Basis for the potent inhibition of influenza virus infection by equine and guinea pig alpha 2-macroglobulin.

The unique properties of equine and guinea pig sera which make them potent inhibitors of influenza virus adsorption and infection have been investigated. The inhibitory activities of both sera are found to reside entirely in their respective alpha 2-macroglobulins, high molecular weight glycoproteins which bind to viral hemagglutinins via sialic acids of their N-linked carbohydrate groups. Structure analysis has shown that both proteins contain 4-O-acetyl-N-acetylneuraminic acid (4-O-Ac-NeuAc) (Hanaoka, K., Pritchett, T. J., Takasaki, S., Kochibe, N., Sabesan, S., Paulson, J.C., and Kobata, A. (1989) J. Biol. Chem. 264, 9842-9849). These 4-O-acetylated sialic acids have been found in few species, making their coincidence with high inhibitory potency in equine and guinea pig alpha 2-macroglobulin striking. However, 4-O-Ac-NeuAc does not appear to increase the avidity of interaction with influenza virus since isolated oligosaccharides of equine alpha 2-macroglobulin are no more potent inhibitors of adsorption than isolated oligosaccharides of human alpha 2-macroglobulin, which is a relatively poor inhibitor and contains only NeuAc. Since 4-O-Ac-NeuAc is resistant to cleavage by viral sialidase it may serve to protect the inhibitor from inactivation. These and supporting results suggest that the key property of equine and guinea pig alpha 2-macroglobulin which make them high potency inhibitors is a spatial arrangement of sialic acid containing oligosaccharide groups which allows optimal interaction with multiple hemagglutinins. The implications of these results for the design of low molecular weight inhibitors of influenza virus infection are discussed.

Basis for the Potent Inhibition of Influenza Virus Infection by Equine and Guinea Pig a2-Macroglobulin* (Received for publication, November 21, 1988) Thomas J. PritchettS  The unique properties of equine and guinea pig sera which make them potent inhibitors of influenza virus adsorption and infection have been investigated. The inhibitory activities of both sera are found to reside entirely in their respective az-macroglobulins, high molecular weight glycoproteins which bind to viral hemagglutinins via sialic acids of their N-linked carbohydrate groups. Structure analysis has shown that both proteins contain 4-0-acetyl-N-acetylneuraminic acid (4-0-Ac-NeuAc) (Hanaoka, K., Pritchett, T . J., Takasaki, S., Kochibe, N., Sabesan, S., Paulson, J. C., and Kobata, A. (1989) J. Biol. Chern. 264,[9842][9843][9844][9845][9846][9847][9848][9849]. These 4-0-acetylated sialic acids have been found in few species, making their coincidence with high inhibitory potency in equine and guinea pig azmacroglobulin striking. However, 4-0-Ac-NeuAc does not appear to increase the avidity of interaction with influenza virus since isolated oligosaccharides of equine az-macroglobulin are no more potent inhibitors of adsorption than isolated oligosaccharides of human az-macroglobulin, which is a relatively poor inhibitor and contains only NeuAc. Since 4-0-Ac-NeuAc is resistant to cleavage by viral sialidase it may serve to protect the inhibitor from inactivation. These and supporting results suggest that the key property of equine and guinea pig az-macroglobulin which make them high potency inhibitors is a spatial arrangement of sialic acid containing oligosaccharide groups which allows optimal interaction with multiple hemagglutinins. The implications of these results for the design of low molecular weight inhibitors of influenza virus infection are discussed.
Sialic acid has been known as an essential receptor determinant of influenza viruses for over 40 years (reviewed in Refs. 1 and 2). Two viral envelope glycoproteins interact with sialic acid containing receptors. These are the hemagglutinin, which is the cell attachment protein, and the sialidase, which presumably aids in the elution of virus from the infected cell and in the destruction of sialic acid containing mucus glycoproteins that can act as receptor analogs and inhibit infection (1,2).
Through analysis of the three-dimensional structure of the hemagglutinin complexed with a receptor analog, sialyllactose, Weiss et al. (3) have recently shown that sialic acid fills the receptor binding pocket of the hemagglutinin. Yet influ-* This work was supported by United States Public Health Service Grant AI-16165. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisenent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. enza viruses are known to vary widely in their receptor binding properties (1,4,5). Indeed, influenza viruses differ in their recognition of different types of sialic acid, e.g. NeuAc,' etc. (6,8), in their ability to bind various sequences to which sialic acid is attached (9)(10)(11), and in their sensitivity to a variety of glycoprotein inhibitors of infection (12-15). The basis for such variation in receptor binding properties at the atomic level is at present largely unexplained (3).
Potent glycoprotein inhibitors of influenza virus infection were discovered in horse and guinea pig serum following the Asian influenza epidemic of 1957, when a new hemagglutinin of the H2 serotype was introduced into the human virus population (12)(13)(14). Although they were potent inhibitors of the new Asian flu viruses (H2N2), they were not inhibitors of the earlier influenza A (HlN1) or B viruses (14). Extensive investigation of the nature of the inhibitor in horse serum over the next 15 years revealed that the majority of the inhibitory activity was found in the a-macroglobulin fraction and was likely a2-macroglobulin (12,13,(16)(17)(18)(19). In addition, Levinson et al. (19) concluded that the inhibitor contained an unusual sialic acid, 4-O-Ac-NeuAc, which was responsible for its unique inhibitory properties. Purified equine az-macroglobulin has subsequently been shown to be a potent inhibitor of both hemagglutination and infection of influenza viruses containing the H2 and H3 hemagglutinins (15); the latter being introduced into the human virus population in 1968 as the Hong Kong strain (H3N2).
In view of the potential for designing low molecular weight inhibitors of influenza virus infection (3,20), we have sought to establish the unique properties of the glycoproteins of equine and guinea pig sera which make them potent inhibitors, while glycoproteins of other animal sera such as human and bovine are poor inhibitors. In this report, it is shown that the potent inhibitory activity of both equine and guinea pig serum reside entirely in their respective an-macroglobulins and that the low inhibitory activity of human, bovine, and chicken sera is also reflected in the low inhibitory potency of their purified an-macroglobulins.
In the accompanying report by Hanaoka et al. (21) which describes the structures of the N-linked carbohydrate groups of equine, guinea pig, and human az-macroglobulins, the single distinguishing feature of equine and guinea pig structures is the presence of 4-0-Ac-NeuAc as 30-50% of the total sialic acid, as reported earlier for the equine inhibitor (19). In contrast, human an-macroglobulin contains only NeuAc. The correspondence of 4-0-Ac-NeuAc in an-macroglobulins with The abbreviations used are: 4-O-Ac-NeuAc, 4-0-acetyl-N-acetylneuraminic acid; SDS, sodium dodecyl sulfate; aZM, a2-macroglobulins; PAGE, polyacrylamide gel electrophoresis; Sia, sialic acid; HAI, hemagglutination inhibition; SA, sialic acid. G. N. Rogers, T. J. Pritchett, and J. C. Paulson, unpublished work.

9850
high inhibitory potency is striking since this sialic acid has been found in few species (22) and has not previously been reported in guinea pig. However, as described here, 4-0-Ac-NeuAc does not appear to play a direct role in the high inhibitory potency of the equine and guinea pig az-macroglobulins. Indeed, despite the difference in the inhibitory potency of the equine and human az-macroglobulins, their isolated Nlinked carbohydrate groups have equal potency for inhibition of influenza virus adsorption to erythrocytes, as do free NeuAc and 4-0-Ac-NeuAc. In contrast, on a sialic acid basis, equine az-macroglobulin is a 4,000,000-fold more potent inhibitor than its free N-linked oligosaccharides.
The results suggest that the overriding factor in the inhibitory properties of animal az-macroglobulins is their high valency, coupled with a spatial arrangement which allows optimal interaction with multiple influenza virus hemagglutinins. The significance of the coincidental occurrence of the rare sialic acid 4-0-Ac-NeuAc on the as-macroglobulins of unusually high inhibitory potency may be related to its inhibition of the viral sialidase activity.

RESULTS
Choice of Sera-Our initial goal was to identify the basis for the potent inhibitory properties of several animal sera, by purifying several "active" glycoproteins and compare their structures with corresponding glycoproteins from "low potency" sera. Of animal sera examined to date, equine, guinea pig, and hedgehog sera have been found to exhibit the highest inhibitory potency for influenza virus adsorption to cells (12,14,23). T o determine the degree to which high inhibitory potency was unique in the animal kingdom, 47 sera from mammals, birds, a reptile, and fish were screened for the ability to inhibit hemagglutination by a human influenza virus, A/Memphis/102/72 (H3N2). While most sera surveyed possessed low inhibitory activity, potency varied widely within each order and family (Table I; see miniprint supplement). The highest inhibitory potency was found with sera from horse, guinea pig, East African bongo, and the fish Heterodontis Francisi, which gave HA1 titers of 1024-4096. These results indicate that potent HA1 activity is a property exhibited by the sera of relatively few species.
For further characterization equine and guinea pig sera were chosen as high potency sera because of their ready availability. Human serum was chosen as a low potency inhibitor for comparison. Equine and human sera have additional biological significance, since horses and humans are both natural hosts of influenza.
as-Macroglobulin Is the Only High Potency Inhibitor in Horse Plasma-Several investigations have reported that purified equine as-macroglobulin is a potent inhibitor of human influenza virus attachment to cells, an activity mediated by its sialic acid containing carbohydrate groups (15,19). Since serum glycoproteins may carry similar carbohydrate structures it was important to ascertain whether or not the inhibitory activity of equine sera was due to several glycoproteins or an-macroglobulin alone. Accordingly, the inhibitory activity of equine plasma was followed by inhibition of hemagglutination by A/Memphis/102/72, during purification of az-macroglobulin.
Portions of this paper (including "Experimental Procedures" and Tables I and V) are presented in miniprint at the end of this paper.
Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.
Purification was achieved with a two-step procedure involving Cibicron Blue Sepharose 6B chromatography (24) followed by zinc chelate affinity chromatography (25,26). Essentially all of the hemagglutination inhibition (HAI) activity ( Fig. 1, uertical bars) coeluted with az-macroglobulin during Cibacron Blue Sepharose chromatography, which removed the majority of plasma proteins (Fig. 1). With further purification of the az-macroglobulin by zinc chelate affinity chromatography, all the HA1 activity bound and eluted with asmacroglobulin (Fig. 2). This method resulted in an az-macroglobulin preparation in which 90-95% of the total protein migrated as a single band with an apparent molecular weight of 180,000 on SDS-PAGE under reducing conditions (Fig. 2, insert, lane 7) and which contained virtually all the HA1 activity ( Fig. 2, vertical bars). The results show that 0 2macroglobulin is the only potent glycoprotein inhibitor of influenza virus present in horse plasma.
Purification of Guinea Pig and Human as-Macroglobulin-Using essentially the same procedure, an-macroglobulin could also be purified from guinea pig serum and human plasma. Analysis of purified guinea pig az-macroglobulin by SDS-PAGE showed characteristic properties of az-macroglobulins purified from serum ( Fig. 3a) (27). Without reduction (lane 3) it appeared as two closely spaced major bands with relative molecular weights ( M J greater than that of myosin heavy chain. With reduction (lane 2), the majority of the protein appeared as two bands with M, of 180,000 and 166,000 corresponding, respectively, to the electrophoretically slow form (the only form seen when as-macroglobulin is isolated from plasma) and the electrophoretically fast form generated when az-macroglobulin reacts with active proteases released when blood is clotted to obtain serum (27). Several bands of lower M, are probably degradation products (27) since these bands were absent when the sample was run without reduction (lane 3 ) . In this regard, a similar preparation of equine az-macroglobulin was obtained when isolated from horse serum rather than horse plasma (not shown).
Purification of az-macroglobulin from human plasma yielded a preparation similar to that obtained from equine plasma. Analysis by SDS-PAGE under reducing conditions was performed essentially as previously described (52). A 4% (w/v) acrylamide stacking gel and a 7% resolving gel were used. Protein samples (10 pg) were heated at 100 "C for 3 min in sample buffer containing (final concentrations) 10% (v/v) glycerol and 2% (w/v) SDS (nonreduced) or glycerol and SDS plus 2.5% 2-mercaptoethanol (reduced) before being loaded onto the gel. The protein bands were visualized by staining with Coomassie Brilliant Blue R (Sigma). revealed that greater than 95% of the protein was in a single band with M , of 173,000 (Fig. 3B, lane I).
Inhibitory Activity of Purified cup-Macroglobulins-As observed in the purification of equine cup-macroglobulin, essentially all of the HA1 activity of guinea pig serum and most of the HA1 activity in human plasma copurified with a2-macroglobulin. This can be seen in Table I1 which compares the HA1 activity of plasma (or serum) with that of purified apmacroglobulin at 3 mg/ml, the reported concentration in serum (28). The activity of equine and guinea ap-macroglobulin accounted for the very high potency of their sera, giving titers of 4096 and 2048, respectively. Human cup-macroglobulin was much lower in potency, with an HA1 titer of 64. In all three cases, however, the inhibitory titer of the purified apmacroglobulin was equal to or greater than that obtained using the corresponding serum. The purified ap-macroglobulins from two other low potency sera, bovine and chicken, also exhibited very low inhibitory potency.

TABLE I1
Comparison of the inhibition of hemagglutination by serum and purified nZ-macroglobulins Hemagglutination inhibition (HAI) was performed as described under "Experimental Procedures." The initial concentration of equine, guinea pig, human, bovine, and chicken az-macroglobulin was adjusted to 3 mg/ml, approximately the same concentration as in unfractionated and serum (28). Neutralization of Infection by Purified ap-Macroglobulirzs-Potent serum inhibitors of influenza viruses have been reported to be potent inhibitors of infection (14,15). TO verify the ability of purified ap-macroglobulins to neutralize viral infectivity, influenza A/Memphis/102/72 (H3N2) was exposed to increasing concentrations of human, equine, and guinea pig ap-macroglobulin just prior to adsorption to Madin-Darby canine kidney cells. As shown in Fig. 4, equine and guinea pig ap-macroglobulins were powerful inhibitors of viral infection, causing 50% inhibition of plaque formation at concentrations of 0.04 and 0.18 pg/ml, respectively. Human azmacroglobulin was a much less potent inhibitor of infection, with 50% inhibition at 5.4 pg/ml, 30-120-fold greater than the equine and guinea pig ap-macroglobulins (Fig. 4).
Examination of the Role of 4-0-Acetyl-NeuAc in the Inhibitory Potencies of ap-Macroglobulins- Levinson et al. (19) provided early evidence that the inhibitory activity of equine ap-macroglobulin toward influenza isolates with the HZ hemagglutinin was dependent on the presence of 4-0-acetyl sialic acids. As described by Hanaoka et al. (21), both equine and guinea pig ap-macroglobulin contain 4-0-Ac-NeuAc as 30-50% of their total sialic acids, consistent with the possibility that 4-0-Ac-NeuAc is the common denominator in the potent inhibitory activity of the two proteins. To examine this point, the effects of periodate oxidation, mild base treatment, and digestion with Clostridium perfringens sialidase on the HA1 activities of equine, guinea pig, and human azmacroglobulin were monitored. The point of attack of each treatment on the structure of 4-0-Ac-NeuAc is illustrated in Fig. 5, and the results are summarized in Table 111.
Very mild conditions of periodate oxidation selectively cleave adjacent hydroxyl groups of the polyhydroxy side chain of sialic acid with the loss of one or two carbons (29-31). The complete abolition of HA1 by periodate suggest that the inhibitory activity is dependent upon sialic acid and that the polyhydroxy side chain is unsubstituted at the 8 and 9 positions (22). Subsequent reduction with sodium borohydride (29) restored inhibitory activity to approximately 20% of its original value, providing evidence that the hemagglutinin can bind to sialic acid with a shortened side chain terminated

TABLE 111
Effects of chemical treatments and bacterial sialidase digestion on the hemagglutination inhibition (HAI) potency of purified a,-macroglobulins The effect of each treatment on the structure of NeuAc glycosides is illustrated in Fig. 5. Hemagglutination inhibition assays were performed as detailed under "Experimental Procedures." a,-Macroglobulins were adjusted to an initial concentration of 3 mg/ml prior to assay except for NaBHr-treated samples (1 mg/ml). In this case actual titers were multiplied times three for comparison. with a hydroxyl group, but not with a C-7(C8) aldehyde group. The HA1 activities of equine and guinea pig as-macroglobulin were essentially stable to sialidase digestion, showing only a %fold drop in each case (Table  111). Colorimetric analysis revealed that 30 and 50% of total sialic acids remained glycosidically bound to equine and guinea pig apmacroglobulin, respectively, consistent with the presence of sialidase-resistant 4-0-acetyl-NeuAc (32) on the N-linked carbohydrate groups of these proteins (21). However, if these two glycoproteins were first treated with mild base, which removes 0-acetyl groups ( Fig. 5; 32), the inhibitory activity was reduced only 4-8-fold, in repeated experiments (see Table  111). Subsequent sialidase treatment quantitatively removed sialic acids and completely abolished the inhibitory activity of both the equine and guinea pig a2-macroglobulins.
Taken together these results show that sialic acids mediate the interaction of equine and guinea pig an-macroglobulin with influenza virus. While their 4-0-Ac-NeuAc content alone is sufficient to account for their potent inhibitory activities (see activity after sialidase treatment, Table 111), the two glycoproteins remain 16-32-fold more potent inhibitors than human ap-macroglobulin following removal of 0-acetyl groups (see base treatment, Table 111).
In contrast to the equine and guinea pig proteins, the low inhibitory activity of human a2-macroglobulin, which contains only NeuAc, was abolished by sialidase digestion prior to base treatment (Table III), and colorimetric analysis revealed that approximately 99% of the total sialic acids of human an-macroglobulin had been removed.  Table I11 suggested that the equine and guinea pig az-macroglobulins retained most of their inhibitory potency after 4-0-acetyl groups were removed. The absence of GalNac in the sugar composition of these proteins excludes sialic acid containing 0-linked carbohydrate groups as a source of the inhibitory p~t e n c y .~ To ascertain directly whether or not the N-linked carbohydrate groups of equine and human a,-macroglobulins differed in their avidity for the H3 hemagglutinin, free carbohydrate groups were isolated following N-glycanase digestion. These were then compared for inhibition of viral adsorption using an assay developed for low molecular weight sialosides (20). As shown in Fig. 6, the isolated oligosaccharides of human az-macroglobulin were actually slightly more potent than those of equine az-macroglobulin (Fig. 6), despite the fact that the equine az-macroglobulin oligosaccharides retained the majority of their 4-0acetyl groups during the release and isolation process, as determined by sialidase resistance (not shown). Using the concentration required for 50% inhibition as a basis of comparison, the simplest possible sialoside, a-methyl-NeuAc was comparable in inhibitory potency (Table IV). Moreover, the inhibitory potencies of free NeuAc and 4-0-Ac-NeuAc were essentially equal (only 5% the active a-anomer; 20). Taken together, these results shown that neither the 4-0-Ac-NeuAc substituent, or any other structural feature of the N-linked carbohydrate groups account for the high inhibitory potency of equine az-macroglobulin.

az-Macroglobulin Inhib
Also shown in Table IV for contrast are the inhibitory activities of the intact human and equine az-macroglobulins. These were 4,000-4,000,000 times more potent inhibitors, respectively, than were the oligosaccharides of these proteins (Table IV). The result emphasizes the importance of structural features contributed by polypeptide in the inhibitory properties such as valency, size, and the arrangement of the carbohydrate groups on the polypeptide backbone.
Comparative Inhibitory Potencies of a Variety of Glycoproteins with Known Carbohydrate Structures-Results pre-No 0-linked carbohydrate groups are present on equine, human, or guinea pig a2M as judged by the lack of N-acetylgalactosamine during amino sugar analysis (T. Pritchett and J. Paulson, unpublished data). Each glycoprotein, oligosaccharide, or free sialic acid was examined for its ability to inhibit A/Memphis/102/72 adsorption to resialylated erythrocytes modified to contain 18 nmol/ml packed cells NeuAc in the NeuAca2,6Gal linkage as described under "Experimental Procedures." * Inhibitory potency is expressed relative to a-methyl-NeuAc. e Isolation of N-linked of oligosaccharides az-macroglobulin is de-NeuAc was from Sigma and 4-0-acetyl-NeuAc was isolated by H. scribed under "Experimental Procedures."

litors of Influenza Virus
Higa as described previously (7). e Native glycoprotein.
sented thus far support the conclusion that the high inhibitory potencies of equine and guinea pig cYz-macroglobulin are not mediated by a unique feature of the carbohydrate moieties of these glycoproteins. Further evidence that inhibitory potency does not necessarily correlate with carbohydrate structure can be seen in a comparison of the relative abilities of several glycoproteins with known terminal carbohydrate sequences to inhibit hemagglutination by A/Memphis/102/72 as summarized in Table V (miniprint supplement).
Based on previous studies, A/Memphis/102/72 exhibits preferential binding to sialosides with the terminal SAa2,6Gal linkage (10). Yet, glycoproteins which contain the terminal sequence SAa2,6Gal/31,4GlcNAc on N-linked oligosaccharides, differ in by 33,000-fold in their inhibitory potency. Molecular size may be an important factor, since among glycoproteins with similar carbohydrate groups, those with larger size appear to have higher inhibitory potency. However, there are notable exceptions. Human and equine az-macroglobulin have similar size, but as already demonstrated differ 40-100-fold in inhibitory potency. a2-Acid glycoprotein and fetuin are also similar in size but differ 75-fold in inhibitory potency. This difference cannot be explained by the additional 0-linked carbohydrate groups of fetuin, since sialylated antifreeze glycoprotein derivatives with the identical sequences exhibited negligible inhibition a t 100-fold higher concentrations.
Relative Rates of Inactivation of Equine a2-Macroglobulin and De-0-Acetylated Equine a2-Macroglobulin by Viral Sialidase-The presence of 4-0-Ac-NeuAc in both equine and guinea pig a,-macroglobulin raises the question of its possible biological relevance. The 4-0-acetyl group is well known to confer resistance to digestion by a variety of bacterial and viral sialidases (19,22). To determine if this was the case for a recent N2 sialidase, A/Memphis/102/72 (H3N2) was assessed for its ability to inactivate the inhibitory activity of equine a,-macroglobulin with and without 4-0-A-NeuAc (native and base-treated, respectively; Table IV). Native and base treated equine a2-macroglobulin were incubated with concentrated virus and their inhibitory activities were monitored over a 24-h period, as shown in Fig. 7. The inhibitory activity of the base-treated glycoprotein was rapidly inactivated, with a half-time of inactivation of approximately 10- Reaction mixtures (200 pl) containing a*-macroglobulin (0.4 mg) and virus (6 units of sialidase activity) were incubated at 37 "C and pH 6.5. At the indicated times, aliquots (20 pl) were withdrawn, sialidase activity was inhibited by the addition of 110 mM 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, and incubation was continued for 60 min at pH 5.0 to inactivate viral hemagglutination activity (53). Following this, each aliquot was adjusted to a volume of 25 rl (pH 7.4) and tested for the ability to inhibit hemagglutination by 4 hemagglutination units of A/Memphis/ 102/72. Hemagglutination inhibitor titer is expressed as the reciprocal of the highest dilution of test substance causing inhibition of native human erythrocyte agglutination.
15 min. Inhibitor with intact 4-0-acetyl sialic acids was about 10-fold more resistant to inactivation, with a half-time of inactivation on the order of 2 h. Thus, the presence of 4-0acetyl groups on the sialic acids of an-macroglobulin appear to render its inhibitory activity resistant to inactivation by viral sialidase, as noted previously with the human H2N2 virus by Levinson et al. (19).

DISCUSSION
The high inhibitory potency exhibited by equine and guinea pig sera is found in relatively few species and can be accounted for entirely by a single glycoprotein, a2-macroglobulin. The inhibition of influenza virus adsorption to cells is mediated through the binding of the hemagglutinin to terminal sialic acids on N-linked, carbohydrate groups (Table III). 4 However, there appears to be no unique feature of the carbohydrate structures of equine and guinea pig an-macroglobulins which can account for their potent inhibitory properties. Although both of these glycoproteins contain 4-0-Ac-NeuAc as 30-50% of the total sialic acids, the 4-0-acetyl substitutions do not appear to be involved in binding affinity for the following reasons. 1) The free sialic acids, NeuAc and 4-0-Ac-NeuAc have approximately equal inhibition of influenza virus adsorption to erythrocytes. 2) Isolated N-linked carbohydrate groups from equine and human an-macroglobulin, with and without 4-O-Ac-NeuAc, respectively, also have equal inhibitory potencies and are indistinguishable from the simple sialoside a-methyl-NeuAc. 3) Crystal structure localization of sialic acid in the receptor binding pocket of the H3 hemagglutinin by Weiss et al. (3) shows that the 4-OH projects out of the pocket into solvent. Thus, there is no possibility for an 0-acetyl substitution at that position to influence the binding interaction with the hemagglutinin.
The fact that equine an-macroglobulin exhibits 4,000,000fold higher inhibitory potency than the free N-linked oligosaccharides indicates the important role of the polypeptide chain. The importance of valency has been noted previously by Gottschalk et al. (16) and Fazekas de St. Groth and Gottschalk (33) and is supported by studies showing dramatic increases in inhibitory potency of glycoproteins following chemical cross-linking or aggregation (34, 35). However, human and equine az-macroglobulins have similar size and number of sialic acids per mol, yet differ 50-100-fold in inhibitory potency. Thus, it appears that the major difference between these two glycoproteins reflects a more favorable distibution of the N-linked carbohydrate groups on the surface of the equine protein, allowing for a higher valency interaction with the hemagglutinins of the virus.
Although Pepper (17) and Levinson et al. (19) concluded that 4-0-Ac-NeuAc was required for the inhibitory activity of equine an-macroglobulin against influenza H2 viruses, their data are largely consistent with the results reported here. Their conclusion was based on the sialidase resistance of the inhibitory activity and a 30-fold drop in inhibitory potency following mild base treatment. The results in Table I11 are similar except a smaller decrease in HA1 was observed following base treatment in this report (4-%fold). This drop is likely due to a base-mediated alteration in the conformation of the protein that disrupts the complementarity between the glycoprotein carbohydrate groups and the viral hemagglutinins.
The presence of a*-macroglobulins in pleural fluids (36) and their active secretion by alveolar macrophages (37) make them candidates as physiologically relevant inhibitors. In this regard, all equine H3 isolates examined to date  are 100-1000-fold less sensitive to inhibition of hemagglutination by horse serum than human influenza viruses (10,15).5 It is well documented that equine an-macroglobulins can mediate the selection of inhibitor resistant receptor variants from inhibitor sensitive H2 and H3 human influenza isolates (10,12,13,15). Thus equine an-macroglobulin or some other glycoprotein inhibitor could account for the selection and maintenance of the inhibitor insensitive phenotype of equine H3 influenza viruses.
The coincidence of finding 4-0-Ac-NeuAc in the two anmacroglobulins that exhibit high inhibitory potency is striking. Prior to the report of Hanaoka et al. (21) which demonstrates 4-0-Ac-NeuAc in guinea pig az-macroglobulin, this sialic acid had been found only in horses, donkeys, and the echidna, a monotreme (22). A report by Shortridge and Landsdell (23) suggests that this coincidence extends to inhibitors of hedgehog serum. Indeed, the inhibitor potency of hedgehog serum is similarly resistant to digestion by bacterial sialidase and destroyed by mild periodate treatment, hallmarks for the presence of 4-0-Ac-NeuAc (32). Although the 4-0-acetyl substituent does not influence the avidity of the interaction with the hemagglutinin, it is well known to inhibit bacterial and viral sialidase activity (22; Fig. 7). Thus, the 4-0-acetyl substitution would protect these an-macroglobulins from inactivation by sialidase during infection. Because virulent infections are capable of killing large percentages of the populations of some species (38,39), it is possible that glycoproteins with high inhibitory potency and 4-0-acetyl substituents to protect against inactivation could have arisen by coevolution as a protection against virulent influenza viruses.
With increased understanding of the receptor specificities of influenza viruses and the recent localization of sialic acid in the receptor binding pocket of the hemagglutinin crystal structure, attention has been focussed on the possibility for design of low molecular weight receptor analogs as inhibitors of infection by influenza virus (3,20). We begun this work to identify structural features of the most potent natural inhib-itors which might be useful in the design of such analogs. The results reveal that the single most important factor is not a unique structural feature of the carbohydrate sequence, but the effective valency of the interaction of the glycoprotein with the virus. Accordingly, rational drug design should include the synthesis and testing of multivalent sialoside analogs.