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Department of Microbiology and Infection, Nagoya City University Graduate School of Medical Science, Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
Correspondence
Makoto Ujike
ujike{at}nih.go.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Division of Respiratory Viral Diseases and SARS, Department of Virology III, National Institute of Infectious Diseases, Murayama Branch, Gakuen 4-7-1, Musashi-Murayama, Tokyo 208-0011, Japan. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Each HA monomer is synthesized as a single polypeptide chain (HA0) and cleaved into two subunits, HA1 and HA2, by proteolytic enzymes. This processing creates the hydrophobic N terminus of the HA2 subunit, referred to as the fusion peptide. After the entry of influenza viruses into host cells, an acidic environment in the endosome leads to ‘spring-loaded’ conformational changes in HA (Bullough et al., 1994; Carr & Kim, 1993; Gruenke et al., 2002; Qiao et al., 1998). According to current models, these conformational changes induce the formation of an extended 
-helical coiled coil in the stem regions of HA2 and cause the previously buried fusion peptide to move toward the top of the HA to interact with the target membrane. Following the insertion of the fusion peptide into this membrane, the HA2 ectodomain bends like a hairpin. The fusion peptides and membrane-anchoring regions are thereby situated close to each other at one end (Borrego-Diaz et al., 2003; Chen et al., 1999; Park et al., 2003). This close contact initiates membrane fusion. Therefore, the HA2 ectodomain is the main contributor to the early step of fusion. The subsequent fusion process can be divided into three steps: lipid mixing between outer leaflets of the viral membranes (hemifusion), fusion-pore formation by merging of the inner lipid leaflets, and pore dilation (Spruce et al., 1989).
HA2 is composed structurally of a large ectodomain carrying the fusion peptides, a transmembrane (TM) domain and a short cytoplasmic tail (CT). Recently, many studies have shown that the TM domain and CT are involved in later steps of fusion, fusion-pore formation and pore dilation (Armstrong et al., 2000; Fischer et al., 1998; Kemble et al., 1994; Kozerski et al., 2000; Markosyan et al., 2000; Melikyan et al., 1995, 1999, 2000; Naeve & Williams, 1990; Ohuchi et al., 1998; Sakai et al., 2002; Ujike et al., 2004; Wagner et al., 2005). Mutant HAs in which the TM domain and CT are replaced with a glycophosphatidylinositol anchor promote hemifusion, but not full fusion (Kemble et al., 1994; Melikyan et al., 1995), and mutant HAs with a TM domain that is too short mediate only hemifusion (Armstrong et al., 2000). Both findings indicate that the TM domain plays an important role during the transition from hemifusion to complete fusion. On the other hand, a CT is not essential for HA-mediated fusion, because mutant HAs lacking this structure can support full fusion (Simpson & Lamb, 1992; Ujike et al., 2004). Nevertheless, altering the CT of HA can affect fusion activities. Chimeric HAs with parallel replacement of both the TM domain and CT of wild-type (wt) HA with corresponding domains of a non-viral protein (CD4) inhibited pore dilation (Kozerski et al., 2000). Elongation of the CT by the addition of one to six amino acid residues downstream of the C terminus can abolish fusion activity (Ohuchi et al., 1998). In addition, the mutants of several HA subtypes lacking palmitic acid(s) (PA) in the TM domain and CT have exhibited strongly impaired pore formation or syncytium formation (Fischer et al., 1998; Naeve & Williams, 1990; Sakai et al., 2002; Ujike et al., 2004; Wagner et al., 2005). These findings show that modification of the TM and/or CT domains in HA has a strong effect on fusion activities. However, these domains do not need to fulfil stringent sequence requirements for fusion activity, because TM domains and CTs derived from other viral or non-viral proteins (polyimmunoglobulin receptor) can substitute effectively for that of HA and still cause fusion (Melikyan et al., 1999; Schroth-Diez et al., 1998). Thus, confusion arose as to why these domains consist of relatively conserved amino acids or which amino acids of these domains in wt HA contribute to fusion activities. Melikyan et al. (1999) addressed this issue and found that a point mutation at a semi-conserved glycine residue within the TM domain of subtype H2 HA inhibited pore formation completely, indicating that certain TM residues can play a critical role in the process of fusion. This finding is the first evidence that the fusion activities of HA are dependent on the TM sequence. In contrast, the involvement in fusion of certain single residue(s) within the CT has not been identified.
A comparison of three amino acids at the C terminus on the CT of influenza A and B HA (AHA and BHA, respectively) revealed that this region consists of highly conserved hydrophobic amino acids (Kawaoka et al., 1990; Krystal et al., 1982; Nobusawa et al., 1991; Röhm et al., 1996). Twelve subtypes of AHA have an ICI sequence in this region. Other AHA subtypes have an FCI or VCI sequence, whist BHA has an ICL sequence. It is of interest that the endmost C-terminal amino acid of the CT is I in all AHA subtypes, but L in BHA. All sequences form a highly hydrophobic cluster within the endmost region of the CT C terminus, not only through hydrophobic amino acids, but also by the cysteine residue having been modified by hydrophobic PA. However, the role of the highly conserved hydrophobicity of the HA C terminus remains unclear.
In the present study, we examined the effect of changing the hydrophobicity of the amino acid at the endmost portion of the C terminus of BHA-CT on fusion activity. Our results showed that a point mutation of the C terminus had no significant effect on the ability to induce hemifusion or to form fusion pores; however, the hydrophobicity of the single amino acid at this position played an important role in syncytium formation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Site-directed mutagenesis.
cDNA of wt HA (B/Kanagawa/73) was subcloned into the EcoRI and XbaI sites in the pME18s expression vector (BHA/pME18s) as described previously (Luo et al., 1999, 2002). Site-directed mutagenesis was carried out with standard PCR protocols by using the overlap-extension technique (Horton & Pease, 1991). Mutant HAs carrying a single amino acid change were constructed by replacing the L residue with other residues, using BHA/pME18s as a template. A mutant HA with an L deletion was created by insertion of a stop codon at its position. All mutant HAs were sequenced to confirm the presence of the desired mutations in the respective HAs.
Expression of wt and mutant HAs.
Transient expression of wt and mutant HAs on COS cells was performed by using Lipofectamine 2000 (Invitrogen) as described previously with slight modifications (Ujike et al., 2004). In brief, COS cells (1.6x105) were grown overnight and the growth medium was replaced with 0.3 ml DMEM and 30 µl Opti-MEM (Invitrogen), containing 360 ng cDNA and 0.9 µl Lipofectamine. At 6 h post-transfection, the transfection medium was changed to 7.5 % FBS/DMEM. At 48 h after transfection, the respective HA-expressing cells were analysed.
Fluorescence-activated cell-sorting (FACS) analysis.
HA-expressing cells were subjected to FACS analysis as described previously (Ujike et al., 2004). In brief, these cells were removed from the dish by using 0.02 % EDTA and 0.05 % trypsin and washed with PBS containing 0.5 % BSA. The cells were incubated with anti-B/Kanagawa/73 hyperimmune rabbit serum and a fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin. Unbound antibodies were washed out at every step with 0.5 % BSA/PBS. The cells were then suspended in 500 µl PBS and analysed with a FACScan (Becton Dickinson).
Metabolic labelling and immunoprecipitation.
HA-expressing cells were incubated in methionine- and cysteine-free DMEM (DMEM/met–cys–) for 1 h at 40 h post-transfection, labelled with Tran[35S] label [2.5 µCi (92.5 kBq)] in 500 µl DMEM/met–cys– for 3 h and chased with DMEM for 4 h. The Tran[35S]-labelled cells were then treated with 10 µg (L-tosylamido-2-phenyl)ethyl chloromethyl ketone-treated trypsin (TPCK/trypsin) ml–1 for 15 min at 37 °C to cleave HA0 into HA1 and HA2. For labelling with [3H]PA, HA-expressing cells were incubated in labelling medium (7.5 % FBS/DMEM supplemented with 5 mM sodium pyruvate) for 1 h at 29 h post-transfection and subsequently labelled with [3H]PA [250 µCi (9.25 MBq)] in 500 µl labelling medium for 18 h. Cells were washed once with cold PBS and lysed in cold RIPA buffer [0.15 M NaCl, 50 mM Tris/HCl (pH 7.4), 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % SDS]. Lysates were centrifuged at 15 000 r.p.m. (RT 15A8 rotor; Hitachi) for 30 min and the supernatants were immunoprecipitated overnight at 4 °C with anti-B/Kanagawa/73 rabbit serum and protein G–Sepharose beads. The pellets were then washed four times with RIPA buffer. The immunocomplexes of Tran[35S]-labelled cells were released from the beads by boiling for 5 min in sample buffer (50 mM Tris, 2 % SDS, 0.1 % bromophenol blue, 10 % glycerol, 1 % 2-mercaptoethanol) and those of the [3H]PA-labelled cells were released by incubating for 3 min at 80 °C in the sample buffer containing 20 mM dithiothreitol instead of 2-mercaptoethanol. These samples were analysed by SDS-PAGE in a 10–20 % gradient gel and visualized by autoradiography using Kodak X-OMAT AR film or a Fujix BAS 2500 system.
Haemadsorption test.
HA-expressing cells were washed once with DMEM and incubated with Vibrio cholerae neuraminidase (VCNA; 25 mU ml–1) at 37 °C for 2 h, because BHA requires VCNA pretreatment for its haemadsorption activity (Luo et al., 1999). After the pretreatment, the cells were washed with DMEM and overlaid with 1.0 % human red blood cells (hRBCs). After incubation for 20 min at 4 °C, unbound erythrocytes were removed by washing with DMEM. Haemadsorption was quantified by measuring A540 after haemolysis with distilled water.
Labelling of hRBCs with R18 and calcein–AM.
hRBCs were colabelled with the membrane probe octadecylrhodamine B (R18) and the aqueous dye calcein–AM (Molecular Probes, Inc.) as described by Kemble et al. (1993) and Morris et al. (1989). In brief, a 10 µl aliquot of 2 mM R18 in ethanol was added to 5 ml 1 % hRBCs in PBS under vortexing. The mixture was incubated in the dark for 30 min at room temperature (r.t.) and then incubated for 20 min at r.t. with 7.5 % FBS/DMEM to absorb unbound R18. After the incubation, R18-labelled hRBCs were washed three times with PBS and resuspended in 1.25 ml PBS (4 % R18-labelled hRBCs in PBS). A 5 µl aliquot of 4 mM calcein–AM in DMSO was added to 500 µl 4 % R18-labelled hRBCs and the suspension was incubated in the dark for 1 h at 37 °C. The remaining procedure was the same as for the R18 labelling. The double-labelled hRBCs were suspended in a desired volume of DMEM.
Cell–hRBC dye-transfer experiment.
HA-expressing cells were pretreated with TPCK/trypsin and VCNA as described above and overlaid with colabelled hRBCs. To induce fusion, HA-expressing cells adhering to the colabelled hRBCs were incubated with acidic medium [20 mM citrate acid and 125 mM NaCl (pH 5.0)] for 5 min at 37 °C and neutralized with PBS containing 1 mM CaCl2, 1 mM MgCl2 and 20 mM raffinose to prevent colloidal–osmotic swelling of the hRBCs that could be induced by HA-mediated leakage (Melikyan et al., 1999). After incubation for 20 min at 37 °C, the dye transfer of R18 and calcein from hRBCs to HA-expressing cells was observed under a fluorescence microscope (x200) and photographed.
Syncytium-formation assay.
The syncytium-formation assay was performed as described previously with slight modification (Nobusawa et al., 1995; Ujike et al., 2004). HA-expressing COS cells were pretreated with TPCK/trypsin and exposed to warmed acidic medium [20 mM citrate acid and 125 mM NaCl (pH 5.0)] for 5 min at 37 °C. The acidic medium was replaced with 7.5 % FBS/DMEM and cells were incubated for 3 h. After incubation, the cells were fixed with cold ethanol and stained with fivefold-diluted Giemsa's solution. To evaluate syncytium-formation activity quantitatively, different areas of the culture dish were selected at random under an inverted microscope (x100) and photographed. Nuclei in syncytia (more than two nuclei) were counted in four selected fields. Syncytium-formation activity was calculated from the number of nuclei in syncytia divided by the number of HA-expressing cells, as estimated from the results of FACS analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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summarizes the characteristics of the substituted amino acids. The names of the HA mutants and their amino acid changes are shown in Fig. 1. These mutant and wt HAs were expressed transiently in COS cells by using the pME18s expression vector.
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(a) shows that the total synthesis of mutant HA proteins did not vary significantly from that of wt HA and that mutant HAs were cleaved from HA0 into HA1 and HA2 as efficiently as wt HA. In addition, the levels of surface expression of wt and mutant HAs on COS cells were examined by FACS analysis. As shown in Table 1
, the percentage and mean fluorescence intensity of cells expressing HA were similar in the wt and mutant HAs, suggesting that these HAs were expressed at similar densities and levels on cell surfaces.
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, the amounts of erythrocytes bound to the COS cells were not significantly different among the cells expressing wt and mutant HAs. These data indicated that a point mutation or deletion at the endmost portion of the C terminus of the BHA-CT had no effect on the total cell-surface expression or receptor-binding activity of each HA.
Acylation of wt and mutant HAs
The CT of BHA has two acylation sites at positions 578 and 581 that are modified with PA (PA578 and PA581). Our previous results showed that PA578 potentially had an inhibitory effect on pore and syncytium formation and that PA581 suppressed the potential inhibitory effect of PA578 (Ujike et al., 2004). Therefore, the level of acylation of mutant BHAs is an important factor in fusion activities. To determine whether the acylation levels of the mutant HAs were comparable to that of wt HA, HA-expressing cells were labelled with [3H]PA. As shown in Fig. 2(b), the amounts of PA incorporated by wt and mutant HAs were not significantly different. In addition, the amounts of PA incorporated by all of the mutant HAs were considerably larger than that of the mutant without PA581 of wt HA (C6 mutant) (Fig. 2b, right lane), indicating that the mutants could incorporate PA581 efficiently. These findings showed that both acylation sites of the mutant HAs had incorporated PAs in the same efficient manner as that of wt HA.
Hemifusion and pore-formation abilities of wt and mutant HAs
To determine whether a point mutation at the C terminus of the BHA-CT affected hemifusion and fusion-pore formation, we measured the low pH-triggered fusion of COS cells expressing wt and mutant HAs to hRBCs colabelled with the lipidic fluorescent dye R18 and the water-soluble fluorescent dye calcein. The transfer of both dyes from hRBCs to COS cells expressing the respective HAs was observed under a fluorescent microscope. As shown in Fig. 3, no significant differences in the transfer of either dye were found among wt and one-point mutant HAs. Moreover, the mutant with a deletion of L at the C terminus of the BHA-CT induced transfer at levels similar to that of wt HA. These results showed that a point mutation and deletion at the C terminus had no measurable effect on the induction of hemifusion and pore formation.
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). The mutant lacking an L at the C terminus of the BHA-CT showed a significant ability to induce syncytium formation (L*=68 %), indicating that L at this position was not indispensable for BHA-mediated fusion activities. However, mutant HAs with a point mutation at position L exhibited different syncytium-forming abilities that depended on the substituted amino acid. The mutants R, K, S or W showed strongly reduced abilities to form syncytia compared with wt HA (R=21 %; K=18 %; S=26 %; W=34 %). On the other hand, mutants V and I supported syncytium formation completely, to an extent similar to that of wt HA. As shown in Table 1, the hydrophobicity values of the substituted amino acids among mutants with impaired syncytium formation were lower than that of L of the wt BHA, whereas those of mutants supporting syncytium formation completely were equal to or higher than that of wt HA. These results showed that the hydrophobic amino acids at the endmost portion of the C terminus of the BHA-CT supported the ability to form syncytia and that L at this position was not essential for BHA-mediated fusion.
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). We hypothesized that the structure of the fusion-deficient mutant HA-CTs could not counteract the negative effect of PA578. Thus, it was thought that the removal of PA578 from these mutant HAs would restore their fusion activities. To prove this hypothesis, we introduced an amino acid change of cysteine at position 578 by replacing cysteine with serine in mutants R and K (R-C9 and K-C9). On analysis, both mutants were expressed properly and both had incorporated PA581 efficiently to an extent comparable to that of C9, which has a single acylation site at position 581 of wt HA (Fig. 6a). Moreover, the abilities of both mutant HAs to form syncytia were restored significantly (R-C9=65 %; K-C9=61 %) (Fig. 6b). These results implied that the hydrophobicity of the amino acid at the endmost portion of the C terminus of the BHA-CT plays an important role in the suppression of the negative effect of PA578 on fusion activities.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Recently, many researchers have emphasized that the CT of HA plays an important role in the later steps of the fusion process. Elongation of the CT in influenza HA by adding several amino acid residues downstream of the C terminus was shown to impair fusion activities (Ohuchi et al., 1998). Chimeric HAs with replacement of the CT with corresponding domains of non-viral proteins inhibited pore dilation (Kozerski et al., 2000). The mutants of several HA subtypes changing acylation site(s) in the CT exhibited strongly impaired pore or syncytium formation (Fischer et al., 1998; Naeve & Williams, 1990; Sakai et al., 2002; Ujike et al., 2004; Wagner et al., 2005). These findings indicated that the CT of influenza HA is involved in regulation of its fusion activity. However, the dependence of fusion on the CT sequence or the involvement of specific amino acid(s) had not been elucidated. In this study, we provide the first evidence that the CT of influenza BHA regulates fusion activity in a specific amino acid-dependent manner. In addition, we found that the hydrophobicity of a single amino acid at the endmost portion of the C terminus of the BHA-CT played an important role in syncytium formation. On the other hand, L at the endmost portion of the C terminus was not essential for BHA-mediated fusion, because the mutant HA with the deletion of L induced full fusion. One possible explanation is that the CT has not evolved simply to optimize fusion. It must fulfil other functions as well as including virus maturation and packaging, as has been reported for the AHA-CT (Chen et al., 2005; Zhang et al., 2000a, b). The BHA-CT appears to have retained the L residue of its C terminus for optimal function during its evolution.
Why does a point mutation of the endmost portion of the C terminus of BHA-CT have such a strong effect on fusion activity? As the endmost region of the C terminus of the wt BHA-CT comprises highly conserved hydrophobic amino acids, it is likely that this local region forms the specific structure that promotes the orientational shifting or tilting of the TM domains required for proper fusion activity (Kozerski et al., 2000; Melikyan et al., 1999). Thus, it is conceivable that a point mutation at the endmost portion of the CT destabilizes this specific structure, which causes the constraint of the TM domains to interfere with the fusion processes. As for our present results, the highly hydrophobic amino acids V and I at the BHA-CT C terminus, like the L of wt BHA-CT, would maintain the correct structure and promote full fusion. The deletion of L would neither support this structure nor destabilize it and thus the effect on the fusion activity was moderate. In contrast, the amino acids R, K and S, without sufficient hydrophobicity, or the larger-sized amino acid W at the C terminus would destabilize this structure and could not promote full fusion. Thus, it is likely that the hydrophobicity of a single amino acid at the C terminus is one of the important factors in maintaining the correct structure of the BHA-CT. In addition, we also showed that deletion of PA578 in mutants with K or R at the C terminus of the CT restored their fusion activity significantly. Because PA578 of the BHA-CT potentially had an inhibitory effect on fusion activities, it seems that the BHA-CT of the fusion-deficient mutants with a point mutation at the C terminus could not counteract the inhibitory effect of PA578; this would lead to a conformational constraint of the BHA-CT that would hinder the movement of the TM domains. Therefore, it appears that the C-terminal region of the wt BHA-CT forms a specific sequence-dependent structure, which would facilitate the movement of the TM domains required for proper fusion activity. Further experiments on the CT C terminus of several types of HA will probably contribute to a better understanding of the role of the HA-CT in fusion activity.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 14 September 2005;
accepted 2 February 2006.
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