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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We previously investigated the antigenic structure of the influenza A/Kayano/57 (H2N2) virus HA by using anti-HA monoclonal antibodies (mAbs) and escape mutants selected by these antibodies and identified six distinct antigenic sites, designated I-A to I-D, II-A and II-B (Tsuchiya et al., 2001 ). We demonstrated that most of the escape mutants selected by mAbs to site I-A, I-B or I-C acquired a novel glycosylation site at position 160, 187 or 131, respectively, showing that influenza A/H2N2 viruses have the potential to acquire at least one additional oligosaccharide on the tip of the HA (Tsuchiya et al., 2001
). Interestingly, however, examination of the available HA amino acid sequences of influenza A/H2N2 viruses showed that none of the HA molecules had obtained a new glycosylation site on the tip and had only one carbohydrate chain at position 169 or 170 [two glycosylation sequons overlap each other at residues 169172 (NNTS)]. We recently showed that HA glycosylation-site mutants, which acquired one to three oligosaccharide chains at position 160, 187 or 131 by site-directed mutagenesis, are transported to the cell surface efficiently but exhibit a moderate or drastic decrease in both receptor-binding and cell-fusing activities (Tsuchiya et al., 2002
).
Potential sites for glycosylation occur when the Asn residue is in the consensus sequence AsnXSer/Thr, where X can be any amino acid except Pro (Kornfeld & Kornfeld, 1985 ). The HA of influenza A/H2N2 virus contains five N-linked glycosylation sites and two glycosylation sequons overlap at positions 2023 (NNST) and 169172 (NNTS) (Waterfields et al., 1980
; Brown et al., 1981
) (Fig. 1
). The overlapping glycosylation sequons at positions 2023 are also present in H1, H6, H8, H11 and H12 subtype HAs and those at positions 169172 are present in H13 subtype HA (Nobusawa et al., 1991
). In the present study, we explored the role of these two oligosaccharide chains in the antigenic properties, intracellular transport and biological activities of the HA protein by eliminating each of the overlapping glycosylation sequons by site-specific mutagenesis. The data obtained suggest that the oligosaccharide chains linked to Asn 20 or 21 and Asn 169 or 170 are not essential for the intracellular transport and biological activity. It is reasonable to conclude that the two overlapping glycosylation sequons present at positions 2023 and 169172 are conserved among all of the HAs of influenza A/H2N2 viruses because conservation of the amino acid sequence itself in these glycosylation sequons is critical for the formation of the proper conformation, intracellular transport and biological activities of the H2 subtype HA.
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Methods |
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Antibodies.
Antibodies to egg-grown influenza A/Kayano/57 virions were raised in rabbits as described previously (Yokota et al., 1983 ). Neutralizing mAbs to the influenza A/Kayano/57 virus HA were prepared previously (Tsuchiya et al., 2001
) and were demonstrated by operational mapping to be directed against six nonoverlapping or partially overlapping antigenic sites, designated I-A to I-D, II-A and II-B (Tsuchiya et al., 2001
).
Plasmid construction and oligonucleotide-directed mutagenesis.
The wt HA gene cDNA of influenza A/Kayano/57 virus was generated from viral RNA using AMV Reverse Transcriptase XL (Life Sciences) and oligonucleotide primers complementary to positions 125 of RNA segment 4 and amplified by PCR using a plus-sense primer, corresponding to positions 4161 and containing a NotI site, and a minus-sense primer, corresponding to positions 17591731 and containing a SpeI site. The PCR product was cut with NotI/SpeI and subcloned into the NotISpeI sites of the transient expression vector, pME18S (Takebe et al., 1988 ). The mutated HA gene cDNAs with mutation(s) in two overlapping glycosylated sequons located at positions 2023 (NNST) or 169172 (NNTS) were constructed with mutant primers. The mutant primers were designed so that the Asn-, Thr- or Ser-encoding codon would be replaced by an Ala-encoding codon. In a particular mutant, 20-NNTA (see Fig. 7
), the Ser-encoding codon was changed into a Thr-encoding codon. PCR products were excised by digestion with NotI/SpeI and inserted into the NotISpeI sites of pME18S. The recombinant pME18S plasmids were digested with XhoI, self-ligated and then used for transfection. Nucleotide sequences of all mutant cDNAs in pME18S were confirmed by dideoxynucleotide chain-terminating sequencing.
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Endoglycosidase H (endo H) digestion.
Immunoprecipitated HA proteins were digested with endo H (30 mU) for 16 h at 37 °C, as described (Hongo et al., 1997 ), precipitated with acetone and analysed by SDSPAGE.
Trypsin treatment of transfected cells.
Transfected COS-1 cells were labelled with [35S]methionine for 20 min at 48 h post-transfection and chased for 4 h in DMEM containing an excess of nonradioactive methionine. During the last 15 min of the chase, cells were treated with DMEM containing TPCKtrypsin (5 µg/ml) to cleave the surface-expressed HA into its two subunits, HA1 and HA2. The reaction was terminated by adding soybean trypsin inhibitor (5 µg/ml). Cells were then immunoprecipitated with rabbit antiserum and the immunoprecipitates obtained were analysed by SDSPAGE.
Haemadsorption test.
At 48 h post-transfection, transfected COS-1 cells were washed once with DMEM and incubated with 5 mU/ml Arthrobacter ureafaciens neuraminidase (Roche Diagnostics) for 1 h at 37 °C. Cells were washed three times with DMEM and incubated at 4 °C for 10 min with 1% suspension of guinea pig erythrocytes. Monolayers were then washed extensively with PBS (pH 7·4) deficient in Ca2+ and Mg2+ ions. To quantify the extent of haemadsorption, erythrocytes were lysed by adding 1 ml of distilled water and the released haemoglobin was measured by reading the optical density at 540 nm.
Cell-fusion assay.
Cell-fusion assays were carried out according to the procedures described previously (Muraki et al., 1999 ). Transfected COS-1 cells were treated with TPCKtrypsin (5 µg/ml) for 15 min at 37 °C at 48 h post-transfection 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 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.
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Results and Discussion |
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To investigate the antigenic properties of the individual HA mutants, COS-1 cells expressing each of the six mutant HAs were labelled with [35S]methionine, immunoprecipitated with each of the six anti-HA mAbs directed against six different antigenic sites (I-A to I-D, II-A and II-B) and the resulting precipitates were analysed by SDSPAGE. As demonstrated in Fig. 2(B), all mutants 20-NNAT, 20-NNSA and 20-NNAA, like wt HA, reacted with all of the mAbs used. Interestingly, however, it became evident that 169-NNAS, unlike wt HA and 169-NNTA, was unreactive with any of the anti-HA mAbs used and that the reactivity of 169-NNAA was weak compared with wt HA and 169-NNTA, suggesting that the amino acid change at position 171 (Thr
Ala) results in an extensive, conformational change of the HA molecule and that this drastic, conformational change is restored partially by an amino acid substitution at position 172 (Ser
Ala).
To see whether the six HA mutants described above acquire resistance to endo H, indicative of the conversion of carbohydrate chains from high mannose-type to complex one, which takes place in the Golgi cisternae (Tarentino & Maley, 1974 ), COS-1 cells expressing wt HA or each of the mutant HAs were labelled with [35S]methionine for 20 min at 48 h post-transfection and then chased for 4 h. Immediately after a pulse, or after a subsequent chase, cells were immunoprecipitated with rabbit antiserum and the resulting precipitates were treated with endo H followed by analysis with SDSPAGE. As can be seen in Fig. 3(A)
, mutants 20-NNAT, 20-NNSA and 20-NNAA became endo H-resistant during a chase, showing that all these mutant HAs are transported to the medial Golgi compartment. In contrast, 169-NNAS remained endo H-sensitive even after a 4 h chase, although 169-NNTA acquired resistance to endo H. Interestingly, however, most of the 169-NNAA molecules, like 169-NNTA, became endo H-resistant, suggesting that the amino acid change at position 171 (Thr
Ala) affects the transport of the HA molecule from the endoplasmic reticulum to the Golgi apparatus.
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The results described above raised the possibility that, with respect to the overlapping glycosylation sequons present at positions 169172, conservation of the amino acid sequence itself rather than that of N-glycosylation is critical for the formation of the proper conformation and intracellular transport of the HA molecule. To further confirm this notion, an additional six mutant HAs (20-ANST, 20-NAST, 20-AAST, 169-ANTS, 169-NATS and 169-AATS), in which Asn residues were replaced by Ala residues, were constructed. COS-1 cells expressing wt HA or each of these mutant HAs were labelled with [35S]methionine, immunoprecipitated with rabbit antiserum and then analysed by SDSPAGE (Fig. 4A). The expression levels of mutant HAs were comparable to that of wt HA, as described above. Clearly, the amounts of 169-NATS and 169-AATS recovered in the immunoprecipitates were much lower than those of wt and the other mutant HAs. To validate this observation, immunoprecipitation was carried out with six anti-HA mAbs to six different antigenic sites (Fig. 4B
). 169-NATS and 169-AATS, in contrast to wt HA and the other mutant HAs, were completely unreactive with any of the anti-HA mAbs used, showing that the amino acid change at position 170 (Asn
Ala) prevents the formation of all of the neutralizing epitopes tested. Thus it is reasonable to conclude that the overlapping glycosylation sequons at positions 169172 is conserved among all of the HAs of influenza A/H2N2 viruses because conservation of the amino acid sequence itself in these glycosylation sequons is essential for the formation of the proper conformation of H2 subtype HA. This is not incompatible with the previous report showing that the presence of oligosaccharides on the globular head of HA is not necessarily required for influenza virus replication, since most of the HAs of influenza viruses isolated from aquatic birds do not contain oligosaccharides on their tips (Inkster et al., 1993
; Matrosovich et al., 1999
).
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Biological activities of mutant HAs
To investigate the receptor-binding activity of the 12 mutant HAs constructed here, COS-1 cells transfected with wt cDNA or each of the mutant cDNAs were examined for haemadsorption at 48 h post-transfection according to the procedures described in Methods. As shown in the left column of Table 1, the extent of haemadsorption of cells expressing each of the mutants 20-NNAT, 169-NNTA and 169-NNAA was comparable to that of cells expressing wt HA. The fact that 169-NNAA exhibited a high level of haemadsorption activity indicates that an oligosaccharide chain linked to Asn at position 169 or Asn at position 170 is not necessary for the H2 subtype HA to show receptor-binding activity. The other mutant HAs displayed lower levels of haemadsorption activity than that of wt HA (277% of wt). The levels of haemadsorption activity of these mutant HAs appeared to be comparable to those of their cell surface expression. For example, although 20-NNSA and 20-NNAA were transported to the cell surface, the amounts of these mutant HAs cleaved after TPCKtrypsin treatment were lower than that of wt HA (20-NNSA=40% of wt; 20-NNAA=36% of wt) (Fig. 3B
). Furthermore, 169-NNAS, 169-NATS and 169-AATS, which failed to be transported to the cell surface, showed little or no haemadsorption activity.
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All of these data taken together suggest that even if oligosaccharide chains linked to Asn residues 20 or 21 and Asn residues 169 or 170 are eliminated, the antigenic properties, intracellular transport and biological activities are not influenced strongly. Rather, the reason why the two overlapping glycosylation sequons present at positions 2023 and 169172 are conserved completely among the H2 subtype HAs is the requirement of conservation of the amino acid sequences at these positions. If changes in amino acid sequences at these two positions occur, the H2 subtype HA protein undergoes a conformational change that results in a decrease in intracellular transport and biological activity.
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Acknowledgments |
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Footnotes |
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References |
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Received 22 April 2002;
accepted 22 July 2002.