VIROLOGY 182, 879-882 (1991) Recognition by Human A and B Influenza Viruses of 8- and 7-Carbon Analogues of Sialic Acid Modified in the Polyhydroxyl Side Chain M. N. MATROSOVICH,’ A. S. GAMBARYAN, F. N. REIZIN, AND M. P. CHUMAKOV Institute of Poliomyelitis and Viral Encephalitides, USSR Academy of Medical Sciences, Moscow Region 142 782, USSR Received October 23, 1990; accepted January 23, 199 1 To evaluate the recognition by influenza viruses of the C9-C7 polyhydroxylated moiety of sialic acid (SA) receptor determinant a novel assay has been developed based on the assessment of binding by the solid-phase immobilized virus of the enzyme-labeled sialyglycoprotein fetuin treated by periodate or periodate/borohydride to contain an 8-car- bon aldehyde, 7-carbon aldehyde, or corresponding hydroxyl analogues of SA. Some features of recognition by human influenza viruses of these SA analogues were type and subtype specific, especially marked differences being found between type A and type B viruses. At the same time a significant diversity was observed among virus strains belonging to the same subtype. The assay described provides a new tool for the differentiation of influenza viruses according to receptor binding properties and for an investigation of molecular interactions in the receptor binding site of the virus. 0 1991 Academic Press, Inc. The attachment of influenza viruses to host cells is mediated by the binding of viral hemagglutinin (HA) to sialic acids of cell surface glycoproteins or ganglio- sides (see (1, 2) for a review). Attempts are in progress to evaluate in molecular terms the interactions of indi- vidual parts of the cellular receptor with the receptor- binding site (RBS) of viral HA (3-6). The polyhydroxy- lated side chain of SA (carbon atoms 7, 8, and 9) was known for a long time to be involved in binding since treatment of soluble sialylglycoproteins or erythrocytes with periodate which selectively oxidized the glycerol moiety of SA changed their ability to interact with the virus (7-1 I). In all of the previous studies, though, ei- ther products of receptor oxidation were not precisely characterized or only a limited number of viruses was examined. The present work was initiated to investigate in more details the role of the glycerol side chain of SA in the influenza virus receptor binding. For this purpose the sialylglycoprotein fetuin was labeled with horseradish peroxidase (HRP), chemically modified to contain C7 or C8 carbon analogues of SA (Fig. l), and the binding of thus obtained fetuin-HRP conjugates by different influ- enza A and B virus strains was studied using a two- stage solid-phase assay. At the first stage of the assay the virus adhered spe- cifically to the wells of microtitration plates coated with unlabeled fetuin. The solid-phase attached virions were then allowed to bind the native or modified fetuin- HRP conjugate, with binding being evaluated by rou- ’ To whom requests for reprints should be addressed. tine assay of HRP activity. Rather distinctive curves of binding versus periodate concentration during the oxi- dation step were obtained for different viruses. In Fig. 2 some characteristic curves are presented and are in- terpreted as follows. The composition of sialylglyco- conjugate oxidized by periodate under mild controlled conditions is known to depend upon the periodate concentration used (9, 73, 14). At low periodate/SA molar ratios (~1 :l) the content of unoxidized SA is high, and the C8-A aldehyde is preferentially formed, in excess of periodate (>lO:l) the glycoconjugate con- tains predominantly the C7-A analogue (see Fig. 1). The use of intermediate periodate/SA ratios gives a mixture of all three forms of glycosidically bound SA, the native one, C8-A, and C7-A (9, 14). The left side of the binding curves of oxidized conjugates reflects, therefore, a mixed effect of increase in their C8-A con- tent and concomitant decrease in the content of native SA residues, whereas the right side of the curves in the plateau region show the binding of conjugates con- taining predominantly the C7-A aldehydic analogue of SA. The curves of reduced conjugates reflect recogni- tion by the viruses of corresponding hydroxyl ana- logues. Taking the above-mentioned into account, it may be concluded from Fig. 2a that A/Mis/85 virus binds C8-A residues of oxidized fetuin more avidly than parent SA moieties, the reduction of C8-A to C8-H lowers the binding significantly, and the binding of con- jugates bearing the C8-H, C7-A, and C7-H SA ana- logues is negligible in comparison to unmodified con- jugate. The SA analogue recognition “formula” for A/ Mis/85 virus is, thus, C8-A > SA > C8-H = C7-A = C7-H. The main difference in recognition of native 879 0042-6822/91 $3.00 CopyrIght 0 1991 by Academic Press. Inc. All rrghts of reproduction in any form resewed.