1 Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain
2 National Institute for Medical Research, Mill Hill, London NW7 1AA, UK
Correspondence
José A. Melero
jmelero{at}isciii.es
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ABSTRACT |
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These authors contributed equally to this work.
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INTRODUCTION |
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The HRSV F protein is a type I glycoprotein that is synthesized as an inactive precursor (F0) of 574 aa. This precursor is cleaved by furin-like proteases during maturation to yield two disulfide-linked polypeptides, F2 from the N terminus and F1 from the C terminus. In contrast to other paramyxovirus F proteins that are cleaved only once, the F0 precursor of HRSV and the related bovine RSV are cleaved twice, after residues 109 (site I) and 136 (site II), which are preceded by furin-recognition motifs (González-Reyes et al., 2001; Zimmer et al., 2001
) (see Fig. 1a
for a diagram of the primary structure). The F proteins of all paramyxoviruses have three main hydrophobic regions: one at the N terminus, which acts as the signal peptide for translocation into the ER; another region, the membrane anchor or transmembrane domain, near the C terminus; and a third region at the N terminus of the F1 chain, called the fusion peptide because it is thought, by analogy with other fusion peptides (Durrer et al., 1996
), to be inserted into the target membrane during the process of membrane fusion. The mature F protein is a homotrimer in which two heptad-repeat sequences, HRA and HRB, adjacent to the fusion peptide and to the transmembrane region of each monomer, respectively, are important motifs for the formation and stability of the trimers. HRA and HRB peptides form trimeric complexes in solution (Lawless-Delmedico et al., 2000
; Matthews et al., 2000
) and X-ray crystallography of these complexes reveals an internal core of three HRA
-helices bounded by three antiparallel HRB
-helices, packed into the grooves of the HRA coiled-coil trimer (Zhao et al., 2000
).
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We reported previously that purified HRSV F protein formed rosettes of rods with two different shapes, cones and lollipops (Calder et al., 2000). The rosettes were formed by aggregation of individual rods through their transmembrane regions. A membrane-anchorless form of F that lacked the transmembrane region and the cytoplasmic tail (
) was seen mainly as unaggregated cones, although a significant proportion (
20 %) of rosetted lollipops could also be observed. Preparations of both F and
contained, in addition to molecules cleaved to F1 and F2 chains, uncleaved F0 molecules and partially processed intermediates called F
1109 and F2* (Fig. 1
). F
1109 was generated when F0 was cleaved only at site I and F2* when F0 was cleaved only at site II (González-Reyes et al., 2001
). Site II immediately precedes the fusion peptide. When cleavage of the
monomers was completed, the trimers
aggregated in rosettes of lollipop-shaped spikes (Ruiz-Argüello et al., 2002
). Aggregation of
may occur through interactions of the fusion peptides, which may be exposed after completion of cleavage. As exposure of the fusion peptide is related to activation of the F protein, cones and lollipops may represent the pre- and post-activated forms of the F protein. Here, we provide evidence that the fusion peptide is involved in aggregation of
, but that cleavage of the F protein trimer does not have a significant effect on the thermostability of the F protein molecule.
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METHODS |
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A pTM1-derived plasmid carrying a full-length cDNA insert of the HRSV F protein gene under the T7 polymerase promoter has been described previously (González-Reyes et al., 2001). The
137146 deletion was introduced into this plasmid as indicated above for the
gene.
HRSV (Long strain) was grown in HEp-2 cells and purified from the culture supernatant as described previously (García-Barreno et al., 1988). Briefly, virus was precipitated with 6 % PEG 6000, resuspended in PBS and centrifuged in a 2060 % sucrose gradient. The visible virus band was collected, diluted with PBS and spun through a 30 % sucrose cushion. The virus was finally resuspended in PBS and kept at 80 °C until use.
Protein purification.
HEp-2 cells grown in DMEM with 2 % fetal calf serum were infected with vaccinia viruses (m.o.i. of 0·5) expressing the
proteins described above. Culture supernatants were harvested 48 h post-infection, clarified of cell debris by low-speed centrifugation, concentrated 100-fold and buffer-exchanged with PBS by filtration through polyethersulfone membranes (Vivaflow; Sartorius) with 100 kDa exclusion pores. Concentrates were loaded onto immunoaffinity columns comprising an anti-F mAb (2F) bound to Sepharose [
5·0 mg antibody (g resin)1]. Columns were washed with 20 vols PBS and eluted with 20 vols 0·1 M glycine/Tris, pH 2·5. Fractions containing the F protein, as assessed by measurement of A280, were neutralized with saturated Tris, concentrated and buffer-exchanged with buffer A [10 mM Tris/HCl (pH 7·5), 150 mM NaCl] with Vivaspin (Sartorius). Purity was assessed by SDS-PAGE and Coomassie blue staining and protein concentration was estimated by measurement of A280 with a calculated extinction coefficient of 0·7.
Trypsin digestion and sucrose-gradient centrifugation.
Proteins in buffer A were incubated with the indicated amounts (see figure legends) of L-[(toluene-4-sulphonamido)-2-phenyl] ethyl chloromethyl ketone (TPCK)-treated trypsin (Sigma) for 1 h at 4 °C. A mock-digested sample was included as a control. Proteins were loaded onto preformed 525 % sucrose gradients in buffer A and centrifuged in a Beckman SW40 rotor at 39 000 r.p.m. for 15 h at 4 °C. Fractions (1 ml) were collected from the top of the tube. Aliquots of each fraction were analysed by SDS-PAGE and Western blotting as described previously (González-Reyes et al., 2001). The antisera used for Western blotting were raised in rabbits inoculated with the synthetic peptides aa 255275 or 104117 of the F protein (Ruiz-Argüello et al., 2002
).
Syncytium-formation assay.
BSR-T7/5 cells (Buchholz et al., 1999) (a BHK-derived cell line that expresses T7 RNA polymerase constitutively) growing in microchamber culture slides were transfected with 0·5 µg pTM1-derived plasmids as indicated in the figure legends. Transfections were done by the calcium phosphate method (MBS Mammalian Transfection kit; Stratagene). At 48 h post-transfection, cells were fixed with cold methanol for 5 min followed by cold acetone for 30 s. Fixed cells were processed for indirect immunofluorescence as described previously (García-Barreno et al., 1996
).
Electron microscopy (EM).
Protein samples in buffer A were absorbed onto carbon films and stained with 1 % sodium silicotungstate (pH 7·0). A JEOL 1200 electron microscope, operated at 100 kV, was used to view the samples. Micrographs were taken under minimum-dose, accurate defocus conditions to preserve details to 1·5 nm (Wrigley et al., 1986
).
Fluorimetry and circular dichroism.
Proteins (0·10·2 mg ml1) in 0·1 M sodium phosphate buffer, pH 7·0, were placed in the cell of a spectrofluorimeter (Perkin-Elmer LS3B). In some cases, 2 mM dithiothreitol (DTT) and 6·6 M urea were added to the samples. Starting at 20 °C, the temperature was raised at a rate of 1 °C min1 to reach 100 °C and then cooled down to 20 °C at the same rate. At different temperatures, samples were excited at 280 nm and emission spectra were recorded between 300 and 380 nm. Spectra were corrected for values obtained with buffer alone with or without the indicated agents.
Similarly, proteins (0·40·5 mg ml1) in 0·1 M phosphate buffer, pH 7·0, with or without 2 mM DTT and 6·6 M urea were heated in the cell of a spectropolarimeter (Jasco 715) at a rate of 1 °C min1 from 20 to 95 °C. Spectra were recorded between 190 and 260 nm at different temperatures and corrected for buffer controls without protein. Samples were cooled down to 20 °C and final spectra were recorded.
Antibody binding.
Proteins (0·10·2 mg ml1) or purified virus (0·51·0 mg ml1) were distributed in tubes and diluted 1 : 10 in 0·1 M phosphate buffer, pH 7·0. Starting at 30 °C, the sample temperature was raised at a rate of 1 °C min1 in a thermoblock. Tubes were taken at different temperatures and kept at 4 °C until the end of the incubation period. Samples were then diluted 1 : 100 in PBS and used to coat the wells of microtitre plates. Binding of mAbs 2F, 47F and 56F, specific for the HRSV F protein (García-Barreno et al., 1989), was assessed by indirect ELISA using peroxidase-labelled anti-mouse immunoglobulin antiserum and o-phenylaminediamine as substrate, following the manufacturer's instructions (Amersham). A492 was determined and expressed as a percentage of the values obtained at 30 °C.
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RESULTS |
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proteins with either the wild-type sequence or the
137146 deletion were purified by immunoaffinity chromatography. Immunoblot analysis of the anchorless wild-type protein with an antiserum raised against a synthetic peptide of the F1 chain (
-F255275) revealed the presence of a major band, corresponding to the mature F1 chain, and minor bands that corresponded to the uncleaved F0 precursor and to the intermediate F
1109 (cleaved only at site I) (Fig. 1b
). The latter bands were particularly prominent in a Western blot with an antiserum raised against a synthetic peptide spanning residues 104117 (
-F104117) that recognized only epitopes located in the segment between the two cleavage sites. This antibody also highlighted the F2* intermediate. The same bands were observed in Western blots of the
137146 mutant, but their proportions relative to F1 were different from the wild-type, suggesting that the efficiency of cleavage by furin was altered after elimination of the first half of the fusion peptide. However, when the two proteins were treated with trypsin under controlled conditions, the F0, F
1109 and F2* intermediates were cleaved at the remaining furin sites, whereas the F1 polypeptide remained essentially unchanged, although low levels of new bands moving faster than F1 were also observed (Fig. 1b
).
To test the effect of trypsin on the aggregated state of and
137146, the two proteins were centrifuged in 525 % sucrose gradients or observed by EM with negative staining, before and after trypsin digestion (Fig. 2
). Wild-type
sedimented mainly in fractions 56, although trailing towards higher-density fractions was also observed. After trypsin digestion, there was a clear shift in the sedimentation profile of
towards fractions of higher sucrose density. These results correlated with the observations made by EM. Whilst
was seen mainly as unaggregated cone-shaped rods in mock-digested samples, this protein was seen to aggregate in rosettes of lollipop-shaped spikes after trypsin cleavage. The anchorless mutant,
137146, sedimented in fractions 57 of the sucrose gradient (Fig. 2
), but, in contrast to wild-type
, the sedimentation profile did not change following trypsin digestion. By EM, mutant
137146 was seen to be unaggregated in both mock- and trypsin-digested samples.
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The thermostability of the anchorless, uncleaved and the cleaved
108130 proteins was assessed by three different types of assay: (i) spectrofluorimetry, which is responsive to changes in the environment of tryptophans in the protein structure; (ii) circular dichroism, which is sensitive to changes in secondary structure, mainly
-helix content; and (iii) binding of mAbs. In some experiments, wild-type
treated with trypsin (see Fig. 2
) was included as another form of aggregated, anchorless protein to compare with mutant
108130.
Fluorimetry.
Fig. 5(a) shows the spectra of uncleaved
recorded at 20 °C or after heating at 100 °C and cooling to 20 °C. A significant reduction in intensity of the spectra and a displacement of
max to higher wavelengths was noticed after heating. However, no changes in
max were observed until the sample was heated up to 85 °C; then, a rapid increase in
max occurred until the temperature reached 100 °C (Fig. 5a
'). The shift in
max towards higher wavelengths was not reverted when the protein was cooled down stepwise to the starting temperature (20 °C), indicative of irreversible changes induced by heating. However, the increase in
max was higher when 6·6 M urea and 2 mM DTT were included in the buffer (Fig. 5b
) and took place at lower temperatures (Fig. 5b'
) than in the absence of these agents. Thus, as assessed by fluorimetry, heating had a limited effect in denaturation of uncleaved
at high temperatures, but the addition of urea and DTT facilitated more extensive denaturation and significantly reduced the melting temperature (Tm) of this protein.
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Circular dichroism.
Fig. 6(a) shows the circular-dichroism spectra of the uncleaved
protein and mutant
108130. Both spectra were very similar, indicating a relatively high
-helix content for both proteins, with that for the uncleaved
estimated at 33 %.
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Antibody binding.
We have previously reported a set of mAbs (García-Barreno et al., 1989) that delineated different antigenic sites in the F protein primary structure (López et al., 1998
) and were shown to be located in the F protein molecule by immuno-EM (Calder et al., 2000
).
The reactivity of three mAbs representing antigenic sites I (2F), II (47F) and IV (56F) was tested after heating both uncleaved (Fig. 7a
) and mutant
108130 (Fig. 7b
) at different temperatures. Binding of the three antibodies was unchanged until the two proteins were heated up to 100 °C. Then, a significant reduction in antibody binding was observed, which was not recovered by cooling the samples. This reduction in reactivity was not observed with a polyclonal rabbit antiserum raised against purified and denatured protein, excluding inhibition of antigen binding to the plates after heating (not shown).
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The antibody-binding assay allowed us to test the thermostability of full-length F protein inserted into the viral membrane. Thus, a preparation of purified HRSV was heated stepwise and binding of the three anti-F mAbs mentioned above was quantified by ELISA. The results obtained (Fig. 7c) indicated that thermostability of the F protein present in the virus preparation was essentially the same as that of the purified, anchorless F proteins.
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DISCUSSION |
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As the fusion peptide is the main hydrophobic sequence remaining in the anchorless F protein, we assumed that the fusion peptide was exposed after proteolytic cleavage, leading to aggregation of . The results presented here support this notion. Thus, deletion of the first part of the fusion peptide (mutant
137146) abrogated aggregation of
, even after completion of cleavage by trypsin digestion (Fig. 2
). Moreover, the results of Fig. 3
indicated that deletion of aa 137146 in the full-length F protein abrogated its capacity to induce syncytium formation. It is unclear at present whether the deletion of aa 137146 inhibited the transition from cones to lollipops of the
protein after trypsin digestion. A more detailed analysis is now required to clarify this point and to identify residues of the fusion peptide that are indispensable for both aggregation of
and membrane fusion.
We have postulated that cones and lollipops may represent the pre- and post-activated forms of the F protein. Other viral proteins involved in membrane fusion, such as influenza virus haemagglutinin (HA), adopt a metastable conformation before being activated for membrane fusion (Skehel & Wiley, 2000). Once activated, these proteins experience conformational changes that result in the formation of a more stable post-active and inactive conformation when the fusion process is completed. The best-studied example is the X-31 influenza virus HA, with a Tm of 6365 °C at neutral pH (estimated by circular dichroism or trypsin sensitivity), but of greater than 90 °C after activation by exposure to acidic pH (Ruigrok et al., 1986
). Thus, it was important to compare the thermostability of
molecules before and after proteolytic cleavage.
was very resistant to heat denaturation before and after cleavage, as assessed by spectrofluorimetry, circular dichroism and antibody binding. This finding does not mean that the free energies for unfolding at the temperature of fusion are the same for the two forms, as enthalpy and heat-capacity changes may be affected by the proteolytic cleavage. The extreme thermostability of HRSV F protein may also be unique among paramyxoviruses. For instance, in the case of Sendai virus, heating of virus preparations induces a conformational change in the F protein at a temperature of 55 °C, as noted by changes in protease sensitivity. Fusion of Sendai virus with liposomes can be induced at the same temperature, suggesting that the conformational change is related to activation of the F protein for membrane fusion (Wharton et al., 2000
).
The question arises, are the uncleaved and cleaved forms of the F protein the pre- and post-active forms of this molecule? In other words, is proteolytic cleavage the triggering event for activation of the F protein of HRSV? If proteolytic cleavage is the activating event for the F protein, the pre-active conformation may not necessarily need to be in a less-stable configuration, as activation involves hydrolysis of peptide bonds. This situation is clearly different from that of influenza virus HA, where proteolysis of a precursor and subsequent exposure to an acidic environment induce the conformational changes leading to membrane fusion.
However, if the F protein is cleaved by furin inside the infected cell, it might be expected to be inactivated before reaching the cell surface, if the cleaved form of the F protein represents the post-active form of this molecule. Perhaps activation of the F protein may be modulated by the presence of partially cleaved intermediates in the same oligomer, retaining the F trimer in a pre-active state until complete cleavage at the time of virus entry, as such intermediates have been found not only in purified F protein, but also in purified virus (González-Reyes et al., 2001). Alternatively, other components in the infected cell or in the virus particle may interact with the F protein to maintain it in the pre-active state, even after proteolytic cleavage. The latter situation may apply for other paramyxoviruses, where an interaction of the attachment protein with the F protein has been reported and both proteins are needed for membrane fusion (reviewed by Lamb, 1993
). In contrast, HRSV F protein alone can induce syncytium formation efficiently (Fig. 3
) and viruses with F protein as their only glycoprotein infect certain cell types as efficiently as wild-type viruses (Karron et al., 1997
; Techaarpornkul et al., 2001
). Whether these differences between other paramyxoviruses and HRSV reflect the different pathways of proteolytic activation, which, in HRSV F protein, involves cleavage at two different sites compared with other paramyxoviruses, in which the F protein is cleaved only once, is unknown.
Regardless, the results presented here indicate that the changes associated with cleavage of the F protein that lead to aggregation of the anchorless form of the molecule through exposure of the fusion peptide do not significantly alter its thermostability. It is worth mentioning that the thermostability of the F protein in the virus particle, assessed by loss of antibody binding, was very similar to the thermostability of purified anchorless F protein, lending support to the relevance of the results obtained with for the situation expected in the case of the infectious virus.
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ACKNOWLEDGEMENTS |
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Received 26 May 2004;
accepted 3 September 2004.