Receptor specificity of H5 influenza virus escape mutants

Virus Research 100 (2004) 237–241

Receptor specificity of H5 influenza virus escape mutants N.A. Ilyushina a,∗ , I.A. Rudneva a , A.S. Gambaryan b , A.B. Tuzikov c , N.V. Bovin c a

Laboratory of Virus Physiology, The D.I. Ivanovsky Institute of Virology, Gamaleya Street 16, 123098 Moscow, Russia b M.P. Chumakov Institute of Poliomyelitis and Viral Encephalitides, Russian Academy of Medical Sciences, P/O Institute of Poliomyelitis, 142782 Moscow, Russia c Shemyakin Institute of Bioorganic Chemistry, 16/10 Miklukho-Maklaya, 117997, Russia Received 1 July 2003; received in revised form 22 December 2003; accepted 29 December 2003

Abstract The binding of viruses to synthetic polyacrylamide (PAA)-based sialylglycoconjugates was used to characterize the receptor specificities of antibody escape mutants of the influenza virus A/Mallard/Pennsylvania/10218/84 (H5N2). The sialylglycoconjugates that were used carried identical terminal Neu5Ac␣2–3Gal moieties but differed in the structure of the next saccharide residue(s). Our data show that mutations in the vicinity of the haemagglutinin (HA) receptor-binding site (RBS) effect the recognition of the third saccharide residue and change the affinity pattern of binding. The affinity of the majority of the escape mutants for sialyl receptors increased compared to the parental strain. © 2004 Elsevier B.V. All rights reserved. Keywords: Influenza virus; Hemagglutinin; Sialyl receptors

1. Introduction The study of viral mutants able to escape the neutralizing effects of monoclonal antibodies (Mabs) provides a powerful tool for the structural and functional analysis of the influenza virus haemagglutinin (HA). Previous studies have shown that viruses can escape through the acquisition of single mutations in the antigenic sites of the HA molecule causing local changes in the HA structure without destroying its three-dimensional structure (Wiley et al., 1981, Knossow et al., 1984; Jackson and Nestorowicz, 1985; Wharton et al., 1989). However, even minor changes in the HA molecule can have an effect not only on the antigenicity, but also on the other properties of the HA. For example, amino acid changes in the vicinity of the receptor-binding site (RBS) in escape mutants of A/Turkey/Ontario/7732/66 (H5N9) and A/Mallard/Pennsylvania/10218/84 (H5N2) decreased the virus virulence for birds and for mice, respectively (Philpott et al., 1990; Kaverin et al., 2002). There are several mechanisms described for the escape of viruses from the neutralizing effects of antibodies. One of the mechanisms is the blocking of the HA antigenic site by ∗ Corresponding author. Tel.: +7-095-190-2813; fax: +7-095-190-2867. E-mail address: ilyushina [email protected] (N.A. Ilyushina).

0168-1702/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2003.12.032

the emergence of additional carbohydrate chains. Previous studies have shown that H2N2 escape mutants can be generated by the incorporation of additional glycosylation sites at positions 160, 187, or 131. These escape mutants had decreased receptor-binding and decreased fusion activities (Tsuchiya et al., 2002). An additional unusual mechanism of escape from monoclonal antibody inhibition was observed by Fleury et al. (1998). These investigators demonstrated that a selected escape mutant emerged through a mutation that inhibited the HA conformation necessary for the Mab binding. When low affinity Mabs are used in the selection process escape mutants can be obtained that retain the ability to bind the Mab (Thomas et al., 1998). A set of X-31 virus escape mutants possessing these properties was obtained after selection in chicken embryos as well as in MDCK cells. The amino acid substitutions in these escape mutants were in the vicinity of RBS and affected the receptor specificity of the resulting variants. Most variants had an increased affinity toward artificially ␣2–3 sialylated chicken red blood cells and an increased pH requirement for HA conformation transition (Daniels et al., 1987). Thus, in this instance the increased affinity towards host cell provided the escape mechanism. In an earlier study, we identified the location of two antigenic sites on the HA of A/Mallard/Pennsylvania/10218/84 (H5N2) using escape mutants to a panel of Mabs (Kaverin

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et al., 2002). The HA genes of the escape mutants were sequenced and the location of the amino acid substitutions were mapped to the 3D H5 HA model obtained by X-ray analysis (Ha et al., 2001). Several escape mutants exhibited unusual features in their reactions with Mabs in retaining the ability to bind the Mabs as revealed by ELISA yet escaping their neutralizing effects. The observed effects suggest that these escape mutants could have increased affinities for the host cell. We did not, however, observe any increase in the affinity of these escape mutants toward 3 -sialyllactose (3 SL) (Ilyushina et al., 2003). To further characterize the receptor specificity of these escape mutants, we determined their binding affinities to a set of polyacrylamide (PAA)-based sialylglycoconjugates containing a common Neu5Ac␣2–3Gal motif but differing in their inner saccharide core.

2. Materials and methods 2.1. Viruses Avian H5N2 influenza virus A/Mallard/Pennsylvania/ 10218/84 (Mld/PA/84) was obtained from the virus repository at the Virology Department of St. Jude Children’s Research Hospital (Memphis, TN, USA). The virus was adapted to mice by serial lung-to-lung passage (Smirnov et al., 2000). Escape mutants of the mouse-adapted variant, which was designated Mld/PA/84-MA, were selected with a panel of anti-H5 Mabs (Kaverin et al., 2002). The viruses were propagated in 9–10-day-old embryonated chicken eggs. For studies on virus binding, the virus-containing allantoic fluids were clarified from cellular debris by low-speed centrifugation and used without further purification.

The data presented in this report represent the mean of three or four individual experiments for each mutant virus.

3. Results and discussion To characterize the receptor specificity of the Mld/PA/84MA escape mutants, a number of synthetic polyacrylamidebased sialylglycoconjugates were synthesized. The polymers carried the same terminal Neu5Ac␣2–3Gal motif but differed in the structure of the inner saccharide core. The selected sialyloligosaccharides are known carbohydrate chains of natural glycoproteins and glycolipids, which are potential receptors for influenza viruses (Fig. 1). The sialyloligosaccharides used were: Neu5Ac␣2–3Gal␤1–4Glc␤ (3 SL); Neu5Ac␣2–3Gal␤ (3SiaGal); Neu5Ac␣2–3Gal␤1–4GlcNAc␤ (3 SLN); Neu5Ac␣2–3Gal␤1–3GlcNAc␤ (SiaLeC ); Neu5Ac␣2–3Gal␤1–3(Fuc␣1–4)GlcNAc␤ (SiaLeA ); Neu5Ac␣2–3Gal␤1–4(Fuc␣1–3)GlcNAc␤ (SiaLeX ). The receptor analogs differ in the presence of the third moiety, in the linkage (1–3 or 1–4) between galactose and the following moiety, in the nature of the third moiety (glucose

2.2. Sialyloligosaccharides and sialylglycoconjugates 3 -Sialyllactose was purchased from Serva (Switzerland). Spacer-armed 3 -sialyloligosaccharides were synthesized as previously described (Nifant’ev et al., 1996; Tuzikov et al., 2000), their polyacrylamide conjugates were synthesized as described (Bovin et al., 1993). 2.3. Binding assays The affinity of the influenza viruses for soluble receptor analogs was evaluated in an inhibition assay based on the binding of solid-phase immobilized virus to horseradish peroxidase labeled bovine fetuin (Gambaryan and Matrosovich, 1992). For the calculation of the dissociation constants (Kd ) of virus/receptor analog complexes, the concentration of the sialic acid residues in the solution was used both for the monovalent sialosides and for the polyvalent sialylglycoconjugates.

Fig. 1. Structure of sialyloligosaccharides.

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Fig. 2. Receptor-binding site of the H5 influenza virus HA complexed with Neu5Ac␣2–3Gal␤1–3GlcNAc␤1–3Gal␤ (LSTa). Escape-mutations are represented in spacefill mode (H3 numbering). The carbohydrate moiety connected to amino acid 131 is represented by using DS ViewerPro 5.0 (Accelrys Inc.) software. SA is sialic acid in LSTa. The arrow indicates the point where the fucose attaches in SiaLeA .

or glycosamine), or in the absence or presence of additional fucose at the third moiety. Fig. 2 shows the receptor-binding site of the H5 HA influenza virus and the positions of the mutations identified in the escape mutants. The H3 HA sequence is also shown for comparison as is the approximate spatial location of the glycosylation site created by the D131N mutation observed in three of the escape mutants. The carbohydrate chain in this position partly covers amino acid residues 156, 157, 129, and is in close proximity to amino acid residues 133 and 194. It is not surprising that the acquisition of this glycosylation site completely prevents the binding of cp46 and cp55 Mabs, since their antigenic determinant includes amino acids 129, 156, and 157 (Kaverin et al., 2002). On the other hand, the Mab 176/26, the binding of which is abolished by amino acid changes in HA positions133 and 194, retains the capacity to bind the D131N m176/26 escape mutant (Ilyushina et al., 2003). The antigenic determinant of this Mab is formed by residues 131, 133, and 194, and appears to be partly covered by the carbohydrate chain at position 131. The affinity of Mld/PA/84-MA and the escape mutants towards the high molecular weight receptor analogs (3 SLN-PAA, SiaLeC -PAA, SiaLeA -PAA, SiaLeX -PAA) and free 3 SL is shown in Table 1. As the binding of all the tested viruses for 3SiaGal-PAA, 3 SL-PAA, and 3 SLN-PAA was practically equal, the data for 3SiaGal-PAA and 3 SL-PAA are omitted. The affinity of the parent

virus for these analogues differs from those of the affinity of the escape mutants. The highest affinity values of the parental virus were found for SiaLeA -PAA, and the lowest for SiaLeX -PAA. Equivalent intermediate affinities were found for 3SiaGal-PAA, 3 SL-PAA, 3 SLN-PAA, and SiaLeC -PAA. The receptor specificity pattern of this virus coincides with the receptor specificity of other duck influenza viruses (Gambaryan et al., 2003). An increase in the affinity toward free 3 SL was only seen in the escape mutants with amino acid substitutions K157M and N129D. The affinity of the other escape mutants towards these analogues was equal to the Mld/PA/84-MA parent virus. The majority of single mutations lead to an increased affinity toward high molecular weight substrates. The exception was the mutant with the D131N mutation which lead to the acquisition of a new glycosylation site. This mutation reduces the affinity of HA toward 3SiaGal-PAA, 3 SL-PAA, 3 SLN-PAA, and SiaLeC -PAA, but not to fucose-containing SiaLeX -PAA and SiaLeA -PAA. These data correlate with the data of Tsuchiya et al. (2002), who described a decrease in the affinity of HA after the emergence of a glycosylation site in H2N2 escape mutants. The N129D and K157M mutations result in an approximately equal increase in affinity toward 3 SLN-PAA, SiaLeA -PAA and free 3 SL. The similarities in the effects caused by these mutations appears to relate to the fact that the substitutions are very closely situated in the

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Table 1 Binding of influenza virus A/Mallard/Pennsylvania/10218/84 (H5N2) escape mutants to receptor analogs Viruses

Substitution in HA1

Kd (mkM Neu5Ac)a 3 SL

Mld/PA/84-MA m46(7) m46(8) m46(7)–24B9 m46(7)–55 m46(7)−55a m46(7)−55−24B9 m55 m55–24B9 m58 m58−24B9 m58(1)−24B9−176/26 m176/26 m24B9 m24B9−176/26

N129D K157M N129D, N129D, N129D, N129D, K156N K156N, D131N D131N, D131N, D131N R144G R144G,

R144K K156T K156T, T189K K156T, P140L N142K S145P S145P, L194I

D131N

2000 1100 1200 1500 1300 1000 1300 1700 1600 2500 2500 2400 2200 2500 2500

3 SLN-PAA ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

150 150 170 150 100 160 200 180 110 300 200 360 360 200 350

8.2 2.5 2.3 3.0 3.0 1.3 1.6 2.0 3.5 14.1 7.2 1.9 13.9 5.5 7.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 0.3 0.3 0.3 0.2 0.1 0.2 0.1 0.3 1.5 0.3 0.1 1.1 0.2 0.3

SiaLeX -PAA 26.0 21.0 21.4 13.2 22.1 13.7 7.7 3.3 2.4 22.1 22.8 8.0 22.7 13.3 14.4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.3 1.6 2.0 1.1 0.8 1.4 0.3 0.5 0.3 1.1 1.2 1.2 2.0 1.2 1.7

SiaLeC -PAA 9.0 8.9 9.0 4.4 4.4 6.4 1.8 1.8 4.6 14.2 5.1 7.0 14.2 3.8 5.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 0.7 0.3 0.3 0.4 0.9 0.1 0.1 0.4 1.2 0.7 0.7 0.6 0.2 0.9

SiaLeA -PAA 3.0 0.8 0.8 3.0 0.4 0.5 0.8 0.5 1.5 2.3 1.3 2.6 2.2 1.2 2.6

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.3 0.1 0.1 0.3 0.1 0.1 0.1 0.1 0.2 0.1 0.2 0.1 0.2 0.1 0.4

a Dissociation constant (K ) was determined as described in Section 2, the lower the K the stronger binding. In each case, K was obtained in four d d d independent experiments.

three-dimensional structure of the H5 HA molecule. Additionally, both mutations increase the negative charge in this region of the HA. The amino acid substitution K156N leads to an increase in the affinity towards all the sialyl receptor analogs. Positions 156 and 157 are not contiguous to the receptor, however, they are situated in the cavity created by amino acids 194 and 195, of which the former interacts with the N-acetyl moiety of sialic acid directly, and the latter is crucial for the maintenance of RBS 3D structure. A comparison of the three-step escape mutant m58(1)– 24B9–176/26 with the two-step escape mutant m58(1)–24B9 allowed us to determine the effect of the L194I amino acid substitution. This substitution reduces the affinity for type 1 structures (based on Gal␤1–3GlcNAc core) SiaLeA -PAA and SiaLeX -PAA and raises the affinity for type 2 structures (based on Gal␤1–4GlcNAc core) 3 SLN-PAA and SiaLeX -PAA. Thus, this mutation is sensitive to the contacts with the third, GlcNAc, moiety. The T189K amino acid substitution effects the binding affinities of the HA in a similar manner to that of the L194I substitution. This conclusion is drawn from a comparison between the m46(7)–55 and m46(7)–55a escape mutants. The T189K substitution changes the receptor specificity toward the type 2 core. This mutation appears to have a direct effect on the binding, as the asialic parts of the 2–3-linked sialosides point towards the “left” side of the receptor-binding pocket of the HA (Eisen et al., 1997). The effects of mutations in antigenic site A (amino acid substitutions 140–145) were studied by using the one-, two-, and three-step escape mutants obtained by selection with the Mab 24B9. Despite the fact that this antigenic site is far from the receptor-binding site, mutations in this region of HA influence the receptor specificity of the influenza viruses. A R144G mutation results in an increase in the affinity to-

wards all the sialyl receptors, a S145P mutation results in an increase towards all the receptors except SiaLeX -PAA, and a P140L mutation results in an increase towards all the receptors except SiaLeA -PAA. The N142K amino acid substitution raises the affinity for SiaLeX -PAA and reduces the affinity for the other substrates. Summarizing the data presented in Table 1, the affinity of the majority of the escape mutants towards sialyl receptors increases in comparison with the parent virus. The effect is dramatic for SiaLeX -PAA as highlighted by the affinity of the m55(2)–24B9 escape mutant which increased 11-fold. The receptor specificity pattern of the escape mutants has also changed very markedly from the parental virus. The affinity ratio to 3 SLN-PAA versus SiaLeX -PAA differs 14-fold in the escape mutants m46(8) and m55(2)–24B9, probably because the fucose moiety prevents binding in the former and allows the binding in the latter. Different escape mutants prefer either (1–3) or (1–4) linkages between galactose and the adjacent saccharide residue. The m24B9–176/26 escape mutant has a higher affinity towards the type 1 core based receptors, whereas the m46(7)–55 escape mutant binds type 2 receptors with a five-fold increase in affinity. Fucose added to N-acetylglucosamine in SiaLeA -PAA by a ␣(1–4) linkage results in an 11-fold increase in the affinity of binding in the m46(7) escape mutant. However, in the m46(7)–24B9 escape mutant the positive effect of fucose on the binding is eliminated by the additional R144K mutation. These changes in the carbohydrate specificity of the haemagglutinin can lead to a change in the preferential choice of host cell receptors. In our earlier studies, we have shown that the receptor specificity of avian influenza viruses is not limited to the recognition of the Neu5Ac2–3Gal motif. Duck influenza viruses differ from chicken influenza viruses by recognition of additional residues in an oligosaccharide, and the difference correlates with the pattern of

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the sialic acid receptors on target cells (Gambaryan et al., 2002, 2003). Although chicken embryo allantoic membrane and cells of duck intestine have Neu5Ac2–3Gal terminated receptors, one cannot exclude that their predominant sialyl receptors differ in the structure of the proximal core. The changes described in the receptor specificity of the escape mutants may correlate with the interaction of HA with the natural receptors of the allantoic membrane. Until now, the description of influenza virus receptor specificity has primarily been limited to the differential recognition of Neu5Ac␣2–3Gal versus Neu5Ac␣2–6Gal moieties. It is now clear that this is a very simplistic comparison and that influenza viruses can identify more distant fragments of the receptor molecule. Our data demonstrate that amino acid changes in the vicinity of the HA receptor-binding site can modulate the receptor specificity of influenza viruses in subtle, but biologically important ways. Acknowledgements These studies were supported by grants 01-04-48017, 02-04-48109, 01-04-49300, and 03-04-06212 MA of the Russian Foundation for Basic research (RFBR), and NATO Collaborative Linkage Grant LST.CLG.979155. We thank Dr. R.J. Webby for editing the manuscript.

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