Effect of addition of new oligosaccharide chains to the globular head of influenza A/H2N2 virus haemagglutinin on the intracellular transport and biological activities of the molecule

Emi Tsuchiya1, Kanetsu Sugawara1, Seiji Hongo1, Yoko Matsuzaki1, Yasushi Muraki1, Zhu-Nan Li1 and Kiyoto Nakamura1

Department of Bacteriology, Yamagata University School of Medicine, Iida-Nishi, Yamagata 990-9585, Japan1

Author for correspondence: Emi Tsuchiya. Fax +81 23 628 5250. e-mail etakasit{at}med.id.yamagata-u.ac.jp


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The haemagglutinin (HA) of influenza A/H2N2 virus possesses six antigenic sites (I-A to I-D, II-A and II-B), and sites I-A, I-B and I-C are located in the regions corresponding to sites A, B and D on the H3 HA. We demonstrated previously that most escape mutants selected by mAbs to site I-A, I-B or I-C had acquired a new oligosaccharide at position 160, 187 or 131, respectively, but this has never occurred during circulation of A/H2N2 virus in humans. Here, to examine whether the H2 HA has the potential to gain two new oligosaccharides on its tip, 31 double escape mutants were isolated by using a single escape mutant with an oligosaccharide at position 160, 187 or 131 as a parental virus and a mAb to an antigenic site different from that to which the mAb used for selection of the parental virus was directed as a selecting antibody, but there were no mutants with two new oligosaccharides. Glycosylation-site HA mutants containing one to three oligosaccharides at positions 160, 187 and 131 were also constructed and their intracellular transport and biological activities were analysed. The results showed that all of the mutant HAs were transported to the cell surface but exhibited a decrease in both receptor-binding and cell-fusing activities. Thus, influenza A/H2N2 virus may have failed to increase the number of oligosaccharides on the HA because, if this happens, the biological activities of the HA are reduced, decreasing the ability of the virus to replicate in humans.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
The haemagglutinin (HA) of influenza A virus is a type I integral membrane protein with an ectodomain composed of a globular head and a stem region (Wilson et al., 1981 ). HA plays a central role in virus entry. First, it attaches the virus to the cell surface by binding to sialic acid-containing cellular receptors, which is followed by internalization of the virus through receptor-mediated endocytosis. Next, HA induces fusion between the viral envelope and the endosomal membrane, delivering the nucleocapsid into the cytoplasm. The HA protein is cleaved into two subunits, HA1 and HA2, by host-derived proteases and this process is essential for virus infectivity, since it exposes the membrane fusion peptide located at the amino terminus of HA2 (for review, see Lamb & Krug, 2001 ).

Influenza A/H2N2 virus caused a serious pandemic in 1957, circulated in the human population until 1968 and then disappeared. Thus, the history of influenza virus of the H2 subtype was shorter than those of currently circulating viruses of the H1 and H3 subtypes. The reason for the short life of A/H2N2 viruses in humans is not known. As one of the approaches to address this question, we previously investigated the antigenic structure of the A/Kayano/57 (H2N2) virus HA by using anti-HA mAbs and escape mutants selected by these antibodies, and identified six distinct antigenic sites, designated sites I-A to I-D, II-A and II-B (Tsuchiya et al., 2001 ). Sequence analysis of the HA genes of the escape mutants revealed that sites I-A, I-B and I-C form a contiguous antigenic area that contains the regions corresponding to antigenic sites A, B and D on the H3 HA and that sites I-D and II-B are the equivalents of sites E and C, respectively (Wiley et al., 1981 ; Daniels et al., 1983 ; Tsuchiya et al., 2001 ), suggesting that the antigenic structure of the H2 HA is largely similar to that of the H3 HA. However, the former differs from the latter in having a highly conserved antigenic site (II-A) in the stem domain.

The A/H3N2 viruses that circulated between 1968 and 1974 mostly had two oligosaccharide chains on the head region of the HA (residues 81 and 165). However, viruses isolated in 1975 (represented by A/Victoria/3/75) had lost a carbohydrate attachment site at residue 81 and had gained two novel sites, at residues 63 and 126. Moreover, 1986 isolates (represented by A/Memphis/6/86) had gained a new glycosylation site at residue 246 and 1997 isolates (represented by A/Sydney/5/97) had acquired two additional sites at residues 122 and 133. Thus, the currently circulating A/H3N2 viruses (if not all) have six glycosylation sites on the head region of HA, although not all of them may be used. These data raised the possibility that the addition of new carbohydrate chains to the HA tip may provide influenza viruses with an increased ability to prevail in humans, by masking the antigenic sites efficiently (Schulze, 1997 ). Interestingly, however, examination of the available HA amino acid sequences of A/H2N2 viruses showed that none of the HAs had gained a new glycosylation site on their heads and they had only one carbohydrate chain, at position 169 or 170 (H3 numbering) [two glycosylation sequons overlap each other at residues 169–172 (NNTS)]. However, we demonstrated recently that most of the escape mutants selected by mAbs to site I-A, I-B or I-C had acquired a novel glycosylation site at position 160, 187 or 131, respectively, showing that A/H2N2 viruses have the potential to acquire at least one additional oligosaccharide on the tip of the HA (Tsuchiya et al., 2001 ).

The aims of the present study were to answer the following questions. (i) Do A/H2N2 viruses have the potential to acquire two additional oligosaccharide chains on the tip of HA? (ii) Why didn’t addition of new oligosaccharide chains to the HA tip of A/H2N2 virus occur during its circulation in humans? The data obtained show that isolation of escape mutants with two additional carbohydrate chains on the head of HA is not possible and that glycosylation-site mutant HAs that had one to three oligosaccharide chains added as a result of site-directed mutagenesis are transported to the cell surface efficiently but exhibit moderate or drastic decreases in both receptor-binding and cell-fusing activities.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Virus and cells.
The A/Kayano/57 strain of influenza A/H2N2 virus was grown in the allantoic cavities of 10-day-old embryonated hens’ eggs. COS-1 cells were grown in DMEM supplemented with 10% FCS. Madin–Darby canine kidney (MDCK) cells were cultured in Eagle’s minimal essential medium (MEM) containing 10% FCS.

{blacksquare} Selection of double escape mutants.
Attempts to isolate a double escape mutant with two new glycosylation sites in the globular head of HA were performed by using a single escape mutant that had gained a glycosylation site at position 160, 187 or 131 as a parental virus. Briefly, serial 10-fold dilutions of each of the single escape mutants were mixed with an equal volume of 1:10 dilution of ascites fluid containing an anti-HA mAb to an antigenic site different from that to which the mAb used for selection of the parental escape mutant was directed. After incubation for 30 min at room temperature, the mixture was inoculated onto a monolayer of MDCK cells and viruses that escaped neutralization were allowed to grow under the agar overlay medium. Five to six days later, the plaques were picked and treated again with a 1:10 dilution of ascites fluid containing the mAb and then plaqued again. Cloned viruses were found to have greatly decreased reactivity with both the mAbs used for selection of the single and double escape mutants, showing that they were double escape mutants. They were used for analysis after growth in eggs.

{blacksquare} Nucleotide sequence analysis.
The nucleotide sequences of the HA genes of double escape mutants were determined as described previously (Tsuchiya et al., 2001 ). In brief, viral RNA was extracted from purified virions by using the RNeasy Mini kit (QIAGEN). RT–PCR was performed using specific primers for RNA segment 4 (primer sequences are available on request). Nucleotide sequences were determined from the PCR products by cycle sequencing using the BigDye Terminator Cycle Sequencing FS ready reaction kit and an ABI PRISM 310 automatic sequencer (Applied Biosystems).

{blacksquare} Antibodies.
Rabbit antiserum against egg-grown A/Kayano/57 virions was prepared as described previously (Yokota et al., 1983 ). mAbs to the A/Kayano/57 virus HA were described previously (Tsuchiya et al., 2001) and were shown by operational analysis to be directed against six non-overlapping or partially overlapping antigenic sites designated I-A to I-D, II-A and II-B (Tsuchiya et al., 2001 ).

{blacksquare} Plasmid construction and site-directed mutagenesis.
The wild-type (WT) HA gene cDNA of A/Kayano/57 virus was synthesized from viral RNA by using AMV reverse transcriptase XL (Life Sciences) and oligonucleotide primers complementary to positions 1–25 of RNA segment 4 and then amplified by PCR by using a plus-sense primer corresponding to positions 41–61 with a NotI site and a minus-sense primer corresponding to positions 1759–1731 with an SpeI site. The PCR product was digested with NotI and SpeI and the resulting DNA fragment was then subcloned into the NotI and SpeI sites of a transient expression vector, pME18S (Takebe et al., 1988 ). Mutated HA gene cDNAs encoding one to three N-glycosylation sites at positions 160, 187 and 131 were generated by PCR with mutant primers (sequences are available upon request), utilizing plasmid pME18S containing the WT HA gene as a template. Mutant oligonucleotide primers were designed such that Pro, Asp and Thr at positions 162, 187 and 131 would be replaced by Thr, Asn and Asn, respectively, to create a carbohydrate addition site at positions 160, 187 and 131. The PCR products were excised by digestion with NotI and SpeI and then inserted into pME18S. The resulting recombinant pME18S plasmids were cut with XhoI, self-ligated and then used for transfection. Nucleotide sequences of all the mutant cDNAs in pME18S plasmids were confirmed by dideoxynucleotide chain-terminating sequencing.

{blacksquare} Transfection, radioisotopic labelling and immunoprecipitation.
Subconfluent monolayers of COS-1 cells in 3·5 cm Petri dishes were transfected with 1 µg of the recombinant pME18S plasmid that contained a WT or mutated HA gene using LipofectAMINE reagent (Invitrogen). At 48 h post-transfection (p.t.), cells were labelled for 20 min with 10 µCi/ml [35S]methionine (ARC) in methionine-deficient DMEM. Cells were then disrupted in 0·01 M Tris–HCl (pH 7·4) containing 1% Triton X-100, 1% sodium deoxycholate, 0·1% SDS, 0·15 M NaCl and a cocktail of protease inhibitors (Hongo et al., 1997 ) and immunoprecipitated as described previously (Sugawara et al., 1986 ) with rabbit antivirus serum or anti-HA mAb. The immunoprecipitates obtained were analysed by SDS–PAGE on 13% gels (unless otherwise noted) containing 4 M urea under reducing conditions.

{blacksquare} Endoglycosidase H (endo H) digestion.
The immunoprecipitated proteins were digested with endo H (30 mU) for 16 h at 37 °C under the conditions described previously (Hongo et al., 1997 ), precipitated with acetone and analysed by SDS–PAGE.

{blacksquare} Trypsin treatment of transfected cells.
At 48 h p.t., transfected COS-1 cells were labelled with [35S]methionine for 20 min and chased for 4 h in DMEM containing an excess of non-radioactive methionine. During the last 15 min of the chase, cells were treated with DMEM containing TPCK–trypsin (5 µg/ml) and the reaction was terminated by addition of soybean trypsin inhibitor (5 µg/ml). Cells were then immunoprecipitated with rabbit antivirus serum and the resulting immunoprecipitates were analysed by SDS–PAGE.

{blacksquare} Haemadsorption test.
Transfected COS-1 cells were washed once with DMEM at 48 h p.t. followed by incubation with 5 mU/ml Arthrobacter ureafaciens neuraminidase (Roche Diagnostics) at 37 °C for 1 h. The neuraminidase-treated cells were washed three times with DMEM and incubated at 4 °C for 10 min with a 1% suspension of guinea pig erythrocytes or a 0·5% suspension of chicken erythrocytes. The monolayers were then washed several times with PBS (pH 7·4) lacking Ca2+ and Mg2+ and examined by phase-contrast microscopy. For quantification of the extent of haemadsorption, COS-1 cells and erythrocytes were lysed in 1 ml distilled water. After removal of cellular debris by low-speed centrifugation, the released haemoglobin was measured by reading absorption at 540 nm.

{blacksquare} Cell fusion assay.
The transfected COS-1 cells were treated with 5 µg/ml TPCK–trypsin in DMEM for 15 min at 37 °C at 48 h p.t. and then exposed to the prewarmed fusion medium (PBS with 10 mM MES and 10 mM HEPES adjusted to pH 5·0) for 5 min. The fusion medium was then replaced with neutral DMEM containing 10% FCS and the cells were incubated at 37 °C for 3 h. Cells were then fixed with methanol and stained with Giemsa solution.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Isolation of double escape mutants with new oligosaccharide chains at two of the three positions 160, 187 and 131 is impossible
In order to examine whether influenza A/H2N2 virus has the potential to gain new oligosaccharides at two of three positions 160, 187 and 131 of HA, we attempted to isolate double escape mutants that exhibited greatly decreased reactivity with mAbs to at least two of the three antigenic sites I-A, I-B and I-C according to the procedures described in Methods. The HA gene sequences of the 31 double escape mutants obtained were determined and their deduced amino acid sequences were compared with those of the single escape mutants used as parental viruses (Table 1).


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Table 1. Amino acid substitutions detected in the HAs of double escape mutants

 
When the single escape mutant 1/119-EM2 (which had an oligosaccharide chain at position 160) and a site I-B mAb (5/77) were used as parental virus and selecting antibody, six double escape mutants were isolated. Five of these six mutants had an amino acid change at position 193 (T->K) and the remaining mutant (1/119.5/77-EM14) had a change at position 118 (F->L) in addition to a T->K change at position 193. It should be noted that none of the six mutants possessed a change that created an oligosaccharide attachment site at position 187, although three of the four escape mutants isolated by using A/Kayano/57 virus seed stock as a parental virus and a site I-B mAb as a selecting antibody had an oligosaccharide chain at this position (Tsuchiya et al., 2001 ). Nine double escape mutants were isolated when 1/119-EM2 was grown in the presence of a site I-C mAb (4/11 or 3/186). These mutants were found to possess amino acid substitutions at one or two of positions 135 (G->V), 193 (T->K), 219 (T->K), 222 (K->N) and 224 (N->D), none of which generated a carbohydrate attachment site at position 131. Our previous report showed that 12 of the 13 single escape mutants selected by site I-C mAbs obtained a novel glycosylation site at this position (Tsuchiya et al., 2001 ).

No double escape mutants could be isolated when 4/79-EM1 or 5/77-EM1 (which had a carbohydrate side chain at position 187) was allowed to replicate in the presence of a site I-A mAb (1/119). When selection was made with a site I-C mAb (4/148 or 3/179B), 10 double escape mutants were obtained but none of them had an amino acid change that created a new glycosylation site at position 131. Eight of the 10 mutants had an amino acid substitution at one of the three positions 159 (S->P or S->L), 193 (T->K) and 227 (G->R). Interestingly, the remaining two mutants (5/77.4/148-EM21 and 5/77.4/148-EM22) had an N->D change at position 187, in addition to a change at position 131 (T->K) or 159 (S->L), which eliminated an N-glycosylation site at position 187 from the HA of 5/77-EM1.

When 3/186-EM1 (which had an oligosaccharide chain at position 131) was grown in the presence of a site I-A mAb (1/119), one double escape mutant (3/186.1/119-EM71) was isolated. This mutant had an amino acid change at position 197 (N->K) but did not obtain a carbohydrate attachment site at position 160. Five double escape mutants were isolated when selection was done by using one of two mutants (3/106-EM1 and 3/179B-EM1) as the parental virus and one of two site I-B mAbs (4/79 and 5/77) as the selecting antibody. Four of these mutants had an amino acid substitution at position 193 (T->K) or 227 (G->R) and the remaining one (3/179B.5/77-EM1003) had two substitutions, at positions 32 (R->Q) and 227 (G->R); none of these changes created a new glycosylation site at position 187. The results, summarized in Table 1, together suggest strongly that human influenza A/H2N2 virus HA can not acquire new oligosaccharide chains at two of the three positions 160, 187 and 131. It seems likely that, if the addition of carbohydrate were to occur at two of these positions, the biological activities of the H2 subtype HA might be reduced.

Expression of HA glycosylation-site mutants with one to three oligosaccharide chains at positions 160, 187 and 131
To investigate the effect of carbohydrate addition to one or more of positions 160, 187 and 131 on the intracellular transport and biological activities of the H2 molecule, seven HA glycosylation-site mutant cDNAs were constructed (Fig. 1). First, to confirm that the newly introduced carbohydrate-addition sites were used, COS-1 cells were transfected with each of the seven mutant cDNAs and labelled with [35S]methionine in the presence or absence of 1 µg/ml tunicamycin (TM), a specific inhibitor of N-linked glycosylation (Takatsuki et al., 1971 ). Cells were then immunoprecipitated with a site I-D mAb (32/105) and the resulting precipitates were analysed by SDS–PAGE (Fig. 2). Of the three single mutants (G1, G2 and G3), two (G1 and G3) showed reduced electrophoretic mobility compared with WT HA when proteins synthesized in the absence of TM were analysed. However, G1 and G3 synthesized in the presence of TM displayed the same mobility as non-glycosylated WT HA (HANG), suggesting that the novel glycosylation sites introduced into G1 (position 160) and G3 (position 131) were both used. In contrast to G1 and G3, the electrophoretic mobility of G2 synthesized in the absence of TM was virtually identical to that of WT HA. However, the non-glycosylated form of G2 migrated slightly faster than non-glycosylated WT HA, suggesting that the conformational change caused by amino acid substitution at position 187 (Asp->Asn) resulted in an increase in electrophoretic mobility, as was observed previously with the HAs of escape mutants selected with site I-B mAbs (Tsuchiya et al., 2001 ). Thus, it is likely that the G2 molecule, although it acquired a new oligosaccharide at position 187, co-migrated with WT HA because of the increased mobility caused by an Asp->Asn change at position 187.



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Fig. 1. HA glycosylation-site mutants used in this study. N-Glycosylation sites are indicated by filled circles. Numbers indicates Asn residues in the first position of the glycosylation sequon.

 


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Fig. 2. Expression of HA glycosylation-site mutants. COS-1 cells transfected with WT or mutated HA gene cDNAs were labelled with [35S]methionine for 20 min at 48 h p.t. in the absence (TM-) or presence (TM+) of TM. Cells were then immunoprecipitated with a site I-D mAb (32/105) and the resulting immunoprecipitates were analysed by SDS–PAGE. The arrowhead indicates the co-precipitated ER-resident chaperon, Binding-Protein (BiP).

 
Of the three double mutants (G1,2, G1,3 and G2,3), G1,3 synthesized in the absence of TM exhibited reduced electrophoretic mobility compared with any of the single mutants. By contrast, the mobilities of G1,2 and G2,3 were nearly identical to those of G1 and G3, which is probably due to the increased electrophoretic mobility caused by the Asp->Asn change introduced at position 187 into mutants G1,2 and G2,3. Therefore, it is reasonable to conclude that all the three double mutants gained two new oligosaccharide chains. A triple mutant (G1,2,3) synthesized in the absence of TM showed electrophoretic mobility nearly identical to that of G1,3, although it migrated more slowly than G1,2 and G2,3. For the reasons described above, it seems that G1,2,3 acquired three oligosaccharide chains.

Reactivity of HA glycosylation-site mutants with anti-HA mAbs
In order to examine the effect of addition of new oligosaccharides to the tip of the H2 molecule on its reactivity with anti-HA mAbs, COS-1 cells expressing each of the seven glycosylation-site mutants were labelled with [35S]methionine and immunoprecipitated with anti-HA mAbs to each of six antigenic sites (I-A to I-D, II-A and II-B) and the resulting precipitates were analysed by SDS–PAGE. The results obtained are summarized in Table 2. All of the mutant HAs, like WT HA, reacted strongly with antibodies to sites I-D, II-A and II-B. However, the reactivity of the mutant HAs with mAbs to sites I-A, I-B and I-C was markedly different from that of WT HA. G1 reacted strongly with site I-B and site I-C mAbs but did not react at all with the site I-A mAb. G2 was unreactive with the site I-B mAb and was also very weakly reactive with the site I-A mAb. G3 reacted strongly with the site I-A mAb but reacted only weakly with the site I-B and site I-C mAbs. The double and triple mutants exhibited no or greatly reduced reactivity with the antibodies to sites I-A, I-B and I-C, suggesting that carbohydrate addition to two or three of positions 160, 187 and 131 may seriously influence the antigenicity of the H2 molecule by masking most of the antigenic epitopes present in sites I-A, I-B and I-C.


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Table 2. Reactivity of glycosylation-site mutants with anti-HA mAbs

 
Intracellular transport of HA glycosylation-site mutants
To examine whether HA glycosylation-site mutants acquire resistance to endo H digestion, indicative of the conversion of oligosaccharides from the high-mannose type to the complex type that occurs in the medial Golgi cisternae (Tarentino & Maley, 1974 ), COS-1 cells expressing WT or each of the seven mutant HAs were pulse-labelled with [35S]methionine for 20 min at 48 h p.t. and chased for 4 h. The HA proteins were then immunoprecipitated with rabbit antivirus serum, digested with endo H and analysed by SDS–PAGE. As shown in Fig. 3(A), all of the glycosylation-site mutants, like WT HA, acquired endo H resistance during a chase, suggesting that all the mutant HAs are transported to the medial Golgi compartment as efficiently as WT HA.



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Fig. 3. Intracellular transport of HA glycosylation-site mutants. COS-1 cells expressing WT or mutant HA were pulse-labelled with [35S]methionine for 20 min at 48 h p.t. and chased for 4 h. (A) Immediately after a pulse (P) or after a subsequent chase (C), cells were immunoprecipitated with antivirus serum. The resulting precipitates were digested (+) or mock-digested (-) with endo H and then analysed by SDS–PAGE. (B) Cells were treated with TPCK–trypsin during the last 15 min of the chase and then immunoprecipitated with antivirus serum. The resulting immunoprecipitates were analysed by SDS–PAGE on 17·5% gels containing 4 M urea.

 
Next, to analyse the efficiency of transport of the HA glycosylation-site mutants to the cell surface, COS-1 cells transfected with WT cDNA or each of the seven mutant cDNAs were pulse-labelled for 20 min and chased for 4 h. Cells were treated with TPCK–trypsin during the last 15 min of the chase to cleave the HA proteins expressed on the cell surface and then immunoprecipitated with rabbit antivirus serum. The immunoprecipitates obtained were analysed by SDS–PAGE. Fig. 3(B) shows that, in every case, the majority of the HA proteins were cleaved by TPCK–trypsin into HA1 and HA2, indicating that all of the mutant HAs were transported to the cell surface as efficiently as WT HA.

Biological activities of HA glycosylation-site mutants
In order to investigate the effect of addition of one to three new oligosaccharides to the tip of the H2 HA on its receptor-binding activity, COS-1 cells transfected with WT or mutant HA cDNA were treated with neuraminidase at 48 h p.t. and examined for haemadsorption according to the procedures described in Methods. The extent of haemadsorption was enhanced significantly by neuraminidase treatment (about 2-fold), as has been observed with the HAs of the H7 and H1 subtypes (Ohuchi et al., 1995 ). The amounts of WT and mutant HAs expressed in transfected cells were determined by measuring the radioactivity of the HA bands after SDS–PAGE of [35S]methionine-labelled immunoprecipitates obtained after treatment with a site I-D mAb (32/105) and were used to calculate the relative specific haemadsorption activity. The results are shown in Fig. 4. When haemadsorption assays were done with guinea pig erythrocytes, little or no difference was observed in the extent of haemadsorption between WT and single glycosylation-site mutants (Fig. 4A). When chicken erythrocytes were used for the assays, however, the haemadsorbing activities of these mutants were reduced by 54–68% compared with that of WT HA (Fig. 4B), indicating that carbohydrate addition to one of the three positions 160, 187 and 131 results in a significant decrease in the receptor-binding activity of the H2 HA. It was also found that all of the double and triple mutant HAs, irrespective of the erythrocyte species used, exhibited drastically reduced haemadsorbing activity (12–27% of WT HA), showing that addition of oligosaccharide chains to two or three of positions 160, 187 and 131 almost completely abolished the receptor-binding activity of the H2 molecule.



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Fig. 4. Receptor-binding activity of HA glycosylation-site mutants. COS-1 cells were transfected with cDNA encoding WT or mutant HA protein. At 48 h p.t., cells were subjected to haemadsorption with guinea pig erythrocytes (A) or chicken erythrocytes (B). Erythrocytes that attached to the HA-expressing cells were lysed in distilled water and the amounts of released haemoglobin were determined. Mean results from three separate experiments are shown.

 
Fig. 5 shows the results of experiments in which cell-fusing activity was compared between WT HA and the HA glycosylation-site mutants. Extensive syncytium formation was observed with COS-1 cells expressing WT HA. Cells expressing any of the single mutant HAs formed a number of syncytia, but the number of cells involved in syncytia was much smaller than was observed with WT-expressing cells. Only a few, small syncytia were seen with cells expressing any of the double mutant HAs, and cells expressing the triple mutant HA showed no syncytium formation. These observations indicate that the addition of an oligosaccharide chain at position 160, 187 or 131 reduces the fusion activity of the H2 HA significantly and that carbohydrate addition to two or three of positions 160, 187 and 131 results in almost complete loss of the activity.



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Fig. 5. Cell-fusing activity of HA glycosylation-site mutants. COS-1 cells expressing WT or mutant HAs were treated with TPCK–trypsin at 48 h p.t., exposed to fusion medium (pH 5·0) and then incubated for 3 h in neutral-pH medium. At the end of the incubation period, cells were stained with Giemsa solution.

 
Reactivity of HA glycosylation-site mutants with human sera
The HA glycosylation-site mutants (particularly the double and triple mutants) showed markedly reduced reactivity with anti-HA mAbs to antigenic sites I-A, I-B and I-C (see Table 2). These antigenic sites, unlike the other sites (I-D, II-A and II-B), are located on the tip of the H2 molecule (Tsuchiya et al., 2001 ) and are therefore likely to be the major antigenic sites of the HA. If this is true, carbohydrate addition to two or three of positions 160, 187 and 131 may change the antigenicity of the HA drastically. To examine this possibility, COS-1 cells transfected with WT cDNA or each of the seven glycosylation-site mutant cDNAs were labelled with [35S]methionine and then immunoprecipitated with each of eight sera collected from donors who were born between 1948 and 1954. The immunoprecipitates obtained were subjected to SDS–PAGE and the radioactivity of the HA bands was measured. The amounts of WT and mutant HAs expressed in transfected cells were also determined as described above, and the values obtained were used to calculate the relative specific reactivity with human sera. As shown in Fig. 6, there was little difference in reactivity with human sera between WT HA and the single mutant HAs. By contrast, reactivity of the double and triple mutant HAs (except G1,2) with most of the human sera tested was much lower than that of WT HA. These observations raised the possibility that, if carbohydrate addition occurs at two or three of positions 160, 187 and 131, the antigenic properties of the H2-subtype HA may change considerably.



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Fig. 6. Reactivity of HA glycosylation-site mutants with human sera. COS-1 cells transfected with WT cDNA or each of the glycosylation-site mutant cDNAs were labelled with [35S]methionine and then immunoprecipitated with each of eight human sera. The immunoprecipitates obtained were subjected to SDS–PAGE and the radioactivity of the HA band was measured. The relative specific reactivity of the HA glycosylation-site mutants was determined as described in Methods.

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
The goal of the present study was to understand why influenza A/H2N2 viruses, unlike A/H3N2 viruses, did not acquire new oligosaccharide chains on the globular head of the HA during 11 years of circulation in humans. We showed previously that most of the A/H2N2 virus escape mutants selected by anti-HA mAbs to site I-A, I-B or I-C acquired a novel glycosylation site at position 160, 187 or 131, respectively, suggesting that A/H2N2 viruses have the potential to gain at least one additional carbohydrate chain on the tip of the HA (Tsuchiya et al., 2001 ). There were no differences in the ability to replicate in eggs or MDCK cells between the parental virus and the escape mutants described above (Tsuchiya et al., 2001 ). In this study, however, it became evident that the addition of an oligosaccharide at position 160, 187 or 131 caused a significant decrease in both the receptor-binding and cell-fusing activities of the H2 HA. This might be a feature unique to the H2 subtype HA. Gallagher et al. (1988) showed that the HA of A/Aichi/2/68 (H3N2), which contained a novel consensus site for glycosylation at position 188, lacked the ability to bind erythrocytes but still retained the full activity to mediate cell fusion. Ohuchi et al. (1997) also demonstrated with the HA of A/fowl plague virus/Rostock/34 (H7N1) that deletion of oligosaccharides in the head region (positions 123 and/or 148) caused an increase in receptor-binding activity without influencing the cell-fusing activity. It remains unclear why the presence of an oligosaccharide chain(s) near the receptor-binding pocket reduces both the receptor-binding and cell-fusing activities of the H2 HA, whereas it causes a decrease in the receptor-binding activity but not the cell-fusing activity of the HAs of other subtypes (H3 and H7). A moderate decrease not only in the receptor-binding activity but also in the cell-fusing activity of H2 HA with an additional oligosaccharide at position 160, 187 or 131 may influence the ability of A/H2N2 virus to replicate in the respiratory tract of humans, without reducing the ability to grow in tissue-culture cells and eggs. If correct, this may explain why A/H2N2 variants with a carbohydrate chain at one of the three positions did not emerge during circulation in a human population.

Attempts to isolate double escape mutants with oligosaccharides at two of the three positions 160, 187 and 131 were unsuccessful (see Table 1). It seems likely that carbohydrate addition to two of the three positions greatly reduces the biological activities of HA and thereby interferes with the ability of the virus to grow in MDCK cells and/or eggs. This is supported by the finding that glycosylation-site mutant HAs having oligosaccharides at two of positions 160, 187 and 131 exhibited much lower receptor-binding and cell-fusing activities than did WT HA (see Figs 4 and 5). Since isolation of double escape mutants was not possible, it is reasonable that natural variants of A/H2N2 virus did not arise with three oligosaccharide chains on the tip of HA, which is supported by the observation that the mutant G1,2,3 almost completely lost both the receptor-binding and cell-fusing activities.

Recently, evidence has been presented that indicates that a balance of receptor-binding and receptor-destroying activities is strictly required for efficient replication of influenza A virus (Mitnaul et al., 2000 ; Wagner et al., 2000 ); a decrease in the receptor-destroying activity of neuraminidase (NA) must be accompanied by a concomitant decrease in the receptor-binding activity of HA. Otherwise, the relatively strong receptor-binding activity would be seriously disadvantageous in the late stage of infection, since it prevents the release of progeny virions from host cells, resulting in the formation of large aggregates of virions on the cell surface (Palese et al., 1974 ; Liu et al., 1995 ; Mitnaul et al., 1996 ). Wagner et al. (2000) reported that the NA activity of A/Hong Kong/8/68 (H3N2) is much higher than that of A/WSN/33 (H1N1), suggesting that the NA of A/H2N2 virus, a progenitor of A/H3N2 virus NA, has a high level of receptor-destroying activity. It seems likely, therefore, that a decrease in the receptor-binding activity of the H2 HA with additional oligosaccharide(s) at positions 160, 187 and 131 may result in the loss of the balance of receptor-binding and receptor-destroying activities, which in turn may decrease the ability of A/H2N2 virus to replicate in vitro and in vivo. The reason why virus with a low activity of HA and a high activity of NA is unable to replicate efficiently remains to be clarified. However, the most plausible explanation may be that such a virus does not undergo tight binding to cellular receptors (which is essential for virus entry), since most of the receptors are destroyed before tight binding of the virus to receptors is established.

It was pointed out previously that addition of new carbohydrate side chains to the head of HA may constitute a potential source of new epidemic strains, since they are likely to possess a growth advantage in a human population that has antibodies to the dominant virus in the parent virus population (Schulze, 1997 ). To our knowledge, however, the impact of carbohydrate addition to the novel sites located on the tip of HA on reactivity with human sera has not yet been investigated. Here, we showed that, while carbohydrate addition to one of positions 160, 187 and 131 did not influence the reactivity of H2 HA with human sera significantly, HA with additional oligosaccharides at two or three of the positions displayed a markedly decreased reactivity with human sera (see Fig. 6), which supports the hypothesis of Schulze (1997) described above.


   Acknowledgments
 
This paper is dedicated to our co-author, Dr Kiyoto Nakamura, who passed away on 20 October 2001. We thank Y. Takebe (National Institute of Infectious Diseases) for providing the expression vector pME18S. This work was supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists, a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology and a research grant provided by the Japanese Ministry of Health, Labour and Welfare.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 21 September 2001; accepted 4 January 2002.