assay. Neuraminidase treatment of MA-104 cells. MA-104 cells suspended in 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-150 mM NaCl-0.5 mM MgCl2-0.1 mM CaCl2 (pH 7.0) were treated for 1 h at 37°C with a mixture of 100 mU of C. perfringens (Sigma type X) and 20 mU of Arthrobacter ureafaciens (Boehringer Mannheim) neuraminidases. Following three washes with MEM, 1-ml aliquots of cells (2 x 106 cells per ml) were sham treated or treated for 1 h at 37°C with 50, 250, or 500 ,ug of mixed gangliosides. The cells were washed three times with MEM prior to evaluation in the standard binding assay. Sialic acid quantification. During the purification process, total sialic acid content was determined by modifications of an HPLC-thiobarbituric acid method (33). Following acid hydrolysis and development of the chromogen as originally described, 100-pA aliquots were injected and chromatographed through a C-18 reverse-phase column (4.6 by 250 mm; Alltech). The solvent (2 x buffer stock-methanol-water [5:3.5:2]) was run isocratically by using a Series 3 dual-pump HPLC apparatus set at a flow rate of 0.7 ml/min. Chromogen peaks were detected by an LC-65T variable-wavelength UV detector (PerkinElmer) set at 549 nm and integrated with a 3392A integrator

(Hewlett-Packard).

Determination of protein. Protein was determined colorimetrically by using a micro bicinchoninic acid assay (43) with BSA as the standard (Micro BCA; Pierce Chemical Co.). RESULTS Rotavirus binding to host cells in suspension requires ds virus particles. In a virus-cell-binding assay involving gently agitated (end over end) cell suspensions and radioiodinated

VOL. 68, 1994

ROTAVIRUS-CELL INTERACTIONS

261

TABLE 1. Validation of the virus-binding assay

v 45. A z 40. uE 35. °0 30- 0 °- 250 z 20.

3 0

Virus

ds ss ds ds ds

15.

m 10.

a.5 0 0

.... 'I'... ' I" " ""I"' 1 'I'

...

1 " "...I....II..1

5 10 15 20 25 30 35 40 4 5 TIME (MIN)

Q 4-c z

0 co

m

0L02 5 10 15 20 25 30 35 NUMBER OF CELLS (x 105)

30x

a z v 0 m cn Dr

25.

2015105; 5. 20 40 60 80 1001201401E VIRUS ADDED (gg)

FIG. 1. Virus binding assay and effect of virus and target cell concentrations on rotavirus binding to MA-104 cells. 251I-labeled ds rotavirus was added to MA-104 cells or enterocytes and incubated at 4°C with end-over-end rotation. Aliquots were analyzed for the amount of cell-bound radioactivity as described in Materials and Methods. (A) Kinetics of t25I-labeled ds rotavirus and 125 I-labeled ss rotavirus binding to MA-104 cells and porcine enterocytes. Solid symbols, ds rotavirus; open symbols, ss rotavirus; * and O, MA-104 cells; 0 and 0, enterocytes. (B) Effect of increasing the concentration of MA-104 cells. 125I-labeled ds rotavirus (15 ,lI, 75 ng) was added to 1 ml of MA-104 cells (2 x 105 to 4 x 10' cells per ml) and incubated under standard conditions for 30 min. (C) Effect of increasing the concentration of rotavirus. 125I-labeled ds rotavirus (10 ,lI, 50 ng) was mixed with 0 to 170 ,ug of unlabeled ds rotavirus, added to 1 ml of MA-104 cells (1 x 1t)' cells per ml), and incubated under standard conditions for 30 min.

virus particles (ds and ss), only complete, ds virus particles bound to MA-104 cells or enterocytes (Fig. IA). The ss particles displayed only background binding (less than 10% that of ds particles). When highly purified virus preparations (containing 80 to 95% ds particles) were used, as much as 50% of the input virus was bound by MA-104 cells in 30 min at 4°C. Preparations containing less than 80% ds particles were considered unusable for the virus-binding assay. A relatively rapid

Pretreatment"

% of ds rotavirus hound'

None None Anti-VP7 EDTA BSA

100 12.5 17.6 22 102

'>25I-labeled ds rotavirus was pretreated with the indicated additives and incubated with MA-104 cells, and virus binding was determined by using the blocking assay as described in Materials and Methods. Virus binding is expressed as a percentage of the control (untreated) ds rotavirus binding (100I%).

decline in the ability of a single preparation of 1215I-labeled ds rotavirus particles to bind to MA-104 cells was noted. Over a period of 120 h the binding of purified and freshly iodinated ds rotavirus decreased from 50% of input radioactivity to less than 5%, at which point it was indistinguishable from background or ss rotavirus binding. Electron-microscopic observation of 125I-rotavirus 120 h after radiolabeling indicated an increased proportion of ss and damaged particles in these preparations (data not shown). Labeled virus preparations were therefore used within 3 days of labeling. Quantitatively similar binding was observed when cells were harvested with EDTA (0.53 mM) alone or by physical dissociation (data not shown). Trypsin-EDTA was used for all subsequent binding assays because this dissociation method resulted in the highest cell viability and produced the most uniform cell suspension. Rotavirus binding is dependent on virus and host cell concentration. The amount of 125I-rotavirus binding was proportional to the target cell and virus concentration used in the incubation (Fig. lB and C). When a virus concentration of 75 ng/ml was used, a linear increase in binding over a relatively narrow range of target cell concentration (Fig. IB) was observed. At cell concentrations greater than 106/ml, virus binding began to plateau. This nonlinearity occurred following binding of greater than 30 to 35% of the input virus and most probably results from the limiting virus concentration used in these experiments. Virus binding to MA-104 cells increased linearly from 0 to 170 ,ug of virus protein per 10" cells (Fig. IC). Estimation of the number of ds particles per nanogram of viral protein, as described in Materials and Methods, indicated that 170 ,ug of virus protein corresponded to approximately 6.8 x 104 particles per cell (4 x 10 ds particles per ng of virus protein). Using this number of input particles resulted in 37.3 ,ug bound per 106' cells or 1.5 x 104 particles bound per cell

(Fig. IC).

In a separate experiment, total viral binding to MA-104 cells was determined by increasing the absolute quantity of 1251_ labeled ds rotavirus added and by dilution of the specific radioactivity of '25I-labeled ds rotavirus with unlabeled virus. Equivalent binding was observed by the same total nanogram amount of input rotavirus regardless of whether it was composed of a mixture of '25I-labeled ds plus unlabeled rotavirus or 125 I-labeled ds rotavirus alone (data not shown). Under the conditions of the assay we were unable to demonstrate saturation of host cell binding sites or compete with binding by addition of increasing quantities of unlabeled virus. Validation of the blocking assay. Table 1 shows the results of experiments designed to validate the blocking assay. The effect of selected treatments on virus binding was investigated in triplicate with several virus and MA-104 cell preparations. As observed during the kinetic assays, ss virus does not bind. Pretreatment of the '25I-labeled ds rotavirus with a monoclo-

262

J. VIROL.

ROLSMA ET AL.

Uo 0

4-

0 0 -

z D 0

m >'

cE

2

D (/lCI)«