©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Biosynthetic Modulation of Sialic Acid-dependent Virus-Receptor Interactions of Two Primate Polyoma Viruses (*)

(Received for publication, September 14, 1994; and in revised form, November 2, 1994)

Oliver T. Keppler (1) Peer Stehling (2) Markus Herrmann (1) Holger Kayser (2) Detlef Grunow (2) Werner Reutter (2) Michael Pawlita (1)(§)

From the  (1)Angewandte Tumorvirologie (ATV), Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, D-69120 Heidelberg, Federal Republic of Germany, and the (2)Institut für Molekularbiologie und Biochemie, Freie Universität Berlin, D-14195 Berlin-Dahlem, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Sialic acids are essential components of the cell surface receptors of many microorganisms including viruses. A synthetic, N-substituted D-mannosamine derivative has been shown to act as precursor for structurally altered sialic acid incorporated into glycoconjugates in vivo (Kayser, H., Zeitler, R., Kannicht, C., Grunow, D., Nuck, R., and Reutter, W.(1992) J. Biol. Chem. 267, 16934-16938).

In this study we have analyzed the potential of three different sialic acid precursor analogues to modulate sialic acid-dependent virus receptor function on different cells. We show that treatment with these D-mannosamine derivatives can result in the structural modification of about 50% of total cellular sialic acid content. Treatment interfered drastically and specifically with sialic acid-dependent infection of two distinct primate polyoma viruses. Both inhibition (over 95%) and enhancement (up to 7-fold) of virus binding and infection were observed depending on the N-acyl substitution at the C-5 position of sialic acid. These effects were attributed to the synthesis of metabolically modified, sialylated virus receptors, carrying elongated N-acyl groups, with altered binding affinities for virus particles.

Thus, the principle of biosynthetic modification of sialic acid by application of appropriate sialic acid precursors to tissue culture or in vivo offers new means to specifically influence sialic acid-dependent ligand-receptor interactions and could be a potent tool to further clarify the biological functions of sialic acid, in particular its N-acyl side chain.


INTRODUCTION

Sialic acid (^1)is the most abundant terminal sugar moiety on the surface of eukaryotic cells. As a component of oligosaccharides on glucoconjugates, sialic acids are crucial for many biological processes (for review see (1) and (2) ) including functional cell surface receptors for viruses such as influenza viruses, reoviruses, paramyxoviruses, coronaviruses, encephalomyocarditis virus, and polyoma viruses(3, 4, 5, 6, 7, 8, 9, 10) .

The most abundant sialic acid, N-acetylneuraminic acid(11) , is synthesized in vivo from N-acetylated D-mannosamine or D-glucosamine as precursors (12) . These compounds are finally converted to CMP-activated N-acetylneuraminic acid, which is transferred in the Golgi apparatus onto oligosaccharide chains of glycoconjugates(12, 13) . It has recently been demonstrated that mammalian cells can take up synthetic N-substituted D-glucosamine and D-mannosamine derivatives and metabolize them in the sialic acid pathway (see Fig. 1)(14, 15) . As a consequence, structurally altered sialic acids with substituted N-acyl side chains were incorporated into various glycoconjugates(15, 16) .


Figure 1: Metabolism of N-propanoyl-D-mannosamine. This chemically synthesized amino sugar analogue follows the metabolic route of N-acetyl-D-mannosamine. It serves as a model for other N-acyl derivatives. For experimental details see Refs. 15 and 16). PEP, phosphoenol pyruvate.



In cells carrying modified sialic acids, we investigated virus-receptor interactions of three different members of the Polyomavirus genus. Polyoma viruses are small DNA tumor viruses with a 45-nm diameter, nonenveloped icosahedral particle consisting of the three nonglycosylated structural viral proteins VP1, VP2, and VP3. The B-lymphotropic papovavirus (^2)(LPV) (17) and the human polyoma virus BK (BKV) (18) both use distinct sialylated receptors for infection(10, 19) , whereas infection by the highly related simian virus 40 (SV40) is sialidase-resistant(20) . Here we describe that biosynthetically generated N-substituted sialic acids can interfere with sialic acid-dependent biological functions in a dominant fashion and specific for the introduced N-acyl substitution.


EXPERIMENTAL PROCEDURES

Synthesis of N-Acyl-D-mannosamine Derivatives

D-Mannosamine hydrochloride was suspended to 10 mM in 30 ml methanol. At 0 °C NaOMe/methanol (to 11 mM) and the corresponding carbonic acid anhydride (to 12 mM) were added. After stirring for 2 h at 0 °C the solution was dried in a vacuum evaporator. The raw product was purified by column chromatography on silica gel 60 (Merck, Germany) with an elution solvent of acetic acid ethyl ester/methanol/H(2)O (5:2:1 to 10:2:1, depending on the polarity of the anhydride). Characterization was carried out by ^1H NMR spectroscopy. N-Acyl-D-mannosamine derivatives were stored at 4 °C and dissolved in PBS as a stock solution of 100-200 mM shortly before addition to cell cultures.

Treatment of Cells with Sialic Acid Precursor Analogues

Human B-lymphoma cell line BJA-B (21) (subclone K88) and African green monkey kidney epithelium cell line Vero (3) were propagated as described(10) . Briefly, BJA-B cells were cultivated at 2 times 10^5 to 1 times 10^6 cells/ml as suspension cultures in RPMI 1640 medium supplemented with 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% (v/v) fetal calf serum. Vero cells were cultivated as monolayers attached to plastic surfaces in Dulbecco's modified Eagle's medium with the same supplements and for harvesting were brought into suspension by incubation for 2-5 min in 0.2% (w/v) trypsin, 0.2% (w/v) versene (EDTA) in PBS. Unless stated otherwise, cells were cultivated for 48 h in the presence of 5 mMD-mannosamine derivative.

Isolation of Cellular Sialic Acid

Cells were washed three times in PBS and lysed by hypotonic shock in distilled water (1 h, 4 °C); this was followed by 1 cycle of freezing and thawing. The crude membrane fraction was pelleted by centrifugation at 4500 times g for 20 min at 4 °C. The pellet was lyophilized and delipidated by stepwise washing three times with methanol/chloroform (1:2, 1:1, 2:1 by volume) for 30 min(22) . After centrifugation at 10,000 times g (30 min, 4 °C) the pellet was hydrolyzed for 2 h with 200 µl of 2 M acetic acid(23) . The pH of the hydrolysate was adjusted to 4, and further purification was carried out on a cation exchanger (AG-50W-X12, H+ form, Bio-Rad, München, Germany).

Separation of N-Acylneuraminic Acids by HPLC

Neuraminic acids were chromatographed using an anion exchange column (Nucleosil SB 5 µm, 250 times 4 mm (inner diameter), Knauer, Berlin, Germany) with pulsed amperometric detection (Dionex Corp., Sunnyvale, CA). Eluent A contained distilled water while eluent B contained 45 mmol/liter NaH(2)PO(4), pH 4.0. The flow rate was 1.5 ml/min, and all separations were carried out using a linear gradient from 100% A to 0% A during a 50-min period. NaOH (0.5 M) was added to the column effluent through a tee at 0.5 ml/min before the pulsed amperometric detection cell detector to minimize base-line drift. Pulse potentials and durations were: E(1) = 0.10 V (t(1) = 540 ms), E(2) = 0.60 V (t(2) = 60 ms), E(3) = 0.60 V (t(3) = 120 ms).

Further purification and final quantification were achieved by reversed phase HPLC with fluorescent labeled compounds. The derivation was performed according to a method by Reuter and Schauer(24) . Labeled neuraminic acids were chromatographed using a reversed phase C18 column (Lichrosorb C18 5 µm, 250 times 4 mm (inner diameter), Knauer, Berlin, Germany) with a fluorescence detector. Eluent A contained distilled water while eluent B contained acetonitrile/methanol (60:40, v/v). The flow rate was 1 ml/min, and all separations were carried out using a gradient that first ran for 20 min in the isocratic mode with 10% B; then was raised to 25% B in 25 min and finally to 50% B in another 15 min.

Characterization of N-Acylneuraminic Acids by Gas Chromatography-Mass Spectrometry

Cellular N-acylneuraminic acids were isolated and purified by hydrolysis and HPLC as described above. Synthesis of O-SiMe(3) ether and O-SiMe(3) ester of neuraminic acids was performed according to the method of Sweeley et al.(25) using a mixture of trimethylchlorosilane, hexamethyldisilane, and pyridine (1:2:10) at room temperature for 1 h, and samples were subsequently applied to gas chromatography-mass spectroscopy as described previously (15, 26).

Virus Infections

Stocks of LPV (strain P12) were extracted from infected BJA-B cells after one cycle of freezing and thawing by hypotonic extraction as described(10) . The SV40 stock was prepared from infected monkey kidney epithelium cells (TC7) displaying a massive cytopathic effect. After freezing and thawing of washed cells and resuspension of the cell lysate, the same extraction procedure was used as for LPV. Stocks of BKV (Gardner strain) were established from infected Vero cells following the method of Sinibaldi et al.(19) .

For virus infection cells were washed once with cold PBS and were then incubated either with LPV (BJA-B cells, 4 °C, 3 h) or with BKV or SV40 (Vero cells, 4 °C, various times). After removing unbound virus from the cultures by three washing cycles with medium, infected cells were cultured at 37 °C. In BJA-B cells without prior treatment by N-acylmannosamines the applied LPV dose yielded 15-20% immunofluorescence-positive cells (see below) 48 h after infection. For BKV the infectious dose was chosen to yield 1.5-2.5% infected Vero cells in order to allow an exact determination of the increase in infection in ManNProp- and ManNBut-treated cells (see Fig. 3, a and c).


Figure 3: Biosynthesis of N-substituted neuraminic acid derivatives in host cell lines treated with N-substituted D-mannosamines. Three representative electron impact mass spectrograms of extracted N-acylneuraminic acids are shown: a, N-acetylneuraminic acid (5-acetamido-3,5-dideoxy-D-glycero-beta-D-galactopyranosyl-2-onic acid) from ManNAc-pretreated (control) Vero cells; b, N-propanoylneuraminic acid (3,5-dideoxy-5[propanoyl-amido]-D-glycero-beta-D-galactopyranosyl-2-onic acid) from ManNProp-pretreated Vero cells; and c, N-butanoylneuraminic acid (5[butanoyl-amido]-3,5-dideoxy-D-glycero-beta-D-galactopyranosyl-2-onic acid) from ManNBut-pretreated BJA-B cells. m/e defines the ionic mass/ionic charge of fragments generated from neuraminic acids during mass spectroscopy. Fragment peaks characteristic for the individual N-acylderivatives (15) are indicated. Note the m/e increase of 14 for each additional methylene group in corresponding fragments. At m/e 285 a fragment peak constant for all N-acylneuraminic acids occurs. The chair conformations of the main peak fragment of N-acetylneuraminic acid (m/e 356) (a), N-propanoylneuraminic acid (m/e 370) (b), and N-butanoylneuraminic acid (m/e 384) (c) are shown to indicate the elongation of the N-acyl group.



Analysis of Virus-infected Cells by Indirect Immunofluorescence

BJA-B cell suspensions were allowed to settle on poly-L-lysine-coated slides (Bio-Rad adhesion slides, Bio-Rad, München, Germany), and Vero cells were cultivated as monolayers on glass coverslips. Cells attached to the glass surfaces were fixed and permeabilized in cold methanol/acetone (1:1, v/v) at -20 °C for 10 min. Viral antigens were first reacted for 30 min at 37 °C with an LPV VP-specific rabbit antiserum(10) , a rabbit antiserum raised against purified BKV particles (kind gift of G. Noss), or mouse monoclonal antibody 1614 against SV40 T antigen(27) , respectively, followed by staining with rhodamine-conjugated goat anti-rabbit immunoglobulins or fluorescein-conjugated goat anti-mouse immunoglobulins (both from Dianova, Hamburg, Germany). Nuclei were counterstained with DAPI (4`,6-diamidino-2-phenylindole, Boehringer Mannheim). All antibody dilutions and washings between antibody incubations were done in PBS.

Determination of LPV Binding to BJA-B Cells

The LPV binding capacity was determined by an indirect, nonradioactive assay as described previously(10) . Briefly, cells were washed once in PBS and then serially diluted in PBS containing 1% (w/v) gelatin. In 96-well tissue culture plates the cell suspension was incubated in a final volume of 180 µl with a constant amount of LPV particles (corresponding to 800 pg of LPV VP1) for 30 min at 37 °C. After low speed sedimentation of the cells, the amount of free LPV VP1 in the supernatant was quantified by LPV VP1-specific enzyme-linked immunosorbent assay.

Sialidase Treatment of Cells

Vero cells (2 times 10^5 on a glass coverslip), which had been cultivated in the presence of N-acyl-D-mannosamines or an equivalent volume of PBS (control), were exposed for 2 h at 37 °C to 200 µl of either sialidase (EC 3.2.1.18, acylneuraminyl hydrolase, neuraminidase) from Vibrio cholerae (Boehringer Mannheim) in PBS (0.2 unit/ml) or PBS alone. After thoroughly washing the cells with cold PBS, BKV infection was carried out as described above.


RESULTS

Biosynthesis of Modified Sialic Acids with Elongated N-Acyl Groups and Their Incorporation into Glycoconjugates

Two cell lines, BJA-B(21) , a human B-lymphoma line susceptible to infection by LPV (10) , and Vero, an African green monkey kidney epithelium line susceptible to infection by BKV (18) as well as SV40(20) , were exposed to three D-mannosamine derivatives containing elongated N-acyl groups (N-propanoyl (ManNProp), N-butanoyl (ManNBut), or N-pentanoyl (ManNPent); Fig. 1and Fig. 4). Cells treated with 10 mM ManNProp for 48 h showed no signs of toxicity or reduced proliferation. In ManNBut- and ManNPent-treated cultures cell proliferation was reduced by 30-40%.


Figure 4: Reduction or enhancement of host cell susceptibility to LPV or BKV infection by pretreatment with sialic acid precursor analogues. a, the chair conformation of the applied N-substituted D-mannosamines is shown with R indicating the modified N-acyl group. b, host cell lines BJA-B and Vero were cultured for 48 h in the presence of the sialic acid precursor analogues ManNProp, ManNBut, or ManNPent or as control an equivalent volume of PBS and subsequently infected with LPV or BKV, respectively. Virus-infected cells were identified by immunofluorescence staining for LPV and BKV capsid proteins 48 h after infection (this time allows the completion of only one viral replication cycle). Similar numbers of cells are present in microphotographs of each panel as determined by nuclear counterstaining (not shown).



To demonstrate the biosynthesis of structurally modified sialic acids and their incorporation into glycoconjugates after pretreatment of BJA-B and Vero cells by either ManNProp, ManNBut, or ManNPent, extracted sialic acids were separated by HPLC (Fig. 2) and identified by gas chromatography-mass spectrometry (Fig. 3), as has been shown previously for serum glycoproteins of ManNProp-treated rats(15) . The resulting sialic acid derivatives carry the identical N-acyl group substitution as the applied sialic acid precursor analogue, thus resulting in an elongation of the physiological N-acetyl group at position C-5 of sialic acid by one, two, or three methylene groups, respectively (see chair conformations in Fig. 4a). The amount of modified sialic acids incorporated in pretreated cells relative to the amount of physiological N-acetylneuraminic acid was quantified after HPLC separation of the extracted sialic acids (Fig. 2). About 50% of physiological neuraminic acid was replaced by the N-acyl-modified neuraminic acid in both of the cell lines tested after treatment with each of the three D-mannosamine derivatives (Table 1).


Figure 2: Detection of biosynthetically modified sialic acids from Vero cells with two different HPLC methods. a-d, anion exchange chromatography of purified neuraminic acids with pulsed amperometric detection. Glucose 6-phosphate (Glc-6-P) was used as an internal standard. See ``Experimental Procedures'' for details. e and f, reversed phase chromatography of fluorescence labeled neuraminic acids. N-glycolylneuraminic acid (NGNA) was used as an internal standard. a and e, reference chromatograms. The chromatograms of cells treated with ManNAc (b), ManNProp (c), ManNBut (d), and ManNPent (f) are shown. NANA, N-acetylneuraminic acid; NPropNA, N-propanoylneuraminic acid; NButNA, N-butanoylneuraminic acid; NPentNA, N-pentanoylneuraminic acid; PAD, pulsed amperometric detection.





Inhibition of LPV Binding and Infection in Host Cells Carrying Sialic Acids with Elongated N-Acyl Groups

It has been recently demonstrated that infection of BJA-B cells by LPV is sialic acid-dependent since enzymatic cleavage of terminal sialic acids by sialidase from V. cholerae reduced cell LPV binding capacity and susceptibility to LPV infection by approximately 80%(10) . In addition, this indicated that a strong reduction in virus binding can limit virus infection.

Cultivation of BJA-B cells in the presence of the sialic acid precursor analogues ManNProp, ManNBut, or ManNPent for 2 days reduced their LPV binding capacity by 75, 86, and 85%, respectively (Fig. 5c). Similarly, the susceptibility of these pretreated cells to LPV infection was impaired by up to 97% in comparison with cells pretreated with control saline buffer (Fig. 4b, Fig. 5, a and b). 50% inhibition of LPV infection was reached at a concentration in tissue culture of approximately 0.4 mM for ManNProp and 0.8 mM for ManNBut (Fig. 5a).


Figure 5: Reduction of LPV infection (a and b) and binding (c) in host cells containing N-substituted sialic acids. a, BJA-B cells were cultured in the presence of different concentrations of ManNProp, ManNBut, the physiological sialic acid precursor ManNAc (Sigma, Germany), or PBS (control) for 48 h and subsequently infected with LPV. 48 h after infection the concentration of viral antigen in cell extracts was quantified by LPV VP1-specific enzyme-linked immunosorbent assay(10) . The values shown represent the arithmetic means from triplicate samples (± S.D.) relative to the concentration of viral antigen present in control cells. b, after ManNProp pretreatment (10 mM) of 6-48 h, cells were infected with LPV and analyzed as described above. For each time point, LPV infection in ManNProp-treated cells is given relative to LPV infection in control cells (set as 100%). c, the LPV binding capacity of BJA-B cells pretreated with different N-acyl-D-mannosamines (5 mM, 48 h) or PBS (control) was tested in a nonradioactive, indirect virus binding assay where a constant amount of virus is incubated with increasing cell numbers(10) . Values represent the amount of virus bound relative to the total virus offered for binding and are arithmetic means (±S.D.) from triplicate samples. The cell numbers required to bind 37.5% of the administered LPV were used to determine LPV binding capacities of pretreated cells relative to control cells.



D-Mannosamine has been reported to inhibit the biosynthesis of glycosylphosphatidylinositol anchors in mammalian cells(28) . In order to determine whether the observed inhibition of LPV infection was specific for the modified N-acyl group and not an effect of the D-mannosamine residue, cells were also pretreated with the physiological sialic acid precursor ManNAc, differing from ManNProp by only one methylene group. Yet, neither LPV binding (Fig. 5c) nor infection (Fig. 5a) was significantly affected by this D-mannosamine derivative.

In order to examine whether pretreatment with ManNProp, ManNBut, or ManNPent, respectively, also had an effect on other steps of the virus replication cycle apart from virus binding, purified LPV DNA was transfected by the DEAE-dextran method. This experiment yielded similar amounts of virus for all cells, irrespective of their pretreatment (not shown), indicating that the impaired susceptibility to infection involves events prior to virus gene expression and DNA replication. Taken together, these experiments strongly suggest that already the first step of LPV infection, i.e. virus attachment, was impaired by the presence of structurally altered sialic acids in membrane glycoconjugates and by this mechanism hindered LPV infection.

The Length of the Sialic Acid N-Acyl Side Chain Determines Enhancement or Inhibition of Human Polyoma Virus BK Infection

BKV infection of Vero cells is sensitive to treatment with sialidase from Clostridium perfringens(19) as well as from V. cholerae, the latter reducing the percentage of infected cells by over 80% (Fig. 6c). For this virus-host cell system, we analyzed the virus attachment phase indirectly. Pretreated cells were exposed to virus for different times at 4 °C, unbound virus was removed, and after 48 h in culture successfully infected cells were scored by immunofluorescence. It is assumed that at 4 °C only virus binding takes place since efficient virus uptake is an energy-dependent process. Surprisingly, ManNProp- and to a lesser extent ManNBut-pretreated Vero cells could be infected more efficiently (up to 7-fold) than controls (Fig. 4b and Fig. 6, a and c). ManNPent, however, rendered cells nearly resistant to infection by BKV. For attachment times of up to 3 h, infection increased more rapidly as a function of time in ManNProp- and ManNBut-treated cells than in controls (Fig. 6a). After an attachment time of 14 h, infection in control cells also reached high levels indicating that despite a slower rate of virus binding, the final level of virus attachment allowed BKV infection to occur.


Figure 6: Sialic acid-dependent BKV infection (a and c) but not sialic acid-independent SV40 infection (b) is affected in Vero cells precultivated in the presence of N-substituted D-mannosamines (5 mM, 48 h). BKV (a) or SV40 (b) (same symbols as in a) was allowed to attach at 4 °C for 6 min to 14 h to pretreated Vero cells. Unbound virus was washed away after the attachment phase, and then cells were incubated at 37 °C for 48 h before the percentage of virus-infected cells was determined by immunofluorescence. c, the increased BKV susceptibility of ManNProp- and ManNBut-pretreated Vero cells is sensitive to sialidase treatment. Values given are the mean of two independent experiments.



It has been shown previously that N-propanoylneuraminic acid, incorporated into glycoconjugates in vivo, can be enzymatically or chemically cleaved(15) . In ManNProp- and ManNBut-pretreated Vero cells V. cholerae sialidase reduced the elevated levels of BKV infection by 88 and 82%, respectively (Fig. 6c), demonstrating the essential role of terminal sialic acids on the cell surface for the enhanced susceptibility to BKV infection.

SV40 Infection, which Uses a Sialidase-Resistant Receptor, Is Not Affected by Sialic Acid N-Acyl Side Chain Modifications

SV40, a polyoma virus with 81% protein sequence identity to BKV in the major capsid protein VP1(29) , can also efficiently infect and replicate in Vero cells but uses a different, sialidase-resistant receptor(20) . In contrast to the marked effects seen for BKV, SV40 infection was unaffected in Vero cells carrying N-substituted sialic acids (Fig. 6b). This further supports that the incorporation of N-substituted sialic acids apparently does not affect transport or replicative steps in the polyoma virus infection cycle but rather interferes with the interaction of specific viruses with their sialylated receptors. These results provide evidence that the biosynthetically introduced hydrophobic modifications at the N-acyl group of sialic acids directly affected the capability of the sialylated receptor on Vero cells to bind BKV, with the N-propanoyl and N-butanoyl substitutions increasing and the even longer N-pentanoyl group drastically decreasing the affinity for BKV particles.

Binding of Lectins and Antibodies to Sialylated Epitopes on Cell Surfaces Carrying N-Substituted Sialic Acids Is Not Altered

In contrast to the effects seen with LPV and BKV, the binding of monoclonal antibodies and lectins, which recognize sialylated membrane components only, was unaffected by ManNProp pretreatment. For three monoclonal antibodies (LN1, HB-4, and HB-6), which recognize different sialylated epitopes on B cell differentiation antigens(30, 31) , binding was not significantly altered on pretreated BJA-B cells as analyzed by flow cytometry (not shown). This suggests that N-acyl elongation might not affect binding of all types of ligands interacting with sialylated receptors. Similarly, fluorescence microscopy of ManNProp-treated BJA-B and Vero cells stained with biotinylated lectins Sambucus nigra(32) or Maackia amurensis(33) , which specifically detect sialic acid residues in alpha-2,6- or alpha-2,3-linkage, respectively, allowed the exclusion of gross alterations of the overall amount or pattern of sialylation.


DISCUSSION

This study demonstrates drastic but selective biological effects of sialic acids modified in their N-acyl side chains, which have been synthesized and incorporated in high amounts into cellular glycoconjugates. These modified sialic acids have been biosynthesized in competition with physiological sialic acid in cells cultivated in the presence of N-substituted D-mannosamines, and amounts equal to the physiological N-acetylneuraminic acid can be reached. The N-substituted sialic acids drastically enhance and/or abolish in a dominant fashion host cell susceptibility to sialic acid-dependent virus infections.

The high amount of modified sialic acids with elongated N-acyl groups synthesized indicates that enzymes and transport mechanisms in the sialic acid metabolic pathway cannot be very selective with respect to the N-acyl group of substrate intermediates. Also, the modified sialic acids and their precursors apparently are tolerated by the cells, at least in tissue culture. At 5 mMD-mannosamine derivative, when about 50% of total sialic acids with an N-pentanoyl instead of the physiological N-acetyl side chain was reached, cell viability was not affected, and cell proliferation was only slightly reduced. Similarly, in vivo treatment of Wistar rats with ManNProp had displayed no signs of acute toxicity. (^3)This indicates that, at least in these established cell lines, either the metabolic and signaling pathways needed for cellular proliferation and survival in tissue culture are rather independent of sialic acids or are not very discriminatory with respect to the N-acyl side chain.

In contrast to this rather inert behavior of central cellular functions, the minor modifications of the sialic acid side chain, i.e. the introduction of uncharged, hydrophobic methylene groups at C-5, had pronounced and specific effects on interactions of host cells with two viruses that are known to depend on cell surface sialic acid for infection. Most probably, the modification of sialic acid forming part of the viral receptor structures was responsible for alteration of the virus infections. The presence of physiological N-acetyl sialic acid and modified N-propanoyl sialic acid in about equal amounts in human B-lymphoma line BJA-B was sufficient to reduce binding of LPV 6-fold and infection over 10-fold. On the other hand, infection by BKV was enhanced 7-fold in monkey epithelium cell line Vero carrying the same modified sialic acid also in about equal amounts as the physiological sialic acid. The kinetic experiment suggests that the increase in infection was mainly due to faster binding, which may reflect an increase either in receptor number or, more probably, in receptor affinity. One could assume that a polyvalent cooperative interaction of the repetitive virus capsid with several cell surface receptor molecules is necessary for particle binding. If so, then even slight receptor affinity changes in either direction could add up to the strong, opposite effects seen here for the two viruses.

The use of these sialic acid precursor analogues allowed the identification of the N-acyl group as a critical determinant of sialic acid-dependent polyoma virus-receptor interaction. This group has previously been implicated in host range determination of a completely different microorganism. Enterotoxigenic Escherichia coli with strain K99 fimbriae, which use sialylated intestinal glycolipids as receptors, specifically recognize N-glycolyl but not N-acetyl substitutions(34) . In addition to viral infections studied here, the principle of biosynthetic structural modification of sialic acid should be applicable to a wide variety of sialic acid-dependent ligand-receptor interactions including other bacteria(35) , parasites(36) , toxins(37) , and lectins(38) .

For the interaction of the influenza A virus hemagglutinin with synthetic sialic acids, N-substitutions were found to reduce (39) or not to alter (40) the binding affinity. Such studies using nuclear magnetic resonance spectroscopy or virus adsorption inhibition assays on erythrocytes could be complemented and extended by assays on virus binding and infection in cultured host cells and animals carrying biosynthetically incorporated N-substituted sialic acids. This should allow a more detailed understanding of the multivalent nature of this virus-receptor interaction.

Synthetic sialic acid derivatives binding with high affinity to influenza A and B virus sialidase (4-amino- and 4-guanidino-2-deoxy-2,3-didehydro-D-N-acetyl sialic acid) (41) or influenza C virus surface glycoprotein (42) can serve as inhibitors of influenza virus infections. The enhanced susceptibility to BKV observed in our study suggests that sialic acid modifications generated in living host cells may also facilitate the in vitro development of soluble high affinity competitors of virus binding as antiviral drugs.

In conclusion, the principle of biosynthetic modification of sialic acid by application of appropriate sialic acid precursors to tissue culture or in vivo offers new means to specifically influence sialic acid-dependent ligand-receptor interactions.


FOOTNOTES

*
The financial support of the Fonds der Chemischen Industrie, Frankfurt/Main and the Maria Sonnenfeld-Gedächtnis-Stiftung is gratefully acknowledged. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to Dr. Dr. Herbert Falk on the occasion of his 70th birthday.

§
To whom correspondence should be addressed. Tel.: 49-6221-424645; Fax: 49-6221-424932.

(^1)
Sialic acid comprises the family of all neuraminic acids substituted in the carbon skeleton or in the N-acyl side chain.

(^2)
The abbreviations used are: LPV, B-lymphotropic papovavirus; BKV, human polyoma virus BK; VP, viral structural protein; PBS, phosphate-buffered saline; ManNProp, 2-deoxy-2[propanoyl-amido]-D-mannose; ManNBut, (2-deoxy-2[butanoyl-amido]-D-mannose; ManNPent, 2-deoxy-2[pentanoyl-amido]-D-mannose; HPLC, high performance liquid chromatography.

(^3)
P. Stehling and W. Reutter, unpublished data.


ACKNOWLEDGEMENTS

We thank R. Brossmer, A. Bürkle, L. Gissmann, H. zur Hausen, G. Herrler, D. Keppler, H.-G. Kräubetalich, R. Schwartz-Albiez, and G. Sczakiel for discussion during preparation of the manuscript, G. Moldenhauer for the gift of antibodies and FACScan analysis, G. Brandner for the gift of SV40 antibody 1614, G. Noss for BKV antiserum, and M. Oppenländer for expert technical assistance.


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