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A synthetic sialic acid, N -acetyl-9-thioacetamidoneuraminic acid (9-ThioAcNeu5Ac), is recognized by influenza C virus as a receptor determinant but—in contrast to the natural receptor determinant, N -acetyl-9- O -acetylneuraminic acid—is resistant to inactivation by the viral acetylesterase. This sialic acid analog was used to analyze the importance of the receptor-destroying enzyme of influenza C virus in keeping the viral surface free of receptor determinants. Enzymatic transfer of 9-ThioAcNeu5Ac to the surface of influenza C virions resulted in the loss of the hemagglutinating activity. The ability to agglutinate erythrocytes was restored when the synthetic sialic acid was released from the viral surface by neuraminidase treatment. Infectivity of influenza C virus containing surface-bound 9-ThioAcNeu5Ac was reduced about 20-fold. Sedimentation analysis as well as electron microscopy indicated that virions resialylated with the esterase-resistant sialic acid analog formed virus aggregates. These results indicate that the receptor-destroying enzyme of influenza C virus is required to avoid the presence of receptor determinants on the virion surface and thus to prevent aggregate formation and a reduction of the infectious titer.

Influenza C virus is able to inactivate its own cellular receptors by virtue of a sialate 9-O-acetylesterase that releases the acetyl residue at position C-9 of N-accetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). The receptor-destroying enzyme activity is a function of the surface glycoprotein HEF and this esterase belongs to the class of serine hydrolases. In their active site, these enzymes contain a catalytic triad made up of a serine, a histidine and an aspartic acid residue. Sequence comparison with other serine esterases has indicated that, in addition to serine-71 (S71), the amino acids histidine-368 or -369 (H368/369) and aspartic acid 261 (D261) are the most likely candidates to form the catalytic triad of the influenza C virus glycoprotein. By site-directed mutagenesis, mutants were generated in which alanine substituted for either of these amino acids. Using a phagemid expression vector, pSP1D-HEF the HEF gene was expressed in both COS 7 and MDCK I cells. The glycoprotein was obtained in a functional form only in the latter cells, as indicated by its transport to the cell surface and measurable enzyme activity. The low level of expression could be increased by stimulating the NF-κB-binding activity of the cytomegalovirus immediate-early promoter/enhancer element of the vector. The esterase activity of the mutant proteins was compared with that of the wild-type glycoprotein. With Neu5,9Ac2 as the substrate, the esterase specific activities of the S71/A mutant and the H368,369/A mutant were reduced by more than 90%. In the case of the D261/A mutant the specific activity was reduced by 64%. From this data we conclude that S71, H368/369 and D261 are likely to represent the catalytic triad of the influenza C virus glycoprotein HEF. In addition, N280 is proposed to stabilize the oxyanion of the presumptive transition state intermediate formed by the enzyme-substrate complex.

The S protein of bovine coronavirus (BCV) has been isolated from the viral membrane and purified by gradient centrifugation. Purified S protein was identified as a viral hemagglutinin. Inactivation of the cellular receptors by sialate 9-O-acetylesterase and generation of receptors by sialylation of erythrocytes with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicate that S protein recognizes 9-O-acetylated sialic acid as a receptor determinant as has been shown previously for intact virions. The second glycoprotein of BCV, HE, which has been thought previously to be responsible for the hemagglutinating activity of BCV, is a less efficient hemagglutinin; it agglutinates mouse and rat erythrocytes, but in contrast to S protein, it is unable to agglutinate chicken erythrocytes, which contain a lower level of Neu5,9Ac2 on their surface. S protein is proposed to be responsible for the primary attachment of virus to cell surface. S protein is proposed to be responsible for the primary attachement of virus to cell surface receptors. The potential of S protein as a probe for the detection of Neu5,9Ac2-containing glycoconjugates is demonstrated.

The receptors for the hemagglutinating encephalomyelitis virus (HEV, a porcine coronavirus) on chicken erythrocytes were analyzed and compared to the receptors for bovine coronavirus (BCV) and influenza C virus. Evidence was obtained that HEV requires the presence of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) on the cell surface for agglutination of erythrocytes as has been previously shown for BCV and influenza C virus: (i) Incubation of red blood cells with sialate 9-O-acetylesterase, the receptor-destroying enzyme of influenza C virus, rendered the erythrocytes resistant against agglutination by each of the three viruses; (ii) Human erythrocytes which are resistant to agglutination by HEV acquire receptors for HEV after resialylation with Neu5,9Ac2. Sialylation of red blood cells with limiting amounts of sialic acid indicated that strain JHB/1/66 of influenza C virus requires less Neu5,9Ac2 for agglutination of erythrocytes than the two coronaviruses, both of which were found to be similar in their reactivity with Neu5,9Ac2-containing receptors.

We describe a synthetic sialic acid analogue, 9-thioacetamido-N-acetylneuraminic acid (9-thioacetamido-Neu5Ac), which is recognized by the receptor-binding activity of influenza C virus, but is resistant to the receptor-destroying enzyme (acetylesterase) of this virus. Following transfer of the analogue to the surface of receptor-negative cells, influenza C virus is able to attach to these cells, but is unable to infect the cells. This result suggests that inactivation of virus receptors by the receptor-destroying enzyme is essential for initiation of infection. Because of their unique properties such analogues promise to be powerful chemotherapeutic agents.

The interaction of infectious bronchitis virus (IBV) with erythrocytes was analyzed. The binding activity of IBV was not sufficient to agglutinate chicken erythrocytes. However, it acquired hemagglutinating activity after treatment with neuraminidase to remove alpha 2,3-linked N-acetylneuraminic acid from the surface of the virion. Pretreatment of erythrocytes with neuraminidase rendered the cells resistant to agglutination by IBV. Susceptibility to agglutination was restored by resialylation of asialo-erythrocytes to contain alpha 2,3-linked sialic acid. These results indicate that IBV attaches to receptors on erythrocytes, the crucial determinant of which is sialic acid alpha 2,3-linked to galactose. In contrast to other enveloped viruses with such a binding specificity (influenza viruses and paramyxoviruses) IBV lacks a receptor-destroying enzyme.

Enteropathogenic transmissible gastroenteritis virus (TGEV), a porcine coronavirus, is able to agglutinate erythrocytes because of sialic acid binding activity. Competitive inhibitors that may mask the sialic acid binding activity can be inactivated by sialidase treatment of virions. Here, we show that TGEV virions with efficient hemagglutinating activity were also obtained when cells were treated with sialidase prior to infection. This method was used to analyze TGEV mutants for hemagglutinating activity. Recently, mutants with strongly reduced enteropathogenicity that have point mutations or a deletion of four amino acids within residues 145 to 155 of the S protein have been described. Here, we show that in addition to their reduced pathogenicity, these mutants also have lost hemagglutinating activity. These results connect sialic acid binding activity with the enteropathogenicity of TGEV.

Summary The receptor-binding activity of strain CU (grown in MDCK I cells) and of strain VA (adapted to Vero cells) of human coronavirus OC43 was analyzed and compared with the binding activity of bovine coronavirus (BCV) and of the OC43 strain provided by the American Type Culture Collection (AT). Results obtained with resialylated erythrocytes indicated that the ability of the viruses to recognize 9-O-acetylated sialic acid in an α2,6-linkage decreased in the following order: AT>CU>BCV>VA. Only minor differences were observed with respect to the α2,3-linkage. The amino acid sequence of the S protein of strain CU and VA was derived from the nucleotide sequence of the cloned gene. Strain VA differed from strain CU in 34 positions, 18 in the S1 and 16 in the S2 subunit.

A synthetic sialic acid analog, N-acetyl-9-acetamido-9-deoxy-neuraminic acid, can be used by influenza C virus as a receptor determinant for attachment to cells, In contrast to the natural determinant, N-acetyl-9-O-acetylneuraminic acid, the synthetic sialic acid is resistant to the action of the receptor-destroying acetylesterase of this virus. The sialic acid analog was enzymatically transferred to the surface of Madin-Darby canine kidney cells that are resistant to infection because of a lack of receptors. Influenza C virus was able to infect the modified cells though requiring a 10-fold larger amount of the sialic acid analogue on the cell surface compared to the natural receptor determinant. The quantitative difference is accounted for mainly by a less efficient binding of influenza C virus to the analog. Thus, in our system, inactivation of the receptor by the viral esterase is not required for the initiation of an influenza C virus infection.

Bovine coronavirus (BCV), human coronavirus OC43 (HCV-OC43) and hemagglutinating encephalomyelitis virus (HEV) are serologically related viruses that all have hemagglutinating activity. The receptor determinant for attachment to erythrocytes has been shown to be N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). We compared the ability of the three coronaviruses to recognize 9-O-acetylated sialic acid and found that they all bind to Neu5,9Ac2 attached to galactose in either A2,3 or A2,6-linkage. There are, however, some differences in the minimum amount of sialic acid that is required on the cell surface for agglutination by these viruses. Evidence is presented that HCV-OC43 uses Neu5,9Ac2 as a receptor determinant not only for agglutination of erythrocytes but also for attachment to and infection of a cultured cell line, MDCK I cells.

Porcine transmissible gastroenteritis virus (TGEV) has been shown to agglutinate erythrocytes using alpha 2,3-linked sialic acid on the cell surface as binding site. The hemagglutinating activity requires the pretreatment of virus with neuraminidase. We obtained evidence that TGEV recognizes not only N-acetylneuraminic acid but also N-glycoloylneuraminic acid, a sialic acid present on many porcine cells.

Two cellular proteins, membrane cofactor protein (MCP) and moesin, were reported recently to be functionally associated with the initiation of a measles virus infection. We have analyzed the interaction of measles virus with cell surface proteins, using an overlay binding assay with cellular proteins immobilized on nitrocellulose. Among surface-biotinylated proteins from a human rectal tumor cell line (HRT), measles virus was able to bind only to a 67-kDa protein that was identified as MCP. The virus recognized different isoforms of MCP expressed from human (HRT and HeLa) and simian (Vero) cell lines. The binding of measles virus to MCP was abolished after cleavage of the disulfide bonds by reducing agents as well as after enzymatic release of N-linked oligosaccharides. By contrast, removal of sialic acid or O-linked oligosaccharides did not affect the recognition of MCP measles virus. These data indicate that the receptor determinant of MCP is dependent on a conformation of the protein that is maintained by disulfide bonds and N-glycans present in the complement binding domains. Our results are consistent with a role of MCP as primary attachment site for measles virus in the initial stage of an infection. The functional relationship between MCP and moesin in a measles virus infection is discussed.

Bovine coronavirus (BCV) initiates infection by attachment to cell surface receptors the crucial component of which is N-acetyl-9-O-acetylneuraminic acid. Inactivation of receptors by neuraminidase treatment and restoration of receptors by enzymatic resialylation of asialo-cells is described as a method to determine (i) the type of sialic acid that is recognized; (ii) the linkage specificity of the viral binding activity; (iii) the minimal amount of sialic acid required for virus attachment. Evidence is presented that both glycoproteins and glycolipids can serve as receptors for BCV provided they contain 9-O-acetylated sialic acid. A model is introduced proposing that after initial binding to sialic acid-containing receptors, the S-protein of BCV interacts with a specific protein receptor. This interaction may result in a conformational change that exposes a fusogenic domain and thus induces the fusion between the viral and the cellular membrane.

The two surface glycoproteins S and HE of human coronavirus OC43 (HCV-OC43) were isolated from the viral membrane and purified. Only the S protein was able to agglutinate chicken erythrocytes, indicating that this viral protein is the major hemagglutinin of HCV-OC43. The receptor determinant recognized by this virus on the surface of erythrocytes is N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) which is also used by bovine coronavirus for attachment to cells. By analyzing erythrocytes containing different amounts of Neu5,9Ac2 in either of two linkage types, it was found that there are subtle differences in the affinity of both viruses for 9-O-acetylated sialic acid. Bovine coronavirus was more efficient in recognizing low amounts of Neu5,9Ac2 alpha 2,3 linked to galactose, whereas HCV-OC43 was superior with respect to the alpha 2,6 linkage. The gene coding for the S protein of HCV-OC43 was cloned and sequenced. A large open reading frame predicts a polypeptide of 150 kDa in the unglycosylated form. A protein of about 190 kDa is expected if the 20 potential glycosylation sites are used for attachment of N-linked oligosaccharide side chains. These predictions were confirmed by in vitro transcription and translation of the gene in the presence or absence of canine pancreatic microsomal membranes. A high degree of sequence homology was found between the S proteins of HCV-OC43 and bovine coronavirus. Structural and functional analyses of more strains should help to identify the location of the sialic acid-binding site.

Porcine transmissible gastroenteritis virus (TGEV) was found to resemble avian infectious bronchitis virus (IBV) in its interaction with erythrocytes. Inactivation of the receptors on erythrocytes by neuraminidase treatment and restoration of receptors by reattaching N-acetylneuraminic acid (Neu5Ac) to cell surface components indicated that alpha 2,3-linked Neu5Ac serves as a receptor determinant for TGEV as has been reported recently for IBV. Similar to IBV, the haemagglutinating activity of TGEV is evident only after pretreatment of virus with neuraminidase indicating that inhibitors on the virion surface have to be inactivated in order to induce the HA-activity of these viruses. A model is presented to explain why the HA-activity of untreated virus is masked and how neuraminidase treatment results in the unmasking of this activity.

The S protein of hemagglutinating encephalomyelitis virus is shown to be a hemagglutinin requiring N-acetyl-9-O-acetylneuraminic acid as a receptor determinant on the surface of erythrocytes. The ability of bovine coronavirus to recognize 9-O-acetylated sialic acid was used to establish a binding assay for the detection of glycoproteins containing this type of sugar. The assay is very fast, because it uses the acetylesterase of the viral HE protein to localize bound virus.

A relatively simple and sensitive method is described which enables the effect of monoclonal antibodies (MAbs) on the receptor-destroying enzyme (RDE) and the haemagglutination (HA) activity of bovine coronavirus (BCV) to be analysed in one assay. A lysate of HRT-18 cells infected with the L9 strain of BCV was found to have a higher RDE:HA ratio than purified virus. At 4 degrees C the lysate induced an HA pattern which completely disappeared upon raising of the temperature to 37 degrees C. This L9-infected cell lysate was used to determine the HA inhibition (HAI) titres of MAbs directed against the surface glycoproteins S and HE of BCV. Thereafter, the test plates were incubated at 37 degrees C to enable the ability of the MAbs to prevent elution of virus from BCV-erythrocyte complexes to be assessed. No inhibition of RDE was detectable with MAbs against glycoprotein S, which had HAI titres ranging from 1:16 to 1:128. On the other hand, MAbs directed against glycoprotein HE had similar HAI titres, but they inhibited elution of 8 HA units of BCV at titres of up to 1:65,000.

Influenza C virus and a group of coronaviruses, a typical representative of which is bovine coronavirus, use the same strategy for binding to cells. Surface components containing 9-O-acetylated sialic acid are recognized as cellular receptors by these viruses. In addition to the receptor determinant both virus groups have a receptor-destroying enzyme in common, which has been identified as a sialate 9-O-acetylesterase. Differences are, however, found in the distribution of these activities on the viral surface proteins. The influenza C glycoprotein HEF is a multifunctional protein, which is responsible for receptor-binding, receptor-inactivation and fusion. In the case of coronaviruses these activities are functions of two different glycoproteins, S and HE.

Influenza C virus uses 9-O-acetyl-N-acetylaneuraminic acid (9-O-acetyl-Neu5Ac) as a receptor determinant for attachment to cells. The virus contains an acetylesterase which releases acetyl residues from position C-9 of sialic acid thereby inactivating the receptors. A synthetic sialic acid analogue, 9-N-acetyl-Neu5Ac, was attached to cell surface glycoconjugates by purified sialyltransferase and analyzed for its ability to substitute the 9-O-acetylated sialic acid. Erythrocytes which have been modified to contain either 9-O-acetyl-Neu5Ac or 9-N-acetyl-Neu5Ac were agglutinated by influenza C virus to the same titer. However, in contrast to the 9-O-acetyl group the 9-N-acetyl residue is resistant to cleavage by the viral acetylesterase. This characteristic property (recognition as a receptor determinant by influenza C virus, but resistance against the action of the receptor-destroying enzyme) makes this synthetic analogue a valuable tool to analyze the role of the receptor-destroying enzyme for an influenza C virus infection.

N-Acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) has been shown to be a high-affinity receptor determinant for attachment of influenza C virus to erythrocytes (G. N. Rogers, G. Herrler, J. C. Paulson, and H-D. Klenk, 1986, J. Biol. Chem. 261, 5947-5951). In this report the nature of the cell surface receptor for influenza C virus on tissue culture cells was analyzed. Pretreatment with either neuraminidase or neuraminate 9-O-acetylesterase was found to render LLC-MK2 cells resistant to infection by influenza C virus as evidenced by the failure to detect virus release into the medium by hemagglutination titration. Susceptibility to infection was fully restored after incubation of neuraminidase-treated cells with bovine brain gangliosides known to contain Neu5,9Ac2. These results indicate that (i) Neu5,9Ac2 is the primary receptor determinant required for influenza C virus to attach to tissue culture cells and to initiate infection and (ii) gangliosides containing this type of sialic acid are potential receptors for influenza C virus. Several cell lines which are resistant to infection by this virus were able to release influenza C virus into the medium provided they were incubated with bovine brain gangliosides prior to virus infection. This result indicates that lack of appropriate receptors on the cell surface is a major reason for the restricted cell tropism of influenza C virus.

Identification of the receptor-destroying enzyme of influenza C virus as a specific neuraminate O-acetylesterase has suggested that 9-O-acetyl-N-acetylneuraminic acid is an essential component of the cell surface receptor of influenza C virus (Herrler, G., Rott, R., Klenk, H.-D., Muller, H.-P., Shukla, A. K., and Schauer, R. (1985) EMBO (Eur. Mol. Biol. Organ.) J. 4, 1503-1506). In this report, three common sialic acids, N-acetylneuraminic acid (NeuAc), N-glycollylneuraminic acid (NeuGc), and 9-O-acetyl-N-acetylneuraminic acid (9-O-Ac-NeuAc) were compared for their ability to mediate attachment of influenza A, B, and C viruses to cells. Human asialoerythrocytes were resialylated to contain the three sialic acids in defined sequence on glycoprotein carbohydrate groups using purified sialyltransferases and corresponding CMP-sialic acid donor substrates. While influenza C virus failed to agglutinate native cells or resialylated cells containing NeuAc and NeuGc, resialylated cells containing 9-O-Ac-NeuAc in three different sialyloligosaccharide sequences were agglutinated in high titer. In contrast, most representative influenza A and B viruses examined preferentially agglutinated cells containing NeuAc and NeuGc and failed to agglutinate cells containing 9-O-Ac-NeuAc. Cells containing 9-O-Ac-NeuAc were sensitive to the action of influenza C virus neuraminate O-acetylesterase which converts 9-O-Ac-NeuAc to NeuAc. This treatment abolished agglutination by influenza C while making the cells agglutinable by several influenza A and B viruses. Finally, the ability of influenza C virus to agglutinate the erythrocytes of various species correlated with the presence of 9-O-Ac-NeuAc. The results provide direct evidence that influenza C virus utilizes 9-O-acetyl-N-acetylneuraminic acid as the primary receptor determinant for attachment to cell surface receptors.

Bacterial neuraminidases destroy influenza C virus receptors of chick erythrocytes and inactivate hemagglutination inhibitors: rat alpha 1-macroglobulin (RMG) and bovine submaxillary mucin (BSM). These data indicate that neuraminic acid may be a component of influenza C virus receptor. The inhibiting activity of RMG and BSM is also eliminated by the receptor-destroying enzyme (RDE) of influenza C virus. After inactivation, the inhibitors (RMG and BSM) contain a reduced amount of N-acetyl-9-0-acetylneuraminic acid (Neu5, 9Ac2) and a larger amount of N-acetylneuraminic acid (Neu5 Ac). Transformation of Neu5, 9Ac2 into Neu5 Ac may also occur upon incubation of free neuraminic acid with influenza C virus. These data indicate that the RDE of influenza C virus is neuraminate-O-acetylesterase (N-acyl-9 4-O-acetylneuraminate O-acetylhydrolase (EC 3.1.1.53). It was shown that inhibition of influenza C virus hemagglutination by RMG and BSM and, apparently, adhesion of the virus to the cell surface involves binding of influenza C virus with Neu5, 9Ac2.

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.