Evidence for a direct role for sialic acid in the attachment of encephalomyocarditis virus to human erythrocytes

Sialic acid residues are required in cellular receptors for many different mammalian viruses. Sialic acid could have a direct role, being an integral part of the virus binding site on the receptor. Alternatively, negatively charged sialic acid could have an indirect role, being responsible for holding the receptor in the required configuration for virus recognition, for instance, by interacting with positively charged amino acid residues found in the polypeptide chain of receptors. We have investigated the role of sialic acid in virus attachment by studying the interaction of the small RNA virus encephalomyocarditis (EMC) with glycophorin A, its receptor on human erythrocytes. In several experiments, influenza virus A was used for control purposes. Blocking positive charges on glycophorin either in lysine residues by acetylation or in arginine residues with butanedione did not affect its interaction with EMC virus. In contrast, blocking negatively charged carboxyl groups in sialic acid residues by amidation destroyed the ability of glycophorin to inhibit EMC virus attachment suggesting an important role for this part of sialic acid in EMC virus attachment. Removal of the polyhydroxy side chain in sialic acid residues of glycophorin by mild oxidation with periodate followed by reduction with borohydride had little effect on its interaction with EMC virus. Further, sialic acid species with either an acetyl or glycolyl group attached to the amino group on position 5 interacted equally well with EMC virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of the cell surface 70-kilodalton sialoglycoprotein(s) as a candidate receptor for encephalomyocarditis virus on human nucleated cells.

The attachment of encephalomyocarditis (EMC) virus to human nucleated cells susceptible to virus infection was examined with HeLa and K562 cell lines. Both cell types showed specific virus binding competitively blocked by unlabeled virions. The number of binding sites for EMC virus on HeLa and K562 cells were approximately 1.6 x 10(5) and 3.5 x 10(5) per cell, respectively, and dissociation binding constants were 1.1 and 2.7 nM, respectively. Treatment of cells with cycloheximide after pretreatment with trypsin eliminated EMC virus attachment, suggesting that the virus-binding moiety is proteinaceous in nature. Digestion of cells, cell membranes, and sodium deoxycholate-solubilized cell membranes with proteases or neuraminidases or treatment of cells with lectins demonstrated that the EMC virus-cell interaction is mediated by a sialoglycoprotein. Proteins with a molecular mass of 70 kDa were isolated from detergent-solubilized cell membranes of both HeLa and K562 cells by EMC virus affinity chromatography. The purified proteins, as well as their 70-kDa-molecular-mass equivalents detected in intact surface membranes of HeLa and K562 cells, specifically bound EMC virus in a virus overlay protein blot assay, whereas membranes from nonpermissive K562 D clone cells did not. Western immunoblot analysis with glycophorin A-specific antibody confirmed that the identified 70-kDa binding site on K562 cells is not glycophorin A, which is the EMC virus receptor molecule on virus-nonpermissive human erythrocytes (HeLa cells do not express glycophorin A). These results indicate that EMC virus attachment to permissive human cells is mediated by a cell surface sialoglycoprotein(s) with a molecular mass of 70 kDa.     

Site of attachment of encephalomyocarditis virus on human erythrocytes.

Previous studies of the attachment of encephalomyocarditis (EMC) virus to human erythrocytes concluded that the glycophorins, a family of human erythrocyte sialoglycoproteins, act as EMC virus receptors. Evidence is presented that the major glycophorin species, glycophorin A, is the receptor for EMC virus attachment to human erythrocytes. Comparison of the structures of glycophorins A and B and sialoglycopeptides released by chymotrypsin and trypsin treatment of erythrocytes confirmed our previous suggestion (A. T. H. Burness and I. U. Pardoe, J. Gen. Virol. 64:1137-1148, 1983) that attachment of EMC virus to glycophorin A involves the region containing amino acids 35 to approximately 70 (numbered from the NH2 terminus), four of which (amino acids 37, 44, 47, and 50) are glycosylated. In addition, we provide evidence that the segment containing amino acids 35 to 39 with an oligosaccharide side chain on threonine-37 is particularly important for EMC virus attachment.     

Early events of polyoma infection: adsorption, penetration and nuclear transport

Polyoma virions have different attachment proteins which are responsible for hemagglutination of erythrocytes and attachment to cultured mouse kidney cells (MKC). Virion binding studies demonstrated that MKC possess specific (productive infection) and nonspecific (nonproductive) receptors. Empty polyoma capsids have hemagglutination activity and bind to non-specific MKC receptors, but they are not capable of competing for specific virion cell receptors or preventing productive infection. Isoelectric focusing of the virion major capsid protein, VP1, separated this protein into six species (A through F). These species had identical amino acid sequences, but differed in degree of modification (phosphorylation, acetylation, sulfation and hydroxylation). Evidence based upon precipitation with specific antisera supports the view that VP1 species E is required for specific adsorption and that D and F are required for hemagglutination. The virion attachment domain has been localized to an 18 kilodalton fragment of the C-terminal region of VP1. Monopinocytotic vesicles containing 125I-labeled polyoma virions were isolated from infected MKC. A crosslinker was used to bind the MKC cell receptor(s) covalently to VP1 attachment protein, and a new 120 kilodalton band was identified by SDS-PAGE. An anti-idiotype antibody prepared against a neutralizing polyoma monoclonal antiody was used to identify a putative 50 kilodalton receptor protein from a detergent extract of MKC, as well as from MKC membrane preparation.     

Hemagglutination by Rabies Virus

Goose erythrocytes were agglutinated by five strains of rabies virus grown in monolayer cell cultures at pH 6.4 and at 0 to 4 C. Hemagglutination was not affected by the cell type in which the virus was grown. Prerequisites for occurrence of hemagglutination are absence of hemagglutination inhibitors (such as those contained in bovine serum) and a relatively high virus concentration (> 10(6) plaque-forming units of virus per ml). "Soluble" hemagglutinin was not present in crude preparations of extracellular virus. Treatment of purified preparations of extracellular virus with Tween 80 and ether did not result in release of a "soluble" hemagglutinin. The hemagglutinating property of extracellular virus seemed to be conditioned by the integrity of its coat. Preparations of infectious intracellular virus exhibited about 15 times lower hemagglutinating activity than extracellular virus. This decreased hemagglutinating activity did not seem to be caused by binding of hemagglutination inhibitors to the virus particles. Rabies virus can be quantitatively adsorbed onto and eluted from erythrocytes. Erythrocytes pretreated with rabies virus retained their ability to be agglutinated by the same virus strain. The reaction with rabies virus of erythrocytes treated with the receptor-destroying enzyme or KIO(4) was the same as that of nontreated erythrocytes. The hemagglutinating component of rabies virus, therefore, does not exhibit neuraminidase activity. Treatment of extracellular virus by various agents indicated that the hemagglutinating component consists of protein or lipoprotein. Sulfhydryl groups present in the viral hemagglutinin are essential for hemagglutination.     

Selection of influenza A virus adsorptive mutants by growth in the presence of a mixture of monoclonal antihemagglutinin antibodies.

The influenza virus hemagglutinin contains four major regions that are recognized by antibodies able to neutralize viral infectivity. To investigate the effect of an antibody response directed against each of these sites on viral evolution, influenza virus A/PR/8/34 (H1N1) was grown in allantois-on-shell cultures in the presence of a mixture of monoclonal antihemagglutinin antibodies. This selection mixture contained antibodies (two or three antibodies per antigenic site) whose concentrations were adjusted to achieve equal neutralization titers against each of the four antigenic sites. By varying the ratio of input virus to selection mixture concentration, we observed that variant viruses emerged under conditions of partial neutralization. Each of the four variants characterized in detail differed from the parental virus in its interaction with cellular receptors and exhibited minimal changes in antigenicity. Thus, these variants were virtually indistinguishable from wild-type viruses, as assessed by the binding of 103 monoclonal antihemagglutinin antibodies in an indirect radioimmunoassay. Despite this, many of the same antibodies demonstrated decreased titers to the variants in hemagglutination inhibition tests. The magnitude of the differences depended on the indicator erythrocytes used (much greater differences were detected with chicken erythrocytes than with human erythrocytes). Hemagglutination mediated by the variants was more resistant to neuraminidase treatment of erythrocytes than hemagglutination mediated by the parental virus. These findings are consistent with the idea that the variants were initially selected by virtue of their increased avidity for host cell receptors. Sequencing of viral RNA revealed that each of the variants differed from the parental virus by a single amino acid alteration in its HA1 subunit. Two of the changes were close to the proposed receptor binding site on hemagglutinin and could directly alter receptor binding, while a third was located near the trimer interface and may have increased receptor binding by altering monomer-monomer interactions.     

Properties of the erythrocyte receptors for influenza C virus.

Properties of the receptor for influenza C virus were studied. Although the receptor for influenza C virus on chicken erythrocytes was destroyed by the homologous virion, neuraminidase activity could not be detected in any of the influenza C virus strains tested. The receptor activity of chicken erythrocytes for influenza C virus was diminished by formaldehyde treatment but not by periodate oxidation. There was a considerable variation in the pattern and the titer of hemagglutination of influenza C virus when human erythrocytes of different blood types were used; the virus agglutinated most type B erythrocytes but not type A erythrocytes. By using human type B erythrocytes, differences among strains of influenza C virus in the hemagglutinating activity were also demonstrated. These results showed that both the receptor for and the receptor-destroying activity of influenza C virus were completely different from those of influenza A or B virus and also that carbohydrates were not involved in the receptor for influenza C virus.     

Capsid region involved in hepatitis A virus binding to glycophorin A of the erythrocyte membrane

Hepatitis A virus (HAV) has previously been reported to agglutinate human red blood cells at acidic pHs. Treatment of erythrocytes with different enzymes and chemical reagents indicated that HAV attachment is mediated through an interaction with sialylglycoproteins. HAV hemagglutination could be blocked by incubating the virus with glycophorin A, indicating that this sialylglycoprotein is the erythrocyte receptor. The number of receptors used was estimated to be around 500 per cell. At the same time, HAV-induced hemagglutination could also be blocked by either monoclonal antibody H7C27 or an anti-VP3(102-121) ascitic fluid, indicating that lysine 221 of VP1 and the surrounding VP3 residues lining the capsid pit are involved in HAV binding to erythrocytes.     

Artificial mutations and natural variations in the CD46 molecules from human and monkey cells define regions important for measles virus binding.

CD46 was previously shown to be a primate-specific receptor for the Edmonston strain of measles virus. This receptor consists of four short consensus regions (SCR1 to SCR4) which normally function in complement regulation. Measles virus has recently been shown to interact with SCR1 and SCR2. In this study, receptors on different types of monkey erythrocytes were employed as "natural mutant proteins" to further define the virus binding regions of CD46. Erythrocytes from African green monkeys and rhesus macaques hemagglutinate in the presence of measles virus, while baboon erythrocytes were the least efficient of the Old World monkey cells used in these assays. Subsequent studies demonstrated that the SCR2 domain of baboon CD46 contained an Arg-to-Gln mutation at amino acid position 103 which accounted for reduced hemagglutination activity. Surprisingly, none of the New World monkey erythrocytes hemagglutinated in the presence of virus. Sequencing of cDNAs derived from the lymphocytes of these New World monkeys and analysis of their erythrocytes with SCR1-specific polyclonal antibodies indicated that the SCR1 domain was deleted in these cells. Additional experiments, which used 35 different site-specific mutations inserted into CD46, were performed to complement the preceding studies. The effects of these artificial mutations were documented with a convenient binding assay using insect cells expressing the measles virus hemagglutinin. Mutations which mimicked the change found in baboon CD46 or another which deleted the SCR2 glycosylation site reduced binding substantially. Another mutation which altered GluArg to AlaAla at positions 58 and 59, totally abolished binding. Finally, the epitopes for two monoclonal antibodies which inhibit measles virus attachment were mapped to the same regions implicated by mutagenesis.     

Group C rotavirus requires sialic acid for erythrocyte and cell receptor binding.

Current immunological and biochemical information regarding the hemagglutinin and virus-cell interactions of rotavirus is obtained exclusively from studies with group A rotaviruses. In this study, I report that the immunologically and genetically distinct group C rotavirus also possesses a hemagglutinin. The viral hemagglutinin was identified on a cultivable porcine group C rotavirus strain (strain AmC-1) by using agglutinated human and guinea pig erythrocytes. Neuraminidase treatment of fresh human erythrocytes or blocking with glycophorin A or fetuin prevented hemagglutination. Infection of swine testicular cells with group C AmC-1 virus was also prevented by glycophorin A, fetuin, and neuraminidase treatment, suggesting that sialic acid constitutes an essential part of the cell receptor.     

Incorporation of sialoglycoprotein containing lacto-series oligosaccharides into chicken asialoerythrocyte membranes and restoration of receptor activity toward hemagglutinating virus of Japan (Sendai virus)

Bovine erythrocyte sialoglycoprotein (GP-2) (1) containing lactoseries oligosaccharide chains, which showed highly specific inhibition of hemagglutination by HVJ (Hemagglutinating virus of Japan, Sendai virus), was incorporated into neuraminidase-treated chicken erythrocytes which had lost their biological responsiveness to the virus. The GP-2-incorporated erythrocytes were agglutinated and lyzed again by the virus. Incorporation of 1,900 molecules of GP-2 per asialoerythrocyte restored fairly well the susceptibility of the cells to HVJ-mediated agglutination and hemolysis. Treatment of the erythrocytes with neuraminidase again resulted in the complete abolishment of the response to HVJ. The above observations are consistent with the view that exogenous sialoglycoprotein, GP-2, can be functionally integrated into the surface membrane of asialoerythrocytes and serve as the receptor for HVJ during the initial adsorption-fusion phase of the virus infection of the target cells.     

N-acetyl-9-O-acetylneuraminic acid, the receptor determinant for influenza C virus, is a differentiation marker on chicken erythrocytes

Erythrocytes from chicken of different age were analysed for their agglutinability by influenza C virus, which has been shown recently to use N-acetyl-9-O-acetylneuraminic acid as a high-affinity receptor determinant for the attachment to cells. Only with birds not younger than six days complete agglutination of the erythrocytes was observed. The hemagglutination titer which was initially low reached its maximum value at the age of about 20 days. Sialic acid was isolated from erythrocytes, purified and analysed by colorimetry, thin-layer chromatography, high-performance liquid chromatography, and gas-liquid chromatography-mass spectrometry. The sialic acid content of erythrocytes from one-day old and adult chicken was 21 micrograms and 18 micrograms sialic acid/ml packed erythrocytes, respectively. While N-acetylneuraminic acid was the major type of sialic acid on erythrocytes from both one-day old and adult chicken, N-acetyl-9-O-acetylneuraminic acid was only detected on red blood cells from adult animals accounting for 30-40% of total sialic acid. These results indicate that N-acetyl-9-O-acetylneuraminic acid, in addition to serving as a receptor determinant for influenza C virus, represents a developmental marker on chicken erythrocytes.     

Purification and properties of a Ca2+-independent sialic acid-binding lectin from human placenta with preferential affinity to O-acetylsialic acids

A Ca2+-independent sialic acid-specific lectin from two developmental stages of human placenta was similarly purified to apparent homogeneity by DEAE-cellulose chromatography, affinity chromatography on bovine submaxillary mucin, and gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration disclosed a molecular mass of 53 kDa. The specificity of the lectin for O-acetylsialic acids was substantiated by the dependence of hemagglutination on the presence of acetylated sialic acids on the surface of mammalian erythrocytes of various sources, by hapten inhibition in hemagglutination assays with protease-treated rabbit erythrocytes and by hapten inhibition of binding of labeled N-acetylneuraminic acid-bovine serum albumin to the lectin in a solid-phase assay. Bovine and equine submaxillary mucins that contain 9(7,8)-O-acetyl and 4-O-acetylsialic acids were potent inhibitors in contrast to the non-acetylated sialic acids of ovine submaxillary mucin. Absence of inhibitory efficiency of other negatively charged substances like phosphorylated sugars, glucuronic acid, heparin, or oligodeoxynucleotides emphasized the importance of structural features instead of simple ionic interaction. In the presence of acetylation, the pattern of inhibition by gangliosides in the solid-phase assay indicated a preference to alpha-2,8- or alpha-2,6-linked sialic acids in comparison to alpha-2,3-linked moieties. Chemical modification of the lectin by group-specific reagents allowed to emphasize the role of primarily lysine residues, but also, although less pronounced, arginine, tryptophan, and carboxyl groups for ligand binding and/or maintenance of the active conformational state. Application of reagents, specific for histidine or tyrosine residues, failed to affect lectin activity.     

Differentiation of Keratein from Bovine and Canine Hair by Passive Hemagglutination *)

The possibility of keratein species differentiation was examined using the passive hemagglutination test. To the knowledge of the author this approach has not previously been attempted. Keratein was obtained by solubilizing hairs cut from a Jersey cow and a cross-bred dog in disodium sulfide and urea (Goddard & Michaelis 1934). After precipitation with acetic acid the kerateines were redissolved in 0.1 N-NaOH and dialyzed for 48 hrs. against 0.1 M-Na2HPO4, pH 9.0. The nitrogen content was determined by micro Kjeldahl analysis and the keratein content calculated by multiplying the nitrogen figure with the factor 6.25. Antisera against the 2 kerateines were produced in adult rabbits. These were injected with approx. 5 mg keratein once a week for 3 weeks. A 5 mg booster dose was given 4 weeks after the third injection. The potency of the antisera was tested by immuno double diffusion in 1 % agar gel. Suitable sera were used for the passive hemagglutination test (Stavitsky 1954). Goat erythrocytes were coated with the 2 respective kerateines using approx. 0.1 mg keratein per ml of a 2.5 % erythrocyte suspension. After inactivation at 56°C for 30 min. and absorption with 2 volumes of packed goat erythrocytes the antisera were absorbed 3 times with equal volumes of the heterologous keratein containing approx. 0.5 mg protein per ml. Serial 2-fold dilutions of the respective antisera were prepared in 1 % normal rabbit serum in 0.85 % saline. The keratein coated erythrocytes were then suspended in the absorbed and diluted homologous and heterologous antisera. The tests were read after incubation at 20°C for 3 to 4 hrs. From the results listed in Table 1 it may be seen that the hemagglutination titers of the homologous systems are more than 100-fold above their heterologous counterparts.     

Phosphorylation and degradation of ribosomes in starved Tetrahymena pyriformis.

We have characterized an embryonic antigen on the surface of chick erythrocytes using immunochemical electron microscopy. An indirect surface labeling technique (hemocyanin conjugated to goat antirabbit IgG and specific antisera prepared in rabbits) revealed that the antigenic sites, at hatching, nearly saturate the surface of erythrocytes with hemocyanin markers. The number of antigenic sites gradually decreases with age, and the antigen can no longer be detected at 7 months. Further, the antigen has been detected on the very earliest primitive erythrocytes which form in the extra-embryonic mesenchyme before circulation begins. The embryonic antigen appears to be firmly associated with the erythrocyte surface and cannot be removed by extensive washing either with phosphate-buffered saline or with EDTA. Labeling unfixed cells at 37 °C produces clustering of the surface markers, suggesting that the antigen is associated with a membrane component which is fairly free to move in the plane of the membrane. In addition, the erythrocytes from newly hatched chicks were found to agglutinate more readily with several different lectins, particularly Concanavalin A (ConA), than did the erythrocytes from adults. Three times more ConA is bound to chick erythrocytes than to adult erythrocytes, as estimated by electron microscopy. Although this difference in lectin binding suggests that the ConA-binding sites might be related to the embryonic antigen, the sugars known to block lectin-induced hemagglutination had no blocking effect on antiserum-induced agglutination or on antibody binding, as visualized by the electron microscope technique. Also, ConA binding was not inhibited by treatment of the chick erythrocytes with the specific antiserum.     

Expression, purification, and properties of recombinant encephalomyocarditis virus RNA-dependent RNA polymerase.

Encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), which allowed easy purification of the fusion protein by affinity chromatography on immobilized glutathione. Inclusion of a thrombin cleavage site between the GST carrier and the viral enzyme facilitated the release of purified mature EMC virus RNA polymerase from the GST carrier by proteolysis with thrombin. The purified recombinant enzyme has a molecular mass of about 52 kDa and is recognized by polyclonal immune serum raised against a peptide sequence corresponding to the C-terminal region of the protein. The recombinant enzyme comigrates with immunoprecipitated EMC virus RNA polymerase from infected mouse L929 cell extracts when run in parallel lanes on a sodium dodecyl sulfate-polyacrylamide gel. The enzyme exhibits rifampin-resistant, poly(A)-dependent poly(U) polymerase activity and RNA polymerase activity, which are both oligo(U) dependent. Template-size products are synthesized in in vitro reactions with EMC virus genomic RNA or globin mRNA. The availability of recombinant EMC virus RNA polymerase in a purified form will allow biochemical analysis of its role in the replication of the virus as well as structure-function studies of this unique class of enzyme.     

Hemagglutinin activity of human plasma fibronectin.

Purified human plasma fibronectin at concentrations of about 30 microgram/ml was found to agglutinate trypsin-treated erythrocytes from certain species. The hemagglutination reaction was inhibited by specific antibodies to fibronectin, by relatively low concentrations of polyamines and by higher concentrations of basic amino acids and nonacetylated amino sugars. The divalent cations Ca2+ and Mg2+ and the chelating agent ethylenediaminetetraacetate did not affect the reaction. None of the neutral amino acids, neutral sugars or polyanions tested was inhibitory. The results imply that plasma fibronectin is capable of interacting with cell surfaces and support the idea of a similarity between cellular and plasma fibronectins.     

Isolation and characterization of receptor sialoglycoprotein for hemagglutinating virus of Japan (Sendai virus) from bovine erythrocyte membrane

Sialoglycoprotein which exhibits inhibitory activity for hemagglutination by Hemagglutinating Virus of Japan (HVJ, Sendai virus) was isolated from the membrane of bovine erythrocytes. Purification steps for this sialoglycoprotein included extraction with lithium diiodosalicylate, phenol partition, precipitation with ethanol, and chromatography on a phosphocellulose column and an SDS-Sepharose CL-4B column. Purified sialoglycoprotein (GP-2) has high specific activity for inhibiting the hemagglutination with HVJ, and a lesser activity for that with Newcastle disease virus, but it does not inhibit the hemagglutination by influenza A virus. Inhibitory activity of GP-2 on hemagglutination by HVJ is 2,500-fold higher than that of fetuin. Liposomes containing a 10,000-fold larger amount of ganglioside mixture of bovine erythrocytes and those containing a 5,000-fold larger amount of each ganglioside of bovine erythrocytes, N-glycolylneuraminosyl-lactosyl ceramide, sialosyllacto-N-neotetraosyl- and sialosyl-lacto-N-norhexaosyl ceramide, had no inhibitory activity toward hemagglutination with HVJ. GP-2 (mol. wt. 250 K daltons) behaved homogeneously in SDS-polyacrylamide gel electrophoresis. It contained 70% carbohydrate and 30% protein, by weight. N-Acetylgalactosamine, N-acetylglucosamine, galactose, sialic acid (N-glycolylneuraminic acid, 96%; N-acetylneuraminic acid, 4%) were identified as carbohydrate components, in molar ratios of 1.0:4.0:5.2:2.9. All the oligosaccharides of GP-2 appeared to be linked to polypeptide chains by alkali-labile O-glycosidic linkages. Sialidase treatment of GP-2 and conversion of sialic acid residue of the glycoprotein to C8 and C7 analogues resulted in the loss of the inhibitory activity on hemagglutination by HVJ. Oligosaccharides isolated by gel filtration after treatment of GP-2 with alkaline borohydride had also lost the ability to inhibit the hemagglutination by HVJ. The above results indicate that isolated sialoglycoprotein is the endogenous receptor in bovine erythrocyte membrane specific to HVJ, and the hydroxy group linked to the 9-carbon atom of sialic acid and probably also the hydrophobic protein moiety are important for the recognition of HVJ attachment.     

LECTIN-LIKE COMPOUNDS AND LECTIN RECEPTORS IN MARINE MICROALGAE: HEMAGGLUTINATION AND REACTIVITY WITH PURIFIED LECTINS1

We detected lectin-like compounds and lectin receptors in microalgae by hemagglutination, competitive inhibition with sugars, and reactivity with lectins isolated from other sources. Cell extracts from eight species of Dinophyceae and from one species each of Raphidophyceae and Bacillariophyceae exhibited hemagglutination toward trypsinized rabbit erythrocytes. In addition, the culture media of the dinoflagellate Alexandrium cohorticula and the raphidophyte Chattonella antiqua displayed similar hemagglutination. These activities were not inhibited by any monosaccharides or oligosaccharides tested but were inhibited by some specific glycoproteins. This suggests that the active factors were lectin-like compounds. Upon exposing intact, healthy cells of 12 species of Dinophyceae and one species each of Raphidophyceae, Cryptophyceae, Bacillariophyceae, and Chlorophyceae to lectins isolated from either macroalgae or terrestrial plants, most species were adversely affected. The negative effects included one or more of the following: impaired motility, disappearance of motility, agglutination, abnormal morphology, and cell rupture or lysis. Some species, even after freezing, thawing, and washing with saline solution, still agglutinated with macroalgal or terrestrial plant lectins. This study suggests that lectins and carbohydrate-containing lectin receptors may commonly occur on the cell surfaces of various species of microalgae.     

The rhesus rotavirus outer capsid protein VP4 functions as a hemagglutinin and is antigenically conserved when expressed by a baculovirus recombinant.

Rhesus rotavirus (RRV) gene 4 was cloned into lambda bacteriophage, inserted into a polyhedrin promoter shuttle plasmid, and expressed in Sf9 cells by a recombinant baculovirus. The baculovirus-expressed VP4 protein made up approximately 5% of the Spodoptera frugiperda-infected cell protein. Monoclonal antibodies that neutralize the virus bound to the expressed VP4 polypeptide, indicating that the expressed VP4 protein was antigenically indistinguishable from viral VP4. In addition, we have determined that the baculovirus-expressed VP4 protein bound to erythrocytes and functions as the RRV hemagglutinin. The endogenous hemagglutinating activity of the VP4 protein, like the virus, was inhibited by guinea pig antirotavirus hyperimmune serum and by VP4-specific neutralizing monoclonal antibodies. The human erythrocyte protein, glycophorin, also inhibited hemagglutination by RRV or the expressed VP4 protein and appears to be the rotavirus erythrocyte receptor. The baculovirus-expressed VP4 protein was conserved functionally and antigenically in the absence of other outer or inner capsid rotavirus components and represents a logical candidate for future immunological studies.