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
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Abstract |
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Introduction |
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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 169172 (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 didnt 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.
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Methods |
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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.
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). RTPCR 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).
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
).
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 125 of RNA segment 4 and then amplified by PCR by using a plus-sense primer corresponding to positions 4161 with a NotI site and a minus-sense primer corresponding to positions 17591731 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.
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 TrisHCl (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 SDSPAGE on 13% gels (unless otherwise noted) containing 4 M urea under reducing conditions.
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 SDSPAGE.
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 TPCKtrypsin (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 SDSPAGE.
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.
Cell fusion assay.
The transfected COS-1 cells were treated with 5 µg/ml TPCKtrypsin 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.
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Results |
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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 (SP 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 (NK) 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 SDSPAGE (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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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 SDSPAGE. 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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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 SDSPAGE 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 5468% 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 (1227% 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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Discussion |
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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.
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Acknowledgments |
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References |
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Gallagher, P., Henneberry, J., Wilson, I., Sambrook, J. & Gething, M.-J. (1988). Addition of carbohydrate side chains at novel sites on influenza virus hemagglutinin can modulate the folding, transport, and activity of the molecule. Journal of Cell Biology 107, 2059-2073.[Abstract]
Hongo, S., Sugawara, K., Muraki, Y., Kitame, F. & Nakamura, K. (1997). Characterization of a second protein (CM2) encoded by RNA segment 6 of influenza C virus. Journal of Virology 71, 2786-2792.[Abstract]
Lamb, R. A. & Krug, R. M. (2001). Orthomyxoviridae: the viruses and their replication. In Fields Virology , pp. 1487-1531. Edited by D. M. Knipe & P. M. Howley. Philadelphia:Lippincott Williams & Wilkins.
Liu, C., Eichelberger, M. C., Compans, R. W. & Air, G. M. (1995). Influenza type A virus neuraminidase does not play a role in viral entry, replication, assembly, or budding. Journal of Virology 69, 1099-1106.[Abstract]
Mitnaul, L. J., Castrucci, M. R., Murti, K. G. & Kawaoka, Y. (1996). The cytoplasmic tail of influenza A virus neuraminidase (NA) affects NA incorporation into virions, virion morphology, and virulence in mice but is not essential for virus replication. Journal of Virology 70, 873-879.[Abstract]
Mitnaul, L. J., Matrosovich, M. N., Castrucci, M. R., Tuzikov, A. B., Bovin, N. V., Kobasa, D. & Kawaoka, Y. (2000). Balanced hemagglutinin and neuraminidase activities are critical for efficient replication of influenza A virus. Journal of Virology 74, 6015-6020.
Ohuchi, M., Feldmann, A., Ohuchi, R. & Klenk, H.-D. (1995). Neuraminidase is essential for fowl plague virus hemagglutinin to show hemagglutinating activity. Virology 212, 77-83.[Medline]
Ohuchi, R., Ohuchi, M., Garten, W. & Klenk, H.-D. (1997). Oligosaccharides in the stem region maintain the influenza virus hemagglutinin in the metastable form required for fusion activity. Journal of Virology 71, 3719-3725.[Abstract]
Palese, P., Tobita, K., Ueda, M. & Compans, R. W. (1974). Characterization of temperature sensitive influenza virus mutants defective in neuraminidase. Virology 61, 397-410.[Medline]
Schulze, I. T. (1997). Effects of glycosylation on the properties and functions of influenza virus hemagglutinin. Journal of Infectious Diseases 176, S24-S28.[Medline]
Sugawara, K., Nishimura, H., Kitame, F. & Nakamura, K. (1986). Antigenic variation among human strains of influenza C virus detected with monoclonal antibodies to gp88 glycoprotein. Virus Research 6, 27-32.[Medline]
Takatsuki, A., Arima, K. & Tamura, G. (1971). Tunicamycin, a new antibiotic. I. Isolation and characterization of tunicamycin. Journal of Antibiotics 4, 215-223.
Takebe, Y., Seiki, M., Fujisawa, J., Hoy, P., Yokota, K., Arai, K., Yoshida, M. & Arai, N. (1988). SR promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Molecular and Cellular Biology 8, 466-472.[Medline]
Tarentino, A. L. & Maley, F. (1974). Purification and properties of an endo--N-acetylglucosaminidase from Streptomyces griseus. Journal of Biological Chemistry 249, 811-817.
Tsuchiya, E., Sugawara, K., Hongo, S., Matsuzaki, Y., Muraki, Y., Li, Z.-N. & Nakamura, K. (2001). Antigenic structure of the haemagglutinin of human influenza A/H2N2 virus. Journal of General Virology 82, 2475-2484.
Wagner, R., Wolff, T., Herwig, A., Pleschka, S. & Klenk, H.-D. (2000). Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics. Journal of Virology 74, 6316-6323.
Wiley, D. C., Wilson, I. A. & Skehel, J. J. (1981). Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature 289, 373-378.[Medline]
Wilson, I. A., Skehel, J. J. & Wiley, D. C. (1981). Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 resolution. Nature 289, 366-373.[Medline]
Yokota, M., Nakamura, K., Sugawara, K. & Homma, M. (1983). The synthesis of polypeptides in influenza C virus-infected cells. Virology 130, 105-117.[Medline]
Received 21 September 2001;
accepted 4 January 2002.