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1 Department of Biomedical Sciences, Chubu University, 1200 Matsumoto-Cho, Kasugai, Aichi 487-8501, Japan
2 Department of Microbiology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan
3 Department of Microbiology, Suzuka University of Medical Science and Technology, 1001-1 Kishioka-Cho, Suzuka, Mie 510-0226, Japan
Correspondence
Masato Tsurudome
turudome{at}doc.medic.mie-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Lamb & Parks, 2007), which include human parainfluenza virus 3, Newcastle disease virus (NDV) and parainfluenza virus 5 (PIV-5), respectively. The HN protein is responsible for binding to the viral receptor, sialoconjugate, and for enzymic cleavage of the receptor, whilst the F protein is involved in membrane fusion (Karron & Collins, 2007; Lamb & Parks, 2007). As F protein-mediated membrane fusion occurs at neutral pH, envelope–cell fusion takes place at the plasma membrane and the virus-infected cells mediate syncytium formation (or cell fusion) with neighbouring cells. The F precursor, F0, is cleaved by cellular proteases and forms a disulfide-bonded subunit structure consisting of the transmembrane subunit F1 and the peripheral subunit F2. Cleavage of the F protein results in a structural rearrangement of the F protein (Dutch et al., 2001; Hsu et al., 1981; Kohama et al., 1981; Tsurudome et al., 2006; Umino et al., 1990), which involves the generation of a highly conserved fusion peptide at the F1 amino terminus (Hsu et al., 1981; Kohama et al., 1981). The fusion peptide is considered to play a direct role in the fusion event (Gething et al., 1978; Novick & Hoekstra, 1988).
The cleaved F protein is considered to mediate fusion by undergoing a series of conformational changes, as occurs with other class I viral fusion proteins such as the influenza virus haemagglutinin protein (Yin et al., 2006). Interestingly, the F protein requires a fusion-promoting function of the receptor-binding protein, HN, in a type-specific manner (Hu et al., 1992), whilst the other class I fusion proteins reported so far possess both receptor-binding and fusion activities. It is considered that binding of the HN protein to the receptor induces a conformational change in the HN protein that, in turn, triggers the conformational changes in the F protein through an HN–F interaction (Takimoto et al., 2002). The stalk domain of the HN protein is inferred to contain the site that determines the F protein specificity for promoting fusion (Deng et al., 1997; Melanson & Iorio, 2006; Tanabayashi & Compans, 1996; Tsurudome et al., 1995), whilst the F1 middle region in the head domain of the F protein is considered to determine the HN protein specificity (Tsurudome et al., 1998). An interaction between the stalk domain of the attachment protein and the head domain of the F protein has also been reported for canine distemper virus, a member of the genus Morbillivirus in the family Paramyxoviridae (Lee et al., 2008). However, the molecular basis of the HN–F interaction has not been clarified as yet and it is an open question as to how this interaction triggers the conformational changes in the F protein.
It has been shown previously that the F protein of PIV-5 strain W3A does not require co-expression of the HN protein for its fusion activity (Horvath et al., 1992; Paterson et al., 1985), whereas the F protein of strain WR requires the HN protein, as occurs with other paramyxovirus F proteins (Ito et al., 1997). It was also shown that a mutant L22P, in which Leu-22 of the WR F protein was replaced with the W3A F counterpart (Pro), mediated extensive cell fusion independently of co-expression of the HN protein (Ito et al., 1997). This observation that a single amino acid substitution at the F2 amino terminus can bestow HN-independent fusion activity on an otherwise ‘fusion-inactive’ WR F protein led us to compare the structural and functional properties of L22P with those of the WR F protein; this approach was undertaken to provide clues that might be helpful for understanding the molecular mechanism of paramyxovirus fusion. Accordingly, we found that there was a difference in conformation between L22P and the WR F protein either before or after cleavage (Tsurudome et al., 2001, 2006), suggesting that the cleaved L22P is so unstable that it easily undergoes the conformational changes that lead to cell fusion. However, it is not known how Pro-22 destabilizes the protein, although the presence of a Pro residue at position 22 has been shown to decrease the energy required to trigger the presumptive conformational change to the fusion-active state (Paterson et al., 2000). Interestingly, the F protein of PIV-5 strain T1 does not induce cell fusion, even when co-expressed with the W3A HN protein, and chimeric analysis between L22P and the T1 F protein has suggested that Glu-132, which is located immediately downstream of the fusion peptide, also contributes to the HN-independent fusion activity of L22P (Ito et al., 2000). As the T1 F protein counterpart of Glu-132 is a Lys residue, it was anticipated that the presence of a negatively charged amino acid at position 132 might be crucial for the fusion activity.
In the present study, the roles of the amino acids at positions 22 and 132 in the HN-independent fusion activity of the PIV-5 F protein were examined by mutational analyses. The results suggested that the chemical properties of these amino acids, rather than their effects on the conformation of the F protein, may be important for fusion activity.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Recombinant plasmids.
Recombinant SR
plasmids harbouring a cDNA encoding the WR F protein, L22P or mumps virus (MuV) HN protein have been described previously (Ito et al., 1997).
Transient expression of the F proteins.
BHK cells were seeded at 5x105 cells per well in six-well culture plates (Becton Dickinson) and incubated at 37 °C for 24 h in MEM supplemented with 10 % fetal calf serum (FCS). Each recombinant plasmid (2 µg per well) was then added to the cells using the calcium phosphate method (Graham & van der Eb, 1973). After 3 h of incubation at 37 °C, the cells were treated with 15 % glycerol in HEPES-buffered saline [50 mM HEPES (pH 5.7), 0.75 mM sodium phosphate, 140 mM NaCl] at room temperature for 3 min and incubated in MEM fortified with 10 % FCS at 37 °C for 24 h.
Site-directed mutagenesis.
The introduction of mutation-generating synthetic oligonucleotides into the target recombinant plasmid was performed as described previously (Ito et al., 2000; Tsurudome et al., 1995) using a U.S.E. Mutagenesis kit (Amersham Pharmacia Biotech AB) as specified by the manufacturer.
Quantification of cell fusion and surface expression of the F proteins.
Subconfluent cultures of BHK cells in six-well culture plates were transfected with 2 µg each recombinant plasmid per well. After incubation at 37 °C for 24 h, the cells were fixed with 3.7 % formaldehyde and observed by using an inverted phase-contrast microscope (Olympus). The photomicrographs of three randomly chosen fields were subjected to morphometric measurement of syncytia and the mean fusion indices were calculated as described previously (Tsurudome et al., 1995). For quantification of surface expression levels of the F proteins, the same wells used for the morphometric measurement of syncytia were subjected to ELISA. Briefly, the cells were treated with anti-PIV-5 F monoclonal antibody (mAb) 6-7 (Tsurudome et al., 2001), washed three times with PBS and then treated with a peroxidase-conjugated IgG fraction of goat anti-mouse immunoglobulin (Cappel Laboratories). One millilitre of substrate solution (Sakata et al., 1984) was added to each well and the absorbance value at 490 nm was measured. The absorbance value of control cells transfected with plasmid SR was subtracted from the value of each sample and normalized with that produced by L22P-transfected cells.
Cell-surface biotinylation and immunoprecipitation.
Subconfluent cultures of BHK cells in six-well culture plates were transfected with 2 µg each recombinant plasmid per well. After 24 h of incubation at 37 °C, the plates were placed on ice and the cells were washed three times with ice-cold PBS containing 0.1 mM CaCl2 and 1 mM MgCl2 (PBS-CM). The cells were then treated with 0.3 mg Sulfo-NHS-Biotin ml–1 in PBS-CM on ice for 30 min and unbound reagents were quenched by adding ice-cold 0.1 M glycine in PBS-CM. After three washes with PBS-CM, the cells were lysed with 600 µl lysis buffer [25 mM HEPES (pH 7.6), 1 % Triton X-100, 3 mM β-glycerophosphate, 3 mM EDTA, 1 mM PMSF, 137 mM NaCl] per well. Proteins in the lysates were immunoprecipitated with mAb 6-7, subjected to SDS-PAGE and electroblotted onto Hybond-P PVDF membrane (Amersham Biosciences). For detection of the biotinylated proteins by enhanced chemiluminescence (ECL), the membrane was treated successively with streptoavidin–biotin–peroxidase complex (Vector Laboratories) and Western blotting Luminol Reagent (Santa Cruz Biotechnology), followed by exposure to X-ray film (Konica).
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RESULTS |
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and 2a). Importantly, E132K was expressed on the cell surface and cleaved into F1 and F2 as efficiently as L22P (Fig. 2a, b). As similar results were obtained with mutants L22P-E132R and L22P-E132H, we initially postulated that the presence of a basic residue at position 132 might be harmful to the fusion activity. However, substitution of Glu-132 with Ile (L22P-E132I), Ala (L22P-E132A), Asn (L22P-E132N) or Gln (L22P-E132Q) also resulted in a reduction in the fusion activity. Notably, by contrast, substitution of Glu-132 with Asp (L22P-E132D) did not greatly affect the fusion activity (Figs 1 and 2).
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. As shown in Fig. 3, the side chain of Glu-132 proved to be exposed on the trimer surface but no acidic amino acid was present in close proximity to Glu-132. However, as L22P undergoes a structural rearrangement upon cleavage (Tsurudome et al., 2006), there was the possibility that, subsequent to cleavage, Glu-132 would interact with an amino acid that had been somewhere in the neighbourhood before cleavage.
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). We initially intended to substitute these acidic amino acids individually with a basic amino acid (Lys) in the context of L22P. If electrostatic repulsion between Glu-132 and its putative acidic partner were involved in the HN-independent fusion activity of L22P, then substitution of the partner with a basic residue would result in a mutant with reduced fusion activity. However, there was a concern that such a mutant would show reduced fusion activity even if it was not the interacting partner of Glu-132. We thus decided to carry out mutational analysis of L22P-E132K instead of L22P. As L22P-E132K showed remarkably low fusion activity compared with L22P, we anticipated that its fusion activity would be restored when supplied with its ‘basic’ interacting partner by substitution if electrostatic repulsion takes place between these amino acids. Therefore, in order to determine the putative interacting partner of Lys-132, we substituted the eight acidic amino acids individually with a Lys residue in the context of L22P-E132K. We eventually found that, when Asp-416 of L22P-E132K was substituted with Lys, the resulting mutant L22P-E132K/D416K exhibited a prominent fusion activity that was comparable to that of L22P, whereas individual substitution of the remaining seven acidic amino acids with Lys could not restore the fusion activity of L22P-E132K, regardless of the efficient surface expression and cleavage of the mutants (Fig. 4). Thus, Asp-416 seemed the most likely candidate for the interacting partner of Glu-132.
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). Interestingly, when Asp-416 of L22P-E132K was substituted with a basic residue (Arg), the resulting mutant, L22P-E132K/D416R, displayed HN-independent fusion activity at a level comparable to that of L22P-E132K/D416K (Fig. 5). By contrast, when Asp-416 of L22P-E132K was substituted with another acidic residue (Glu), the resulting mutant L22P-E132K/D416E showed much lower fusion activity than that of L22P-E132K/D416K. It is worth noting that substitution of Asp-416 of L22P with a Lys residue resulted in a clear reduction in the fusion activity (Fig. 5a, L22P-D416K), excluding the possibility that the presence of Lys-416 might intrinsically promote fusion activity. Taken together, these results suggested that the presence of basic amino acids at positions 132 and 416 is important for the induction of HN-independent cell fusion by L22P-E132K/D416K, suggesting the importance of acidic amino acids (Glu-132 and Asp-416) in the fusion activity of L22P.
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). As the change from Leu to Pro would result in a decrease in local hydrophobicity as well as an alteration in the local conformation of the F2 amino terminus, we investigated the effect of amino acid substitutions at position 22 on the HN-independent fusion activity by introducing a series of mutations into the WR F protein background (Fig. 6). Intriguingly, substitution of Leu-22 with a small hydrophobic (Ala) or an uncharged polar (Asn) amino acid resulted in the generation of a mutant (L22A or L22N, respectively) with HN-independent fusion activity, although the efficiency was apparently lower than that of L22P. Furthermore, substitution of Leu-22 with an acidic (L22E) or basic (L22K) amino acid could also bestow fusion activity on the WR F protein. By contrast, substitution of Leu-22 with an amino acid with an aliphatic side chain (L22I or L22F) failed to bestow fusion activity on the WR F protein, whereas these mutants exhibited prominent fusion activity, similar to the WR F protein, when they were co-expressed with the MuV HN protein (Fig. 6), which can efficiently promote PIV-5 F protein-mediated cell fusion (Ito et al., 1997). These results indicated that the presence of an amino acid with a hydrophobic side chain at position 22 renders the WR F protein HN-dependent. As substitution of Leu-22 with any non-hydrophobic amino acid readily eliminated the HN dependency whilst substitution with Ile or Phe did not, local hydrophilicity rather than the local conformation of the F2 amino terminus seems to be important for the HN-independent fusion activity.
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). This difference could be detected by using anti-F mAbs 6-7 and 21-1, the latter of which discriminates more clearly between the WR F protein and L22P (Tsurudome et al., 2001). Thus, in order to see whether the conformation of the mutant F proteins that were generated in the current study would be affected by the mutation, we examined the reactivity of mAbs to the F proteins by ELISA using plasmid-transfected and 3.7 % formaldehyde-fixed BHK cells. As shown in Fig. 7(a), the reactivity of mAb 6-7 to the WR F protein was somewhat lower than that to L22P, which is consistent with our previous data from flow cytometry using plasmid-transfected and 3.7 % formaldehyde-fixed HeLa cells (Tsurudome et al., 2001). In contrast, the reactivity of mAb 21-1 to the WR F protein was remarkably low (about 20 %) compared with its reactivity to L22P (Fig. 7b), which is also consistent with our previous data using HeLa cells (Tsurudome et al., 2001). Interestingly, it was shown that the reactivity of mAbs 6-7 and 21-1 to L22A, L22N, L22K, L22E, L22I and L22F was similar to that of the WR F protein, whilst their reactivity to L22P-E132K and L22P-E132K/D416K was similar to that of L22P (Fig. 7). These results thus indicated that, in terms of conformation as detected by the two mAbs, the former six mutants and the latter two mutants are indistinguishable from the WR F protein and L22P, respectively. Hence, the HN-independent fusion activity of L22A, L22N, L22K, L22E and L22P-E132K/D416K (shown in Fig. 6a and Fig. 4a, respectively) could not be explained by the notion that they have a unique conformation shared by L22P. It should also be noted that the reduced HN-independent fusion activity of L22P-E132K (Fig. 2a) was not attributable to its conformation. Taken together, we could not find a clear correlation between the conformation of the newly generated mutant F proteins and their HN dependency.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). According to this model, binding of the HN protein to the receptor induces a conformational change in the HN protein. The F protein is then released from the complex and undergoes conformational changes, followed by adoption of the post-fusion form, which is tightly linked to membrane fusion and pore formation (Lamb & Parks, 2007). In contrast, it is well known that several F proteins can mediate cell fusion in the absence of the HN protein. As described so far, the L22P mutation at the F2 amino terminus bestows HN-dependent fusion activity on the WR F protein (Ito et al., 1997). Interestingly, G3A and G7A mutations in the fusion peptide also cause the WR F protein to be triggered to mediate fusion in the absence of co-expression of the HN protein (Russell et al., 2004). Furthermore, the L539A and L548A mutations in the long cytoplasmic domain of the F protein of SER virus, a porcine PIV-5 closely related to canine T1 (Tong et al., 2002), rescue syncytium formation and eliminate the HN protein requirement for membrane fusion (Seth et al., 2003). In the case of NDV, the L289A mutation in the F protein alters the requirement for the HN protein in fusion (Sergel et al., 2000). Notably, it has been reported that the presence of Pro at residue 22 destabilizes the PIV-5 F protein and thereby decreases the energy required to trigger the conformational changes in the absence of the HN protein (Paterson et al., 2000). However, it has not been clarified how the presence of Pro-22 destabilizes the F protein.
When L22P is expressed alone in HeLa cells, the conformational changes appear to take place immediately after cleavage without any contact with neighbouring cells (Tsurudome et al., 2006). Interestingly, in this context, Ludwig et al. (2008) recently performed electron cryomicroscopy of PIV-5 W3A virions and concluded that the cleaved F protein not in complex with the HN protein adopts the post-fusion form, suggesting that the pre-fusion state of the cleaved W3A F protein may be stabilized by association with HN protein. It is thus conceivable that, in the absence of HN protein, a given F protein with an HN-independent fusion activity may be so unstable after cleavage that it easily undergoes the conformational changes and adopts the post-fusion form without any triggering molecule. However, provided that such an F protein is in complex with the HN protein, its conformational changes would be prevented until the HN protein comes into contact with the receptor on the target membrane, similarly to F proteins with HN-dependent fusion activity (McGinnes & Morrison, 2006). The present study suggests that the hydrophobicity of Leu-22 is attributable to the stability of the WR F protein, as substitution of Leu-22 with any non-hydrophobic amino acids (Ala, Asn, Lys and Glu) eliminated HN dependency, although the fusion activity of the resulting mutants L22A, L22N, L22K and L22E was much lower than that of L22P. Notably, we found that the conformations of these four mutants were similar to that of the WR F protein but were distinct from that of L22P, despite their ability to mediate HN-independent cell fusion (Fig. 7). This apparent discrepancy may be explained by the above observation that these four mutants showed remarkably low fusion activity compared with L22P. Alternatively, as reported previously (Tsurudome et al., 2001), presence of an amino acid other than Pro at position 22 may somehow affect the epitopes for the mAbs, whereas Pro-22 itself does not seem to be a constituent of either of the epitopes. On the other hand, it is of interest that the side chain of Pro-22 of the W3A F protein is apparently exposed on the trimer surface (Fig. 3). Although it is not clear whether the hydrophobic side chain of Leu-22 of the WR F protein is also exposed on the trimer surface, another hydrophobic amino acid, Leu-20, that is shared between the W3A F and WR F proteins is located in close proximity to Pro-22 on the trimer surface of the W3A F protein (Fig. 3). Thus, one possibility is that a hydrophobic interaction between Leu-20 and Leu-22 that somehow stabilizes the F protein could take place on the trimer surface of the WR F protein.
The present study has suggested that the acidic nature of both Glu-132 and Asp-416 contributes to the HN-independent fusion activity of L22P. This assumption was extrapolated from the observation that introduction of the D416K mutation into the L22P-E132K background resulted in efficient promotion of cell fusion. However, this promoting effect of the D416K mutation is difficult to explain, because both the E132K and the D146K mutations in the context of L22P reduced the fusion activity. We thus postulate that Lys-132 and Lys-416 (or Glu-132 and Asp-416) are located in close proximity in the F molecule. If this is the case, then an electrostatic repulsion between Glu-132 and Asp-416 could facilitate Pro-22-mediated destabilization of L22P; substitution of either amino acid with a non-acidic one would thus result in a reduction in HN-independent fusion activity as shown in Fig. 2. However, the structural data shown in Fig. 3 indicate that these amino acids are located on the other side of the fusion peptide on the surface of the uncleaved F trimer and thus are not in contact with each other. Moreover, these amino acids do not seem to be in contact with each other, even in the post-fusion structure of the F protein (Yin et al., 2005). It is conceivable, however, that they might interact with each other if an appropriate intermediate is formed at some stage after cleavage but before formation of the post-fusion structure. Interestingly, data from immunoprecipitation analyses suggest that cleavage site mutants of PIV-5 F protein (FR3 and Se-WR F), which do not have HN-independent fusion activity, undergo conformational changes upon cleavage beyond the cleavage site and fusion peptide (Dutch et al., 2001; Tsurudome et al., 2006). Nevertheless, these data do not provide evidence that would suggest an interaction between Glu-132 and Asp-416. Thus, it remains to be elucidated by which mechanism these acidic amino acids contribute to the fusion activity of L22P.
The conformation of L22P-E132K with impaired fusion activity proved to be indistinguishable from that of the highly fusogenic L22P and L22P-E132K/D416K, presumably reflecting a subsidiary role of Glu-132 in HN-independent cell fusion. However, we cannot exclude the possibility that our mAbs were unable to detect an important difference in conformation between these proteins, if it does exist.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Tsurudome, M., Ito, M., Nishio, M., Kawano, M., Komada, H. & Ito, Y. (2006). A mutant fusion (F) protein of simian virus 5 induces hemagglutinin–neuraminidase-independent syncytium formation despite the internalization of the F protein. Virology 347, 11–27.
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Yin, H. S., Paterson, R. G., Wen, X., Lamb, R. A. & Jardetzky, T. S. (2005). Structure of the uncleaved ectodomain of the paramyxovirus (hPIV3) fusion protein. Proc Natl Acad Sci U S A 102, 9288–9293.
Yin, H. S., Wen, X., Paterson, R. G., Lamb, R. A. & Jardetzky, T. S. (2006). Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation. Nature 439, 38–44.[CrossRef][Medline]
Received 11 August 2008;
accepted 24 October 2008.
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