An analysis of the role of neuraminidase in the receptor-binding activity of influenza B virus: the inhibitory effect of Zanamivir on haemadsorption
C. Luo1,
E. Nobusawa1 and
K. Nakajima1
Department of Virology, Medical School, Nagoya City University, 1 Kawasumi, Mizuho-chou, Mizuho-ku, Nagoya 467, Japan 1
Author for correspondence: Katsuhisa Nakajima.Fax +81 52 853 3638. e-mail nakajima{at}med.nagoya-cu.ac.jp
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Abstract
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We analysed the role of neuraminidase (NA) on haemadsorption by the haemagglutinin (HA) protein of influenza B virus. The influenza B virus mutant ts-7 has a temperature-sensitive mutation in the NA protein. At high temperature, cells infected with this virus did not exhibit haemadsorption activity, but the addition of bacterial neuraminidase (bNA) restored haemadsorption activity. COS cells transfected with HA cDNAs of B/Kanagawa/73 or B/Lee/40 virus showed no evidence of haemadsorption. However, with the addition of bNA or co- transfection with NA cDNA of the B/Lee/40 virus, haemadsorption was observed. Experiments with point-mutated HA cDNAs of B/Lee/40 virus showed that two N-acetyl glycosylation sites at amino acid residues 160 and 217 were responsible for the inability of the HA protein to adsorb to erythrocytes. These results indicated that haemadsorption by the HA protein of influenza B virus required the involvement of NA. Because the NA inhibitor Zanamivir was reported not to penetrate cells, we investigated the action of this inhibitor and found that Zanamivir inhibited haemadsorption on MDCK cells infected with B/Kanagawa/73 or B/Lee/40 virus. After removing Zanamivir by washing, the addition of bNA restored the haemadsorption activity on the infected cells. Scanning electron microscopy indicated that at 0·4 µM Zanamivir, HA protein did not adsorb to erythrocytes but retained the ability to aggregate virions. However, at 4 µM Zanamivir, distinct virion formation could not be observed.
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Introduction
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The haemagglutinin (HA) protein of influenza virus is a transmembrane glycoprotein anchored through a hydrophobic carboxyl terminal region. It is well known that HA protein has two crucial roles in the early stage of virus infection: it binds to a sialic acid- containing receptor on the cell surface; and after receptor-mediated endocytosis of the virus particles, it mediates fusion of the viral envelope and intracellular membrane under acidic conditions (Maeda & Ohnishi, 1980
; Huang et al., 1981
; Lenard & Miller, 1981
). HA protein has many carbohydrate side chains and some of these contain sialic acid as the terminal residue. The neuraminidase (NA) of influenza virus removes sialic acid to release virus particles from host cells or to disaggregate these particles from each other (Palese et al., 1974
; Shibata et al., 1993
). Additionally, sialic acid on some of the carbohydrate chains on the HA protein must be removed to allow for the haemadsorption activity of influenza A H7 (Ohuchi et al., 1995
) and H1 (Tong et al., 1998
) and influenza B viruses (Brassard & Lamb, 1997
). Klenk et al. (1996)
suggested that the carbohydrate on the HA protein modulates binding affinity to the host receptor, since the loss of some of the carbohydrate chains caused the HA protein to bind too strongly to erythrocytes and inhibited the release of influenza A H7 virus from its receptor. As for influenza B virus, however, the molecular mechanism involved in the interaction between HA and NA in haemadsorption has not been clearly resolved. Brassard & Lamb (1997)
tried to locate a carbohydrate correlating with the cleavage of influenza B virus but they did not specifically examine haemadsorption. In the present report, we describe the role of NA in the haemadsorption of influenza B virus and the locations of the carbohydrate chains that affect this activity. It has been suggested that NA might be responsible for the removal of sialic acid from carbohydrate chains on the HA protein in the trans-Golgi or sorting vesicles leading from the Golgi (Wandinger-Ness et al., 1990
). On the other hand, from the results based on reassortant virus experiments, we suggested previously that the removal of sialic acid by NA might take place on the host cell membrane (Tong et al., 1998
). Therefore, we investigated whether desialidation of HA protein of influenza B virus prior to haemadsorption could occur on the cell surface.
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Methods
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Cells and viruses.
COS and MDCK cells (1x105 cells per 18 mm coverslip) prepared on coverslips 18 h earlier were washed with Eagle's minimum essential medium (MEM) without serum (MEM0). They were then inoculated with 50 µl of virus (0·91·2 p.f.u. per cell) and the coverslips were incubated at room temperature for 30 min. After the cells were washed twice with PBS, MEM0 was added and the preparations were incubated at 3437·5 °C as indicated in the text. Coverslips were removed and haemadsorption with 0·5% chicken red blood cells (CRBC) was performed on ice for 15 min. Samples were then washed with cold PBS to remove unadsorbed erythrocytes and fixed with ethanol/acetone. Viruses B/Lee/40, B/Kanagawa/73 and ts-7 (Yamamoto-Goshima et al., 1994
) were propagated in MDCK cells.
Neuraminidase treatment.
Neuraminidase from Vibrio cholera (bNA) (25 mU/ml; Takara Shuzo) was added to the transfected COS cells, ts-7-infected MDCK cells or Zanamivir-treated cells and incubated for 30 min at 37 °C or 37·5 °C.
Construction of an expression system of HA and NA proteins of influenza B virus.
cDNAs of the HA genes from B/Lee/40 and B/Kanagawa/73 viruses were amplified by RTPCR. The sense and antisense primers for the first PCR were 5'-specific CAGCAAGCGTTGCA (116) and 3'-specific CGAGAACAATGATGA (17751757) (Krystal et al. , 1983
), respectively. The second PCR was performed with the 5'-specific primer incorporating an EcoRI site and the 3'-specific primer incorporating an XbaI site. The cDNA was digested with EcoRI and XbaI enzymes. These fragments were ligated and then inserted into EcoRI and XbaI sites in the pME18S expression vector (Morishita et al., 1996
). cDNA of the NA gene from B/Lee/40 virus was amplified by RTPCR. The sense and the antisense primers for the first PCR were 5'-specific AGCAGAAGCAGAGCA and 3'- specific CGAGAACAATGATGA (Show et al., 1982
), respectively. The second PCR was performed with the 5'-specific primer incorporating an EcoRI site and the 3'-specific primer incorporating a BamHI site. These fragments were ligated and inserted into EcoRI and BamHI sites in the pME18S expression vector.
Haemadsorption assay of HA cDNA.
cDNA (200 ng) or mixed cDNA (200 ng of each) in 20 µl Eagle's MEM0 was incubated with 20 µl of diluted lipofectamine (36 µl lipofectamine/400 µl MEM0) for 15 min at room temperature. COS cells (0·5x10 5 cells per 18 mm coverslip) which had been prepared 18 h earlier were washed with MEM0 and then 0·16 ml MEM0 was added. The DNA and lipofectamine mixture (40 µl) was added to the medium and the cells were incubated for 6 h. The medium was changed to MEM containing 10% foetal calf serum (MEM10) and further incubated for 40 h at 37 °C. MEM10 was removed and the cells were washed twice with PBS, then 0·5% CRBC or goose red blood cells (GRBC) was added to the culture, which was then incubated for 15 min on ice. Unadsorbed red blood cells were washed away with MEM0.
Site-directed mutagenesis.
The HA1 region of the HA cDNA (EcoRISphI fragment) was inserted into pUC19. Site-directed mutagenesis was performed using a PCR mutagenic procedure as described previously (Morishita et al., 1996
). The mutant primers were GTCAATGTGGCTGGTGTG (asparagine to alanine at residue 36) TTTAAATGCACAGATCA (asparagine to lysine at residue 63), GACAACGACAAGACAGCA (asparagine to aspartic acid at residue 160), TTCCCTGATCAAACAGAA (asparagine to aspartic acid at residue 217), GGATTAGATAAAAGCGAG (asparagine to aspartic acid at residue 289) and GCCAATGGAGCCAAATAT (threonine to lysine at residue 314). The amino acids were numbered to correspond to the numbering for the H3 subtype according to the alignment of Krystal et al. (1983)
. Bold letters indicate the changed nucleotides used to generate the mutants. After the desired base substitutions were confirmed by sequencing, the DNA fragments were exchanged with the corresponding region in pME18S- HA. Double mutants were prepared by sequentially employing the same method.
Antisera and immunostaining.
Anti-HA antiserum against B/Kanagawa/73 virus was kindly supplied by T. Morishita of Aichi Prefectural Institute of Public Health, Japan. The indirect immunofluorescent staining was carried out as described previously (Nobusawa & Nakajima, 1988
).
Treatment of infected cells with Zanamivir.
Zanamivir (4-guanidino-2,3-dehydro-N-acetylneuraminic acid, GG167) was kindly supplied by Glaxo Wellcome Research and Development, Stevenage, UK. After virus infection, MDCK cells were washed with PBS, after which maintenance medium containing different amounts of Zanamivir was added. The cells were then incubated at 35 °C for 820 h.
Assay of NA activity on the infected cells.
Infected cells were harvested together with maintenance medium and NA activity was assayed using fetuin as a substrate for 18 h incubation at 37 °C as described previously (Palmer et al. , 1975
).
Scanning electron microscopy.
Infected or non-infected cells were incubated with or without Zanamivir. At 20 h post-infection, cells on the coverslip were fixed with 2% osmium tetroxide for 2 h. The cells were subjected to dehydration through a graded series of ethanol and t-butanol. After applying an ion-spatter coating with platinum, cells were scanned at 10 kV with a Hitachi S-4000 scanning electron microscope.
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Results
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Neuraminidase activity is necessary for haemadsorption of influenza B virus-infected MDCK cells
We infected MDCK cells with the ts-7 mutant of B/Kanagawa/73, which has no NA activity at 37·5 °C with these cells (Yamamoto-Goshima et al., 1994
). The infected cells were incubated for 8 h at 34 °C or 37·5 °C prior to carrying out haemadsorption experiments. At 34 °C, infected cells exhibited haemadsorption (Fig. 1b
); whereas at 37·5 °C, they did not (Fig. 1a
). However, when these latter cells were further incubated at 37·5 °C for 30 min with bNA, haemadsorption was observed (Fig. 1c
). Adsorbed red blood cells were measured by absorbance at 540 nm after disruption of these cells with water. The experiments were done twice independently. The A540 values were 0·291 and 0·293 at 34 °C, and 0·039 and 0·039 at 37·5 °C. After incubation with bNA at 37·5 °C for 30 min, the A540 values reached 0·230 and 0·211, respectively.

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Fig. 1. Haemadsorption of MDCK cells infected with the ts-7 strain. NA treatment and the haemadsorption assay using CRBC are described in Methods. MDCK cells were infected with ts-7 (1·2 p.f.u. per cell) and incubated at 37·5 °C (a) or 34 °C (b) for 8 h and the haemadsorption test was then performed. At 8 h post-infection, bNA treatment of infected cells incubated at 37·5 °C was carried out followed by haemadsorption ( c).
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Neuraminidase activity is necessary for the haemadsorption ability of influenza B virus haemagglutinin in HA cDNA-transfected COS cells
In order to locate the carbohydrate chain on the HA protein which inhibits haemadsorption, we used a DNA transfection system. We first checked the growth of influenza B virus in COS cells. The virus yield in COS cells measured by HA titre was one-tenth of that in MDCK cells. However, there was no obvious difference between the two with respect to haemadsorption (data not shown). HA cDNAs of B/Kanagawa/73 or B/Lee/40 viruses were expressed on COS cells. Immunofluorescent staining of HA protein by antiserum against B/Kanagawa/73 showed that 60% of cells were positive but no haemadsorption was observed at all (Fig. 2
a). bNA treatment of the transfected COS cells restored the haemadsorption capacity of HA (Fig. 2 b
) and co-transfection with NA cDNA of the B/Lee/40 virus enabled the haemadsorption activity of the HA protein (Fig. 2c
). The A540 value was 0·13 after co-transfection of HA cDNA and NA cDNA and 0·03 with HA cDNA. After incubation with bNA at 37·5 °C for 30 min, the A540 value reached 0·18. Immunofluorescent staining of transfected COS cells with HA-specific antisera revealed no difference in intensity or positive cell numbers between transfection with HA cDNA only and with HA cDNA and NA cDNA. Co-transfection of NA cDNA of A/PR/8/34 (H1N1) virus also aided the haemadsorption activity of influenza B virus (data not shown). On the other hand, transfection of NA cDNA alone did not result in haemadsorption.

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Fig. 2. Adsorption of CRBC to COS cells transfected with HA and NA cDNA of B/Lee/40 virus and bNA treatment of COS cells transfected with HA cDNA. bNA treatment and the haemadsorption test with CRBC are described in Methods. (a) Haemadsorption of COS cells transfected with HA cDNA. (b) Haemadsorption of COS cells transfected with HA and NA cDNA. (c) Haemadsorption of COS cells transfected with HA cDNA and treated with bNA.
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The position of the glycosylation site responsible for haemadsorption
We prepared several point mutants of HA protein of B/Lee/40 virus, all of which lacked one or two N-glycosylation sites, as indicated in Table 1
. In order to determine the conformational changes affecting haemadsorption, we carried out co- transfections with NA cDNA. Table 1
shows that all single point-mutated HA proteins had no haemadsorption activity at all without co-transfection of NA cDNA of B/Lee/40 virus. We chose to make double mutants involving residues close to the receptor binding site (Krystal et al., 1983
). Of these, only the mutant with point mutations at 160 and 217 had haemadsorption activity (Table 1
). Therefore, desialidation of the carbohydrate chain at both these positions might be necessary for the haemadsorption ability of HA protein of B/Lee/40 virus.
The glycosylation site which interferes with the haemadsorption of HA protein was conserved in all influenza B virus strains examined. We surveyed possible N-acetyl glycosylation sites on the HA protein of 44 strains of influenza B virus using GenBank sequences and sequences of strains from our laboratory. The glycosylation site at residue 160 was conserved in all strains, but that at position 217 was not.
Zanamivir interfered with the haemadsorption activity on influenza B virus-infected cells
Zanamivir is a strong inhibitor of the NA of influenza A and B viruses (Itzstein et al., 1993
). We examined the effect of Zanamivir on the haemadsorption activity on MDCK cells following infection with B/Lee/40 and B/Kanagawa/73 viruses. After infection with B/Kanagawa/73 (1·2 p.f.u. per cell) virus, different amounts of Zanamivir were added to the maintenance medium in which cells were incubated for 8 h at 35 °C. The NA activity and haemadsorption of infected cells are shown in Fig. 3
. The amount of NA activity and haemadsorption activity correlated in a dose-dependent manner with the amount of Zanamivir (Fig. 3a
). When 0·2 µM Zanamivir was used, the HA titre of the maintenance medium could not measured. Infected cells incubated with 0·4 µM Zanamivir were washed in PBS and further incubated with or without bNA for 30 min. Haemadsorption activity was observed only with bNA treatment (Fig. 3b
). Therefore, HA protein was present on the cell surface when they were incubated with Zanamivir. Immunofluorescent staining also showed the presence of the HA protein on the cell surface (data not shown). Similar results were obtained when B/Lee/40 virus was used.

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Fig. 3. Effect of Zanamivir on haemadsorption. (a) Haemadsorption and the NA activities of infected MDCK cells incubated with Zanamivir were examined. Two dishes of MDCK cells were infected with B/Kanagawa/73 (1·2 p.f.u. per cell) for 30 min at room temperature. After washing the cells twice with PBS, maintenance medium containing different amounts of Zanamivir was added to the cells. At 8 h post-infection, one sample was used for haemadsorption and HA titration. Maintenance medium was used for the titration of produced viruses as estimated with HA titres. HA titres in the fluid medium are shown as 16 or <2. Remaining cells were used for haemadsorption with CRBC. Cells were fixed and haemadsorbed cells were counted under a light microscope. The other sample was used for assessment of NA activity. The cells together with maintenance medium were collected and NA activity was measured. (b) Recovery of the haemadsorption capacity with bNA. MDCK cells were infected with B/Kanagawa/73 (1·2 p.f.u. per cell) and incubated with 0·4 µM Zanamivir. At 10 h post-infection, cells were washed twice with PBS and further incubated for 30 min with or without bNA. Haemadsorption was then carried out at 4 °C.
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Virion formation of influenza B virus with Zanamivir
NA protein is thought to play an important role in the release of virus from the cell surface by removing sialic acid from the HA protein to protect against aggregation (Palese et al., 1974
; Shibata et al., 1993
). In this event, HA would be expected to exhibit receptor-binding activity. In Zanamivir-containing medium, infected cells failed to exhibit haemadsorption activity and the cells were examined for virions by scanning electron microscope. In medium lacking Zanamivir, virus particles (Fig. 4
b) were observed, and even when Zanamivir was present at 0·4 µM, aggregates of virion particles were noted (Fig. 4c
, d
). However, with 4 µM Zanamivir, there was no clear evidence of virus particles. Knotty-type structures were seen which covered the cell surface (Fig. 4e
, f
) but it was difficult to say whether these structures were aggregated virions or an abortive virion form. The electron microscopic features of non-infected MDCK cells incubated with 4 µM Zanamivir or without (Fig. 4a
) were not different.

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Fig. 4. Scanning electron microscopy. MDCK cells were infected with B/Kanagawa/73 and incubated at 34 °C for 20 h with or without Zanamivir. The cells were then processed for scanning electron microscopy as described in Methods. (a) Mock- infected cells. (b) Infected cells without Zanamivir. (c , d) Infected cells with 0·4 µM Zanamivir. (e, f) Infected cells with 4 µM Zanamivir. Magnification: x10000 (a, b, c, e); x20000 (d , f). Asterisk in (b) indicates a virus particle and arrows (c,d) indicate virus aggregations.
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Comparison of haemadsorption activity using chick and goose erythrocytes
We previously reported that the HA protein obtained with influenza A (H1 and H3) viruses showed different binding activity to CRBC and GRBC (Morishita et al., 1993
, 1996
; Nobusawa et al., 1996
). However, with the HA proteins of B/Lee/40 or B/Kanagawa/73 viruses, there was no difference in binding activity with CRBC and GRBC, as seen in haemadsorption experiments with MDCK cells in the presence or absence of Zanamivir.
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Discussion
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The results obtained from the present haemadsorption experiments involving infection of MDCK cells with a ts mutant of influenza B virus and with or without the use of Zanamivir, or involving co-transfection of COS cells with HA and NA cDNAs, showed that sialic acid(s) had to be removed from the carbohydrate chains on the HA protein of influenza B virus in order to display haemadsorption activity. Furthermore, with B/Lee/40 virus, carbohydrate chains on the HA protein which interfered with the haemadsorption ability were found to be located at residues 160 and 217. Interference with haemadsorption by the sialic acid on the HA protein was examined using three cDNAs of influenza B viruses, B/Lee/40, MD/59 (Brassard & Lamb, 1997
) and B/Kanagawa/73. We examined another two HA proteins, of B/Aichi/84 and B/Aichi/94 viruses, but they also were incapable of haemadsorption activity without NA treatment. Therefore, it seems to be a common phenomenon with influenza B viruses that the sialic acid of carbohydrate chains on the HA protein interferes with haemadsorption activity. Ohuchi et al. (1995)
showed that sialic acids of carbohydrate chains at residues 123 and 149 interfered with the haemadsorption of H7 HA protein. For influenza B virus, Robertson et al. (1985)
and Oxford et al. (1990)
reported that the loss of glycosylation at residue 187 (H3 alignment) might correlate with egg adaptation. Recently, Gambaryan et al. (1999)
indicated that the loss of glycosylation sites in residue 187 of type B and 163 of type H1 viruses decreased the steric interference with more distant parts of sialic acid (
23 Gal)-containing receptor. Only few influenza B viruses isolated before 1990 had the glycosylation site at residue 187 of the HA protein. After that time, all influenza B viruses appeared to contain the glycosylation site at this position. HA proteins of B/Lee/40 or B/Kanagawa/73 viruses did not have this glycosylation site. Our results indicated that the carbohydrates at residues 160 and 217 were involved with binding of erythrocytes. The residue 160 is located near the receptor binding site and the carbohydrate at this residue is conserved in all strains. However, residue 217 is located relatively far from the receptor binding site compared to residue 160 and the carbohydrate at this residue is not conserved. Therefore, it was suggested that the carbohydrate at residue 160 mainly affected the haemadsorption activity and the carbohydrate at residue 217 might support the effect. The process of desialidation of HA protein by the NA protein is not clear. Our time-course experiments with a recombinant influenza A H1N1 virus suggested that some desialidation might take place after HA and NA proteins moved to the plasma membrane (Tong et al., 1998
). The NA inhibitor Zanamivir affected haemadsorption (Fig. 3
) and Morris et al. (1999)
reported that Zanamivir did not penetrate cells. Therefore, it might be concluded that NA affects the HA protein on the plasma membrane. However, Wandinger-Ness et al. (1990)
suggested that NA could interact with the HA protein in the Golgi apparatus of MDCK cells. Their experiments were carried out to block the movement of glycoproteins from the trans-Golgi and to allow them to accumulate in the trans-Golgi. Under these conditions, HA and NA may interact to remove the sialic acid without having to reach the plasma membrane. The aggregation of virions when 0·4 µM Zanamivir was used showed that even though the HA protein did not adsorb to erythrocytes, it still had the ability to bind to the sialic acid on the HA and/or NA protein. One possible explanation for the discrepancy between haemadsorption and the aggregation of virions is that a strong affinity on the cell surface might be needed to insure that the erythrocyte remains attached. Some sialic acids on the HA protein negatively affect the binding affinity or binding ability to the erythrocyte. We uncovered a similar discrepancy with H1 and H3 influenza viruses with respect to the binding to CRBC and GRBC (Morishita et al., 1993
, 1996
; Nobusawa et al., 1996
). In the presence of 4 µM Zanamivir, virion formation was not clearly observed. Scanning electron microscopy showed knotty structures (Fig. 4 e
, f
) but we do not yet know whether these forms were aggregates of virions or abortive forms of virions. The mechanism responsible for the removal of sialic acid on the plasma membrane was not clear, but a two-step process may be considered. (1) There would be a movement of HA and NA proteins from the trans-Golgi network to the plasma membrane, but it would not occur concomitantly or perhaps both proteins would be too far from each other for enzymatic activity in the trans-Golgi or sorting vesicles. (2) After moving to the plasma membrane, HA and NA proteins would be near enough for the NA enzymatic site to cleave sialic acid from the HA. We previously observed that in reassortant strains of H1N1 virus, the presence of M1 protein correlated with the haemadsorption activity with NA assisting in the reaction (Tong et al., 1998
). However, as for influenza B viruses, we do not have any information about a correlation between M1 protein and haemadsorption. M1 protein is the only protein in the virion which is present in sufficient quantity to form a shell beneath the lipid bilayer. A virion form which lacked an NA tail or HA and NA tails was found to have a grossly altered morphology characterized by a large and irregular shape (Mitnaul et al., 1996
; Jie & Lamb, 1996
). Enami & Enami (1996)
showed that HA and NA proteins stimulate movement of M1 protein to the plasma membrane. Therefore, some interaction of the HA and NA tails with the M1 protein should be considered to occur in virion formation. Finally we can summarize our findings: (1) desialidation of the HA protein by the NA protein of influenza B virus is necessary for haemadsorption activity. (2) Carbohydrates responsible for the activity of the HA protein are located at residues 160 and 217 of the HA protein. (3) Desialidation of the HA protein by the NA protein occurs after they move to the plasma membrane.
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Received 24 May 1999;
accepted 23 July 1999.