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A Synthetic Sialic Acid Analogue Is Recognized by Influenza C Virus as a Receptor Determinant but Is Resistant to the Receptor-destroying Enzyme *

Synthetic sialic acid analogues varying in the substituents at position C-9 were analyzed for their ability to replace the natural receptor determinant for influenza C virus, N-acetyl-9-0-acetylneuraminic acid (Nev5,9Acz). By incubation of erythrocytes with sialyltransferase and the CMP-activated analogues, the cell surface was modified to contain sialic acid with one of the following C-9 substituents: an azido, an amino, an acetamido, or a hexanoylamido group. Among these, only 9-acetamido-N-acetylneuraminic acid (9-acetamido-Neu5Ac) was able to function as a receptor determinant for influenza C virus as indicated by the ability of the virus to agglutinate the modified red blood cells. In contrast to the natural receptors, 9acetamido-Neu5Ac-containing receptors were found to be resistant against the action of sialate 9-0-acetylesterase, the viral receptor-destroying enzyme. No difference in the hemolytic activity of influenza C virus was detected when analyzed with erythrocytes containing either Neu5,9Acz or 9-acetamido-Neu5Ac on their surface. This finding indicates that cleavage of the receptor is not required for the viral fusion activity. The sialic acid analogues should be useful for analyzing not only the importance of the receptor-destroying enzyme of influenza C virus, but also other biological processes involving sialic acid.

to require sialic acid as a crucial part of the cellular receptors for attachment to cell surfaces (4). Among these viruses influenzaviruses, paramyxoviruses, as well as some coronaviruses are unique because they contain a receptor-destroying enzyme. The enzyme of paramyxoviruses and influenza A and B viruses is a neuraminidase which releases terminal N-acetylneuraminic acid (Neu5Ac)' from glycoproteins and glycolipids, thereby abolishing the ability of these molecules to serve as virus receptors. Influenza C viruses inactivate their receptors by an acetylesterase which cleaves the 9-0-acetyl residue from 9-0-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) (5). The 9-0-acetyl group of sialic acid is crucial for the ability of a glycoconjugate to function as a receptor for influenza C virus (6, 7). Some members of the family coronaviridae have been shown recently also to contain an acetylesterase and to recognize Neu5,9Ac2 as a receptor determinant (8)(9)(10)(11)).
Influenza C virus is an RNA virus with a lipid envelope from which surface projections are protruding. These spikes are formed by a glycoprotein, which is designated HEF to indicate that it is responsible for three activities: hemagglutinating (receptor-binding), esterase (receptor-destroying), and fusion activity (12). The fusion is believed to be due to a hydrophobic amino acid sequence, which requires for its activation the proteolytic cleavage of the glycoprotein into the subunits HEFl and HEF2 and the exposure to low pH (13,14). The esterase has been shown to belong to the class of serine hydrolases, which are inactivated by diisopropylfluorophosphate (15). Using this inhibitor which binds covalently to the active site of serine hydrolases, amino acid 71 has been identified as the active-site serine (16,17). Inactivation of the esterase by diisopropylfluorophosphate or other inhibitors does not abolish the hemagglutination activity (15,17). From this finding it has been concluded that the active site of the enzyme and the receptor-binding site of HEF are located on different epitopes. We have analyzed several synthetic sialic acid analogues for their ability to function as receptor determinants for influenza C virus. After enzymatic transfer to surface glycoproteins, 9-acetamido-N-acetylneuraminic acid was able to mediate the binding of influenza C virus to erythrocytes. The synthetic receptor determinant was, however, resistant to the action of the viral acetylesterase. This compound is, therefore, ' The abbreviations used are: Neu5Ac, N-acetylneuraminic acid; useful for studying the importance of the receptor-destroying enzyme. The approach described here is also applicable to other biological systems and should provide a valuable tool to study the importance of sialic acids.

MATERIALS AND METHODS
Viruses-Strain Johannesburg/l/66 of influenza C virus was grown in embryonated chicken eggs as described previously (5).
Influenza A and B strains were grown as reported (18).
To determine the amount of sialic acid incorporated into surface glycoconjugates, trace amounts of radioactively labeled CMP-sialic acids were added to the samples. CMP-Neu5,9Ac2 was not available in radioactively labeled form. As shown in Table I, the kinetic data for the transfer of Neu5Ac and Neu5,9Ac2 by Galpl,4GlcNAc a2,6sialyltransferase are very similar. Therefore, tritiated CMP-Neu5Ac was used as a tracer to estimate the incorporation of 9-0-acetylated sialic acid. Following resialylation, cells were washed three times with PBS. Half of the sample was used for an HA-assay with influenza C virus; the other half of the cells were permeabilized by addition of saponin to a final concentration of 0.1%. After centrifugation for 2 min at 3000 X g, the sediment was suspended in PBS and the radioactivity was determined in a scintillation counter.
Esterase Treatment of Erythrocytes-Erythrocytes from 1-day-old chicken were modified to contain either 9-0-acetyl-N-acetylneuraminic acid or 9-acetamido-N-acetylneuraminic acid on their surface (see above). Aliquots containing 1 ml of a 1% erythrocyte suspension were incubated with purified acetylesterase from influenza C virus (12) at 37 "C. At the times indicated the cells were washed twice with PBS. The erythrocytes were suspended in PBS at a concentration of 1% and used to determine the HA-titer of allantoic fluid containing influenza C virus.
Hemagglutination Assay-The hemagglutination assays were performed in microtiter plates as described previously (20). The hemagglutinating activity (HA-units/ml) is expressed as the reciprocal value of the highest dilution causing complete agglutination of erythrocytes.
Hemolysis Assay-The ability of influenza C virus to cause hemolysis was determined with erythrocytes from 1-day-old chicken, which had been modified to contain either 9-0-acetyl-Neu5Ac or 9-acetamido-Neu5Ac. Aliquots containing 10 pl of packed cells were incubated with 200 pl of a virus suspension, which was obtained by diluting allantoic fluid from infected eggs with PBS. Prior to use the virus had undergone three cycles of freezing and thawing. After 15 min at 4 "C, 1 ml of cold PBS was added and the cells were sedimented. Following removal of unadsorbed virus, the erythrocytes were incubated at 37 "C in a buffer containing 0.125 M NaC1, 0.02 M MES, pH 5.2. After 30 min, the cells were sedimented and the optical density of the supernatant was determined at 530 nm.

Analysis of 9-Substituted Sialic Acids for Their Ability to Function as Receptor Determinants for Influenza C Virus-
From previous studies it has been known that the 9-0-acetyl residue of sialic acid is indispensible for the ability of a glycoconjugate to function as a receptor for influenza C virus (6,7). We wanted to address the question of whether this virus will also recognize receptors containing sialic acid with substituents other than an acetyl group at position C-9. For this purpose synthetic sialic acid analogues were analyzed for their ability to replace the natural receptor determinant Neu5,9Ac2. The analogues are all derivatives of N-acetylneuraminic acid with the hydroxyl group at position C-9 substituted by either of the following residues: an azido, an amino, an acetamido, or a hexanoylamido group (Fig. 1). In order to transfer the artificial sialic acids to cell surface glycoproteins, human asialoerythrocytes were incubated with GalP1, 4GlcNAc a2,6-sialyltransferase and either of the CMP-activated analogues. The modified red blood cells were used for hemagglutination assays (Table 11). As has been shown previously, human red blood cells are agglutinated by influenza C virus only after resialylation with 9-0-acetylated sialic acid (6). Of the erythrocytes containing either of the analogues on their surface, only those which had been resialylated with 9acetamido-Neu5Ac were agglutinated by influenza C virus (Table 11). The inability of the virus to agglutinate cells containing the other analogues is not due to a less efficient transfer of these sialic acids to the cell surface. As shown in Table I, the kinetic parameters for the transfer of the analogues onto glycoproteins are comparable, except for the 9amino compound. The erythrocytes containing 9-azido, 9amino, or 9-hexanoylamido-Neu5Ac were not reactive even if the concentration of CMP-glycoside was increased 10-fold over the minimal effective concentration determined for the 9-acetamido compound (see Fig. 2). None of the influenza A and B viruses tested was able to agglutinate red blood cells modified by any of the artificial sialic acids. This result indicates that the type of the substituent present at position C-9 of sialic acid is crucial for the recognition by influenza C virus. The 0-acetyl group can be replaced by an acetamido group but not by any of the other substituents. 9-Acetamido-Neu5Ac differs from Neu5,9Ac2 only in one respect. The acetyl group is attached to sialic acid via a nitrogen rather   than an oxygen atom (Fig. 1). T o indicate this similarity, in the following the analogue will be designated 9-N-Ac-Neu5Ac and Neu5,9Ac2 will be designated 9-0-Ac-Neu5Ac.
Affinity of Influenza C Virus for 9-N-Ac-NeuSAc-The ability of 9-N-Ac-Neu5Ac to serve as a receptor determinant was further analyzed by determining the minimal amount of the analogue required on the cell surface for agglutination by influenza C virus. For this purpose red blood cells from 1day-old chicken were used, which, in contrast to cells from adult chicken, are resistant to agglutination by influenza C virus because of a lack of 9-0-Ac-Neu5Ac (22). The erythrocytes were resialylated in the presence of various concentrations of CMP-activated sialic acids to obtain cells with different amounts of sialic acid on the surface. Using trace amounts of "-labeled CMP-sialic acids, the actual amount of 9-0-Ac-Neu5Ac and 9-N-Ac-Neu5Ac, respectively, transferred to the cell surface was quantitated. As shown in Fig. 2, the presence of about 5-10 pmol of 9-0-Ac-Neu5Ac on the surface of 5 pl of packed erythrocytes was sufficient to obtain HA titers of influenza C virus which were equal or close to the titers determined with erythrocytes from adult chicken. In the case of 9-N-Ac-Neu5Ac an amount of about 20-50 pmol was required. This result indicates that the sialic acid analogue is recognized less efficiently by influenza C virus than the natural receptor determinant with a 4-&fold difference in the minimal concentration of sialic acid on the cell surface necessary for agglutination. Esterase Resistance of 9-N-Ac-NeuSAc-Having found that 9-N-Ac-Neu5Ac can serve as a receptor determinant for the attachment of influenza C virus to cells we analyzed whether the sialic acid analogue is affected by the receptor-destroying enzyme of this virus. Purified sialic acids were analyzed by HPLC before and after incubation with influenza C virus.
The viral esterase efficiently released the 0-acetyl group of 9-0-Ac-Neu5Ac as indicated by the almost complete conversion of 9-0-Ac-Neu5Ac to Neu5Ac (Fig. 3, peaks B and C). On the other hand, 9-N-Ac-Neu5Ac was unaffected by the treatment with influenza C virus (Fig. 3, peak A). This result indicates that the acetylesterase of influenza C virus can release the acetyl group from C-9 of sialic acid, if it attached via an ester linkage, but not, if it is connected by an amide linkage. We tried to confirm this result in a biological assay. For this purpose erythrocytes from 1-day-old chicken were modified to contain either 9-0-Ac-Neu5Ac or 9-N-Ac-Neu5Ac, respectively, on their surface. Following sialylation, the cells were incubated with purified acetylesterase from influenza C virus. After various times the erythrocytes were analyzed for their agglutinability by influenza C virus. As shown in Fig. 4, an incubation for 2 h made the cells containing 9-0-Ac-Neu5Ac completely resistant against agglutination. Erythrocytes containing the 9-N-Ac-Neu5Ac analogue, on the other hand, were unaffected by an esterase treatment for 3 h. This result is in agreement with the HPLC analysis presented above (Fig. 3). Both the biochemical and the biological assay indicate that the receptor-destroying enzyme of influenza C virus can cleave the 9-0-acetyl group but not the 9-N-acetyl group.
Effect of 9-N-Ac-Neu5Ac on the Fusion Activity-The results presented above show that the receptor-binding and the receptor-destroying activity of influenza C virus respond to 9-N-Ac-Neu5Ac in a different manner. The sialic acid analogue is recognized as a receptor determinant for attachment to cells, but it is not a substrate for the acetylesterase. It was therefore of interest to know, whether or not the third activity of the influenza C glycoprotein HEF is affected by 9-N-Ac-Neu5Ac. The ability of HEF to induce the fusion of the viral membrane with cellular membranes at an acidic pH was determined in a hemolysis assay. Chicken red blood cells were modified to contain 9-0-Ac-Neu5Ac or 9-N-Ac-Neu5Aq re- spectively. The resialylation conditions were such that the minimum concentration of each of the activated sialic acids was used which is required for maximum hemagglutination titers. The erythrocytes were then analyzed to what extent they were lysed after incubation with influenza C virus at a low pH. As shown in Fig. 5, the release of hemoglobin was proportional to the amount of virus used. There was no significant difference between cells containing 9-0-Ac-Neu5Ac or the sialic acid analogue. This result indicates that the fusion activity of influenza C virus is not affected by 9-N-Ac-Neu5Ac and therefore does not depend on a cleavable receptor determinant.

DISCUSSION
Sialic acids play an important role in various biological processes. Sialic acid analogues promise to be valuable tools in analyzing the interactions between glycoconjugates involved in these processes (27)(28)(29)(30). We have used analogues with different substituents at the position C-9 of sialic acid to study the three activities of the influenza C glycoprotein HEF.
With respect to the natural receptor determinant, Neu5,9Ac2, it was previously not known whether the 9-0acetyl group itself is recognized by the receptor binding site of HEF or whether its function is the substitution of the free hydroxyl group at position C-9 of Neu5Ac to increase the hydrophobicity of the sialic acid molecule. Several Neu5Ac derivatives with different substituents at position C-9 were included in our analysis. The amino group introduces a positive charge; the acetamido group is similar to the naturally occurring substituent, the 9-0-acetyl group; the hexanoylam-id0 group is an example of a space-filling hydrophobic substituent; the azido group is a small-sized photolabile residue. None of these analogues was able to mimic a receptor for the influenza A and B viruses tested. In the case of influenza C virus, only that analogue was accepted as a receptor determinant, which resembled Neu5,9Ac2 most closely. It can be concluded, therefore, that the structure of the substituent is critical for the recognition of sialic acid by influenza C virus. It will be of future interest to analyze other analogues with substituents which closely resemble an acetyl group.
Several viruses have been reported to recognize sialic acid preferentially in a certain linkage type, e.g. a2,3-linked to galactose (23). The importance of the regioselective linkage for the recognition of a receptor determinant is also known in the case of influenza C virus. 9-0-Ac-Neu5Ac is a receptor determinant, whereas the isomeric sialic acid acetylated at position C-4 is not. The former type of sialic acid is present on rat al-macroglobulin, a potent inhibitor of the influenza C hemagglutinin (24, 25). The equine counterpart of this serum protein, which contains 4-O-Ac-Neu5Ac, has no inhibitory activity. Our results indicate that influenza C virus also has a preference for an ester linkage compared to an amide linkage. The difference is reflected in a 4-5-fold difference in the minimal concentration of surface-bound sialic acid required for agglutination of erythrocytes. However, if there is a sufficient amount of 9-N-Ac-Neu5Ac present on the cell surface, the virus attachment appears indistinguishable from the binding to receptors containing the natural receptor determinant. This is indicated not only by the HA titers obtained but also by the fact that the hemolysis by influenza C virus at low pH occurred to the same extent.
While 9-N-Ac-Neu5Ac is accepted as a receptor determinant by the receptor binding activity of HEF, it is not a suitable substrate for the acetylesterase activity of this glycoprotein. Both a biochemical (HPLC analysis) and a biological assay indicated that the 9-N-acetyl group is unaffected by the viral esterase, whereas the 9-0-acetyl residue is cleaved very efficiently. The acetylesterase of influenza C virus belongs to the class of serine hydrolases comprising both esterases and proteases (15). Although ester and amide linkages differ in their stability, both groups of enzymes are assumed to work by the same mechanism. The cleavage is accomplished by a charge relay system involving a catalytic triad which is composed of a serine, a histidine, and an aspartic acid (26). In the case of the influenza C glycoprotein, serine 71 has been shown to be the active-site serine (16,17). The histidine and the aspartic acid involved in the catalytic reaction have not yet been identified. At present we do not know whether the resistance of 9-N-Ac-Neu5Ac against the acetylesterase activity of HEF is related to the increased stability of an amide linkage compared to an ester linkage. The resistance might also, at least in part, be due to a reduced affinity for the analogue. In the case of the receptor-binding activity the decreased affinity may be compensated by an increased number of receptors resulting in an efficient attachment of the virus particle to a cell. The enzymatic cleavage of a chemical bond which involves one molecule each of enzyme and substrate may not allow for such a compensation.
The unique properties of 9-N-Ac-Neu5Ac allow to dissect the function of the receptor-binding and the receptor-destroying activity. Inactivation of the influenza C esterase by diisopropylfluorophosphate and isocoumarins has been reported to reduce the infectivity (15,17). As virus attachment to cells is not affected by the inhibitors, it has been proposed that the receptor-destroying enzyme is necessary for virus entry into cells. A step in the infectious cycle following the virus adsorption to the cell surface is the fusion between the viral and cellular membranes. Our results indicate that a noncleavable receptor determinant not only allows attachment to but also hemolysis of erythrocytes. Therefore, the fusion activity does not require the inactivation of the receptors. Either the esterase is involved in another step of virus entry or the inhibitors mentioned above have an indirect effect which is not related to the inactivation of the enzyme.
The results presented here show that 9-N-Ac-Neu5Ac is a valuable tool for analyzing the importance of the receptordestroying enzyme. Applying this approach to cultured cells should provide an answer to the question, whether cleavage of the receptor is required for influenza C virus to infect a cell. The sialic acid analogue will also be used to obtain information about the role of the receptor-destroying enzyme in virus maturation.