Virology 460-461 (2014) 119–127

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Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation Concepción Palomo a,b, Vicente Mas a,b, Mónica Vázquez a,b, Olga Cano a,b, Daniel Luque c, María C. Terrón c, Lesley J. Calder d, José A. Melero a,b,n a

Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid, Spain CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain Unidad de Microscopía Electrónica y Confocal, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain d National Institute for Medical Research, MRC, Mill Hill, London NW7 1AA, UK b c

art ic l e i nf o

a b s t r a c t

Article history: Received 5 March 2014 Returned to author for revisions 28 March 2014 Accepted 1 May 2014

Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV_F occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV_F, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV_F at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. & 2014 Elsevier Inc. All rights reserved.

Keywords: Post-fusion specific antibodies against RSV F glycoprotein

Introduction Human respiratory syncytial virus (hRSV) is the most important viral agent of lower respiratory tract infections (LRI) in infants worldwide (Hall et al., 2009; Nair et al., 2010) and also a common cause of LRI in the elderly and adults with cardiopulmonary disease or in whom immune responses are impaired or reduced (Falsey et al., 2005; Whimbey and Ghosh, 2000). Development of a hRSV vaccine has been hampered to great extent by previous experience with a formalin-inactivated hRSV vaccine evaluated in the 1960s in infants and young children. The vaccine did not protect against a natural infection. Furthermore, 80% of the infected vaccinees required hospitalization (Kim et al., 1969). Recently, promising candidate vaccines are being developed ranging from attenuated live viruses (Luongo et al., 2012) to vectorbased and purified antigens (Anderson et al., 2013). hRSV is the prototype of the Pneumovirus genus within the Pneumovirinae subfamily of the Paramyxoviridae. It is an enveloped non-segmented negative-sense RNA virus which has two major n Corresponding author at: Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain. Tel.: þ 34 918223908; fax: þ34 91 5097919. E-mail address: [email protected] (J.A. Melero). 0042-6822/& 2014 Elsevier Inc. All rights reserved.

surface glycoproteins (G and F) inserted in the viral membrane (Collins and Melero, 2011). Glycoprotein G is responsible for attachment of the virus to the target cell by interacting mainly with cell surface proteoglycans (Hallak et al., 2000, 2007; Martinez and Melero, 2000). The fusion (F) glycoprotein mediates fusion of the viral and cell membrane, facilitating virus entry (Walsh and Hruska, 1983). The F protein also promotes fusion of the infected cell membrane with those of adjacent cells to form large multinucleated syncytia, as concluded from inhibition of syncytia formation with anti-F antibodies added to cell cultures after virus infection (Magro et al., 2010; Walsh and Hruska, 1983). Protection against hRSV infection is provided mainly by neutralizing antibodies, as has been demonstrated by passive-transfer experiments in rodents (Graham et al., 1993; Prince et al., 1985). Likewise, infants at high risk of severe hRSV disease are partially protected from hospitalization by prophylactic administration of immunoglobulins with high neutralization titers (Groothuis et al., 1993) or a neutralizing monoclonal antibody (MAb) specific for the F glycoprotein (Groothuis and Nishida, 2002). High serum neutralizing antibody titers have also been correlated with protection of adult volunteers to a hRSV challenge (Hall et al., 1991; Watt et al., 1990) and protection of children (Glezen et al., 1986) and the elderly (Falsey and Walsh, 1998) to hRSV infections. Most of the neutralizing activity found in human immunoglobulin


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Fig. 1. Generation of α-6HB polyclonal antibodies. (A) Scheme of the F protein primary structure, denoting the following structural motifs: the signal peptide (SP), the F2 chain, the two furin cleavage sites (vertical arrows) and the 27 amino acid intervening peptide (p27), the F1 chain in which the fusion peptide (FP) and the two heptad repeats (HRA and HRB) are represented, the transmembrane domain (TM) and the cytoplasmic tail (Cyt). Cysteines are indicated by black dots. The HRA (red) and HRB (blue) sequences that were fused to GST are indicated below. Note that HRA is followed by a linker sequence (L, black) encoded by one of the primers used in the amplification. The bottom line shows the heptad repeat and linker sequences as they are arranged in the GST-HRA-L-HRB construct. (B) Serial dilutions of rabbit serum raised against purified GST-HRA-L-HRB were tested in ELISA against 0.3 mg of either GST or the different constructs shown in panel A, as indicated. (C) Diagram of the depletion protocol used with purified α-GST-HRA-L-HRB antibodies. These antibodies were loaded first onto a column of GST-HRA-L bound to Sepharose (read beads) and the unbound material loaded onto a column of GST-HRB bound to Sepharose (blue beads). Unbound antibodies were collected and tentatively named α-6HB. (D) ELISA binding of α-6HB antibodies to either GST or the different constructs shown in panel A, as indicated. Results in this and other figures are shown as means 7SEM and are representative of at least three independent assays.

preparations resides on antibodies directed against the F glycoprotein (Magro et al., 2012; Sastre et al., 2005). The hRSV_F glycoprotein is a homotrimer in which each subunit is synthesized as an inactive precursor of 574 amino acids that needs to be cleaved by trypsin-like proteases at two furin sites to become fusion competent (Gonzalez-Reyes et al., 2001; Zimmer et al., 2001) (Fig. 1A). This double cleavage generates two chains (F2 N-terminal to F1) that remain covalently linked by two disulfide bonds and an intervening 27 amino acid peptide (p27) that is released from the mature molecule (Begona Ruiz-Arguello et al., 2002). A hydrophobic fusion peptide is thus located at the N-terminus of F1. The fusion peptide is inserted into the membrane of the target cell during the fusion process. Two heptad repeat sequences are present in F1, the N-terminal heptad (HRA) and C-terminal (HRB) heptad, which are located downstream of the fusion peptide and upstream of the transmembrane (TM) domain, respectively. Recombinant hRSV with F as the sole glycoprotein is still able to infect cells in culture, although less efficiently than the wild type virus and it is attenuated in vivo (Techaarpornkul et al., 2002). Thus, F seems to be able to mediate also attachment to cells, at least in the absence of G. In fact, F has been shown to bind to cell surface proteoglycans (Crim et al., 2007) and other molecules, like surface nucleolin (Tayyari et al., 2011). The F trimer is incorporated into the virus particle in a prefusion metastable conformation. Once the incoming virus is bound to the surface of the target cell, the F protein is activated by still

ill-defined mechanisms in which completion of cleavage at the furin sites may be required at the cell surface or after macropinocytosis (Krzyzaniak et al., 2013; Rawling et al., 2008, 2011). Activation of F leads to a series of conformational changes, including the formation and refolding of a pre-hairpin intermediate by which the viral and cell membranes are brought into proximity. The final adoption of a stable post-fusion conformation by the F protein is associated with merging of the two membranes and formation of the fusion pore (Lamb and Jardetzky, 2007). Three-dimensional structures of soluble hRSV_F ectodomains stabilized in either the pre-fusion (McLellan et al., 2013a, 2013b) or post-fusion conformations (McLellan et al., 2011a; Swanson et al., 2011) have been recently solved. These structures have provided crucial information about the conformational changes that the F protein experiences during membrane fusion. One of these changes involves refolding of HRA sequences that form short αhelices separated by loops in the pre-fusion F into a post-fusion long α-helix which assembles into a trimeric coiled-coil core with equivalent segments from the other subunits. The membrane proximal HRB sequences of each subunit fold back from the prefusion structure to pack in an antiparallel manner against the coiled-coil core, creating a six-helix bundle (6HB). Studies done with peptides and segments of hRSV_F have shown that HRA sequences can associate in α-helical trimeric complexes in solution (Lawless-Delmedico et al., 2000; Matthews et al., 2000a; Zhao et al., 2000). In contrast HRB-derived peptides exist as unstructured monomers but are incorporated into

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hexameric α-helical structures when mixed with HRA peptides (Lawless-Delmedico et al., 2000; Matthews et al., 2000a), resembling the 6HB of the post-fusion F (Zhao et al., 2000). Conformation specific antibodies could be helpful reagents to distinguish the pre-fusion and post-fusion conformations when performing structural and functional studies of hRSV_F. We have used previously rabbit antibodies specific for the 6-HB domain to discriminate between pre- and post-fusion forms of hRSV_F (Magro et al., 2012). We now describe in detail their isolation and structural requirements. In addition, a monoclonal antibody (R145) that recognizes the same 6HB motif is described as well as the location of its epitope by site-directed mutagenesis and electron microscopy.

Results Isolation and characterization of anti-six-helix bundle (α-6HB) specific polyclonal antibodies Based on previous reports describing the cloning and expression of hRSV_F heptad repeat sequences in Escherichia coli (Lawless-Delmedico et al., 2000; Matthews et al., 2000b; Zhao et al., 2000), the following F protein sequences (Long strain) were cloned and expressed, fused to the C-terminus of a GST core (see Fig. 1A): HRA-L comprising amino acids 160–207 followed by a GSSGGV linker (L), (i) HRB comprising amino acids 479–520, and (ii) HRA-L-HRB in which HRB was attached to the C-terminus of HRA-L. As consequence of the cloning strategy, a N-terminus factor Xa site and the tripeptide GIL bridged GST to the heptad repeats. New Zealand white rabbits were immunized with purified GSTHRA-L-HRB and the immune serum was used in an ELISA with either the immunogen or with its purified moieties; i.e., GST-HRAL, GST-HRB or GST. The results obtained (Fig. 1B) demonstrated the presence of antibodies that reacted with each of the antigens tested in the ELISA. To enrich for antibodies that required both heptad repeats for binding, the purified anti-GST-HRA-L-HRB antibodies were loaded successively through Sepharose columns that contained covalently bound either GST-HRA-L or GST-HRB (see Fig. 1C). The antibodies that did not bind to both columns were collected and tested by ELISA for binding to the same antigens noted before. In contrast with the results of Fig. 1B, the antibodies depleted of those binding to either GST-HRA-L or GST-HRB could not bind these antigens but still maintained most of the reactivity with GST-HRA-L-HRB (Fig. 1D) and were tentatively dubbed anti-six-helix bundle (α-6HB) antibodies. It has been reported that certain HRA and HRB peptides could interact in solution, forming a 3:3 hexamer that resembles the s ix-helix bundle of the post-fusion F conformation (LawlessDelmedico et al., 2000; Matthews et al., 2000b; Zhao et al., 2000). Thus, to explore further the structural requirements of the α-6HB antibodies for binding, they were tested in ELISA for reactivity with individual GST-HRA-L or GST-HRB or with combinations of the heptad repeats with themselves or with individual peptides (see Fig. 2A). As shown in Fig. 1D, GST-HRA-L or GST-HRB failed to react with the α-6HB antibodies but reactivity was restored if the complementary heptad repeat was added to the ELISA plates (Fig. 2B). Reactivity of α-6HB antibodies with GST-HRA-L was also restored when the HRB peptide p488–522 was included in the assay, but not with peptide p476–510 (Fig. 2C). Similarly, reactivity of GST-HRB with α-6HB antibodies was restored when the peptide p160–207 was included in the ELISA, but neither peptide p157–191 nor p167–201 restored reactivity (Fig. 2D). It is worth stressing that peptides p488–522 and p160–207 coincide almost exactly with the limits of the HRA and HRB sequences that form the protease


resistant core of a HRA:HRB complex (Matthews et al., 2000a, 2000b; Zhao et al., 2000). We have reported previously that the α-6HB antibodies did not react with a full-length hRSV_F protein stabilized in the pre-fusion conformation and thus lacking the 6HB motif, but reacted efficiently with full-length or soluble forms of F that adopted the post-fusion structure (Magro et al., 2012). To test whether the formation of the six-helix bundle was relevant for this interaction and not only the folding in a post-fusion conformation, three truncated forms of F were assayed for binding to the α-6HB antibodies. FTM- is a well characterized soluble form of hRSV_F generated by introducing a stop codon after amino acid 524 (Bembridge et al., 1999; Calder et al., 2000). Likewise, two shorter versions of anchorless F were generated by introducing stop codons after amino acid 487 (F487stop) or amino acid 478 (F478stop, Fig. 3A). The three soluble F molecules were secreted to the culture supernatants of cells infected with corresponding vaccinia virus recombinants, expressing each protein. After affinity chromatography purification with MAb 2F (Garcia-Barreno et al., 1989), the shape of the three soluble proteins seen by electron microscopy (Fig. 3A) resembled the previously described coneshaped molecules of FTM-(Calder et al., 2000), corresponding to the post-fusion conformation. While soluble FTM- protein is expected to have the six-helix bundle, as found in recently reported atomic structures of very similar proteins (McLellan et al., 2011b; Swanson et al., 2011), F487stop and F478stop lack the HRB moiety of the 6HB (see structure models in Fig. 3A), suggesting that HRB sequences are not required by hRSV_F to fold in a post-fusion like conformation. However, and in contrast with FTM- neither F487stop nor F478stop reacted with α-6HB antibodies (Fig. 3B). Interestingly, this reactivity was gained when F487stop and F478stop proteins were supplemented with increasing amounts of either GST-HRB (Fig. 3C) or p488–522 (Fig. 3D) which, as expected had no effect upon FTM-. In other words, also in the context of purified soluble F proteins folded in a post-fusion conformation, the presence of a six-helix bundle was an absolute requirement for binding to the α-6HB antibodies. Isolation and characterization of a monoclonal antibody (MAb) specific for the six-helix bundle While doing a screening of rabbit hybridoma clones, a monoclonal antibody (MAb R145) was found that reacted with the noted post-fusion FcN and FTM- proteins but not with the pre-fusion FcN/ 2C‐C (Magro et al., 2012) (Fig. 4A). To test if this conformationdependent binding of MAb R145 required the formation of the sixhelix bundle characteristic of the post-fusion F, this antibody was tested in ELISA with the chimeric GST-HRA-L, GST-HRB and GSTHRA-L-HRB antigens. As shown in Fig. 4B, R415 did not react with GST-HRA-L or GST-HRB but reacted efficiently with GST-HRA-LHRB, confirming its specificity for the six-helix bundle structure. MAb R145 was also tested for binding to the FTM-, F487stop and F478stop proteins. As observed with the α-6HB antibodies, R145 bound to FTM- but did not bind to the other two proteins unless they were supplemented with GST-HRB (Fig. 4C). In order to map the R145 epitope in the six-helix bundle structure, a series of point mutants of the GST-HRA-L-HRB construct were tested in ELISA (Fig. 5). While all mutants reacted comparably with the polyclonal α-6HB antibodies (Fig. 5A), the double mutant S509C/L513C failed to bind R145 (Fig. 5B). Interestingly, the single mutations S509C and L513C had no apparent effect on the binding of R145 to GST-HRA-L-HRB. Consistent with the location of those mutations, R145 antibody was seen by electron microscopy (EM) binding at the base of purified FTMmolecules (Fig. 5D), where the six-helix bundle is located in the post-fusion F structure (McLellan et al., 2011b; Swanson et al.,


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Fig. 2. Structural requirements for ELISA binding of α-6HB antibodies. (A) Scheme of HRA and HRB sequences (same as in Fig. 1A) and related peptides, indicating the amino acid limits. (B) A fixed amount (0.3 mg) of either GST-HRA-L or GST-HRB, alone or mixed with increasing amounts of the complementary heptad repeat (HR, abscissas) were tested in ELISA for binding to a saturating amount of the α-6HB antibodies. (C) GST-HRA-L (0.3 mg) was mixed with increasing amounts of the indicated HRB peptides and tested in ELISA for binding to the α-6HB antibodies. (D) GST-HRB (0.3 mg) was mixed with increasing amounts of the indicated HRA peptides and tested in ELISA with the α-6HB antibodies.

Fig. 3. Reactivity of the α-6HB antibodies with soluble constructs of the hRSV_F ectodomain of different lengths. (A) Models of the indicated soluble hRSV_F proteins are shown (left panels), based on the 3D structure of the post-fusion F published by McLellan et al. (2011b), as well as negatively stained electron micrographs (right panels) of the purified proteins. Scale bar: 50 nm. (B) Increasing amounts (abscissas) of purified FTM-, F487stop or F478stop proteins were added to ELISA plates that were incubated with fixed amounts of the indicated antibodies (α-FTM- refers to a rabbit antiserum raised against purified FTM-). (C) and (D) Twenty five nanograms of the indicated F proteins were mixed with increasing amounts (abscissas) of either GST-HRB (C) or p488–522 peptide (D) before being added to ELISA plates and assayed with α-6HB antibodies.

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Fig. 4. Structural requirements of MAb R145 for ELISA binding. (A) ELISA of the previously described FTM-, FcN and FcN2C-C proteins (Magro et al., 2012) with increasing amounts of R145 antibody, as described in Materials and methods section. (B) ELISA of either GST or the different chimeric constructs of Fig. 1A with R145 antibody. (C) Twenty five nanograms of the FTM-, F487stop or F478stop proteins were used to coat ELISA wells, either alone or mixed with increasing amounts of GST-HRB that were then probed with a fixed amount of F145 antibody.

2011). For comparison, as reported previously (Calder et al., 2000) 2F antibody was found binding to the tip of the head in the coneshaped FTM- molecules (Fig. 5E).

Discussion The approach used here to raise polyclonal antibodies specific for the six-helix bundle of hRSV_F could in principle be applied to obtain antibodies against the 6HB motif of other paramyxovirus fusion proteins or even be extended to similar structures present in other type I fusion glycoproteins (Baker et al., 1999). It is remarkable that a significant fraction of the antibodies induced after immunization with GST-HRA-L-HRB required both heptad repeats for binding and were therefore not depleted after adsorption to GST-HRA-L nor GST-HRB (Fig. 1D). This is in contrast to other types of antibodies described by Dutch et al. (2001) that were raised against separate peptides derived from HRA or HRB sequences of the parainfluenza virus 5 (PIV5) fusion protein. These antibodies failed to immunoprecipitate a synthetic protein that consisted of the heptad repeat regions separated only by a small spacer, suggesting that the antibodies were unable to recognize the 6HB core complex. At the time this study was done, we were unaware of the paper published by Yunus et al. (2010) that described antibodies raised in guinea pigs against either a HRA-L-HRB construct or a mixture of HRA and HRB sequences, expressed in E. coli as thioredoxin fusion proteins. Although there were some slight differences in the length of the HRA and HRB segments used in the two studies, the main difference between the strategy used by Yunus et al. and ours resides in removing or not the extra moiety of the fused proteins before immunization. Yunus et al. cleaved off the thioredoxin moiety with the factor Xa protease and used the heptad repeats detached of any extra sequences for both immunization and ELISA binding. We have used the heptad repeat sequences fused to GST as immunogens and have demonstrated that the presence of GST does not interfere with induction of antibodies to the other moiety. Although not formally proven, it is likely that GST may also have an immunogenic carrier effect and thus we favor the use of GST chimeras for immunization, since the anti-GST antibodies can be readily removed afterwards if needed, as shown in Fig. 1. Nevertheless, the results reported by Yunus et al. (2010) are totally compatible with ours, although the use they made of their α-6HB antibodies was different from ours. Interestingly, the

same group has also published the generation of equivalent antibodies elicited against heptad repeat peptides of the transmembrane protein (gp41) of the human immunodeficiency virus type I (HIV1) (de Rosny et al., 2001). Thus, it is likely that the approaches taken by Yunus et al., or by us could be extrapolated to other type I fusion proteins to obtain antibodies specific to their respective post-fusion conformations. Zhao et al. (2000) reported the expression in E. coli of a hRSV_F derived HRA-L-HRB protein (without GST) that assembled into a 6HB with a central protease resistant coiled-coil. It is still unknown the actual conformation adopted by the HRA and HRB sequences in the GST fusion chimeras used in this study but it is likely that they also fold into some coiled-coil structure when they are connected by a linker, as in GST-HRA-L-HRB. This conclusion is supported by the results of Fig. 2 in which GST-HRA-L or GST-HRB proteins failed to react with the α-6HB antibodies but reactivity was gained when either protein was incubated with its complementary GST-heptad repeat or certain peptides. Apparently, GST does not seem to restrict the formation of the HRA-HRB complexes which have been shown to form in solution when heptad repeat peptides are properly mixed (Lawless-Delmedico et al., 2000; Matthews et al., 2000a). Interestingly, the peptides that rescue HRA or HRB reactivity with the α-6HB antibodies match almost exactly the boundaries of the protease resistant core of the sixhelix bundle reported by Zhao et al. (2000). It is this propensity of HRA-HRB sequences to assemble in a coiled-coil structure that may have skewed the antibody response towards the 6HB motif, in contrast to the antibodies reported by Dutch et al. (2001) that were raised against separate heptad repeat peptides. The α-6HB antibodies also reacted with the 6HB motif present in the post-fusion conformation of a soluble hRSV_FTM- protein (Fig. 3). This protein, which folds into the most stable post-fusion conformation has a stem made by the 6HB, as revealed in the atomic structure solved recently by X-ray crystallography (McLellan et al., 2011b; Swanson et al., 2011). Remarkably, the overall post-fusion structure of the F protein ectodomain was also observed by electron microscopy with shortened proteins that contain only the first 487 or 478 amino acids of the F protein sequence. Thus, folding in a post-fusion like conformation does not seem to require HRB, at least when expressed as a soluble protein. Yet, neither F487stop nor F478stop reacted with the α-6HB antibodies unless GST-HRB or HRB derived peptides were included in the ELISA; i.e., the 6HB was essential for reactivity with the α-6HB antibodies either in isolated complexes (Fig. 2) or in the context of the post-fusion F (Fig. 3).


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Fig. 5. Location of the R145 epitope by ELISA and electron microscopy. Either wild type GST-HRA-L-HRB or the indicated mutants of this molecule were tested in ELISA for binding to increasing amounts of either α-6HB antibodies (A) or R145 antibody (B). (C), (D) and (F) Electron microphaphs of purified FTM-either alone (C) or in combination with R145 (D) or 2F (E) antibodies. Scale bar: 50 nm. Galleries of selected images are shown at right of each panel with diagrams of the antibody (grey) and FTM- (black) complexes. Models of the FTM- structure are shown at right indicating residues indispensable for binding either R145 (D) and 2F (E) antibodies (green balls). Arrows denote the location of R145 and 2F epitopes, respectively.

The monoclonal R145 was serendipitously found while searching for antibodies that could bind differentially to previously reported F proteins, folded in the pre- or post-fusion conformations (Magro et al., 2012). Further tests demonstrated that R145 antibody could bind to the soluble post-fusion FTM- protein (Fig. 4) and to HRA-L-HRB complexes. R145 has almost the same requirements as the α-6HB antibodies for binding, except in the case of the double mutant S509C/L513C of GST-HRA-L-HRB. Whether loss of binding of R145 by the double mutant is due to either direct interaction of the antibody heavy and/or light chain with the mutated residues or local misfolding of the HRA-L-HRB construct by the two Cys residues is not known. However, it is worth stressing that ELISA binding of the α-6HB antibodies to the wild type and the double mutant was essentially the same (Fig. 5), ruling out major misfolding of the 6HB mutant motif. In any case, although EM is not a high resolution technique, the images of Fig. 5D locate R145 binding at the tip of the 6HB motif in the post-fusion FTM- molecule, in agreement with the ELISA results of Fig. 5B. The α-6HB and R145 antibodies, together with previously described pre-fusion specific antibodies (Kwakkenbos et al., 2010;

McLellan et al., 2013b), expands the collection of reagents to probe F protein conformations. Monoclonal antibodies that have some conformation dependency for binding to other paramyxovirus F proteins, such as PIV (Connolly et al., 2006) and Newcatle Disease Virus (NDV) (Umino et al., 1990) have been reported. As with R145, these antibodies were also found serendipitously. We propose that a combination of the strategy described here for the generation of the α-6HB antibodies together with hybridoma methodology may be a design-based approach to generate post-fusion specific monoclonal antibodies for structural studies of type I fusion glycoproteins.

Materials and methods Ethics statement Procedures that required the use of animals complied with Spanish and European legislation concerning vivisection and the

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use of genetically modified organisms. Protocols were approved by the “Comité de Ética de la Investigación y del Bienestar Animal” of “Instituto de Salud Carlos III” (CBA PA 19_2012). In particular, we followed the Guidelines included in the current Spanish legislation on protection for animals used in research and other scientific aims: RD 1201/2005, 10th October and the current European Union Directive 86/609/CEE, DOCE 12.12.86 (N.L358/1 to N.L358/ 28) on protection for animals used in experimentation and other scientific aims.

equilibrated in PBS and mounted into columns. Then, the antibodies purified from sera of rabbits inoculated with GST-HRA-LHRB were loaded onto the column of GST-HRA-L-Sepharose. The unbound material was collected and loaded onto the column of GST-HRB-Sepharose. Unbound antibodies (tentatively dubbed α6HB) were collected, dialyzed against PBS and stored at  20 1C until used.

Cloning and expression of HRA, HRB and HRA-L-HRB sequences fused to glutathione-S-transferase

Peptides were synthesized by solid-phase Fmoc chemistry in a ABI 433A synthesizer and purified by HPLC as described before (Magro et al., 2010).

F gene sequences (based on the hRSV Long strain, GenBank accession number P12568) encoding amino acids 160–207 of the HRA domain plus a C-terminal linker GSSGGV were amplified by PCR using the following primers: forward 50 -GGCGGATCCTAGAAGGAGAAGTGAACAAG-30 (underlined is the Bam HI site used for cloning) and reverse 50 -GGGCGTCGACGCCCCCCGACGACCCCACAATAGGTAACAATTGTTTATC-30 (the Sal I site is underlained and the sequence encoding the GSSGGV linker is shown in italics). Similarly, F gene sequences encoding amino acids 479–520 of the HRB domain were amplified using the following primers: forward 50 -GGCGGATCCTCGACCCATTAGTATTCCCC-30 (underlined is the Bam HI site) and reverse 50 -CATCATGTAAATGCTGGTAAATGAGCGGCCGC-30 (underlined is the Not I site). The amplified DNAs were cut with the indicated enzymes and cloned into plasmid pGEX-5X-3 (GE Healthcare), digested with the same restriction enzymes. This created C-terminal glutathione-S-transferase (GST) fusions to the heptad repeat sequences, linked through a factor Xa cleavage site and the tripeptide GIL generated during cloning (in the case of HRA, L is the first residue of the heptad, see Fig. 1A). The plasmid encoding GST-HRA-L-HRB (Fig. 1A) was obtained by inserting a newly amplified HRB, flanked by Sal I and Not I sites into pGEX-5X-3-HRA-L after digestion with these enzymes. The mutants Asn175Ser, Ser186Arg, Ser509Cys and Leu513Cys and the double mutant Ser509Cys/Leu513Cys were obtained by sitedirected mutagenesis of GST-HRA-L-HRB using specific primers and the QuikChange mutagenesis kit following the manufacturer's instructions (Stratagene). The different GST-fusion proteins were expressed in the E. coli BL21 strain grown at 37 1C in autoinducible Overnight Express Instant TB Medium (Novagen). Bacteria were collected by sedimentation, resuspended in PBS with 5 mM dithiothreitol (DTT) and lysed by sonication. After clarification, the bacterial extracts were added to Glutathion-Sepharose 4 Fast Flow (GE Healthcare) and incubated overnight at 4 1C in a mixing rotor. After washing with PBS, the bound GST chimeric proteins were eluted with 50 mM Tris–HCl, pH 8.0, 10 mM glutathione. Rabbit immunization and generation of anti-six-helix bundle (α-6HB) antibodies New Zealand white rabbits were inoculated intradermally at multiple sites with 100 mg of purified GST-HRA-L-HRB emulsified with an equal volume of Freund's complete adjuvant. After three weeks, rabbits were bled repeatedly every three weeks and their sera tested by ELISA for antibodies able to bind GST-derived antigens (Fig. 1B). Total rabbit serum antibodies were then purified by using protein A-Sepharose CL-4B columns as recommended by the manufacturer (GE Healthcare). To eliminate antibodies directed against either GST, HRA-L or HRB, two affinity columns were made as follows: 10 mg of either purified GST-HRA-L or purified GST-HRB were covalently bound to 1 g of CNBr-activated Sepharose beads following the manufacturer's instructions (GE Healthcare). The beads were washed and

HRA and HRB peptides

Vaccinia viruses and recombinant F proteins The previously described recombinant vaccinia virus Vac/FTMencodes a soluble form of the hRSV_F protein (Long strain), generated by introduction of a Ile525Stop (ATC to TAA) mutation that eliminates the transmembrane region and the cytoplasmic tail (Bembridge et al., 1999). Additional recombinant proteins (F487 stop and F478stop) were made by introducing the mutations Phe488Stop (TTT to TGA) or Asp479Stop (GAC to TGA) in the F insert of the pRB21/F plasmid and selecting recombinant viruses by the method of Blasco and Moss (1995). All vaccinia viruses were grown in CV-1 monkey cells and the soluble proteins purified from culture supernatants by antibody (2F) affinity chromatography as described (Begona Ruiz-Arguello et al., 2002). Recombinant vaccinia viruses expressing the full length F proteins FcN and FcN2C-C have been described (Magro et al., 2012). FcN corresponds to the hRSV_ F protein (Long strain) in which all basic residues at cleavage sites I and II were substituted with Asn residues. FcN2C-C has additionally the amino acids Leu481, Asp489, Ser509 and Asp510 substituted with Cys residues. The two proteins (FcN and FcN2C-C) have a 6His tag at the Cterminus which was used for purification from extracts of infected cells, using Ni(2 þ) columns, as described (Magro et al., 2012). Isolation of R145 New Zealand white rabbits were inoculated intramuscularly (i.m.) on days 0 and 28 with 107 pfu of the recombinant VAC/Fc virus. Ten weeks later, the rabbits were inoculated intravenously with the same dose of the vaccinia recombinant. Four days later the rabbits were anesthetized and their splenocytes isolated after washing the spleens five times with RPMI 1640 medium (Lonza) containing penicillin (100 U/ml), streptomycin (100 U/ml) and fungizone (2.5 mg/ml). The spleens were crushed and then filtered through two 100 mm cell strainers. The flow through cells were incubated in Red Cell Lysis buffer (Sigma) for 5 min, then centrifuged and the cell pellet resuspended in RPMI 1640 medium The rabbit splenocytes were fused to the 240E-W2 rabbit myeloma cell line, using procedures and know-how own by EPITOMICS (Burlingame, CA 94010-1303 U.S.A). Supernatants of clones growing in 96 well microtitre plates were screened by ELISA for the presence of conformation specific antibodies using the previously described FcN and FcN2C-C proteins (Magro et al., 2012). The R145 hybridoma produced an antibody that reacted with FcN but not FcN2C-C. This hybridoma was recloned and expanded for high yield production of the indicated monoclonal antibody. Enzyme linked immunosorbent assay ELISAs were performed in 96 well plates coated with the antigens indicated in the figure legends. Nonspecific antibody


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binding was blocked with 2% filtered porcine serum in PBS with 0.05% Tween-20. Antibody preparations were added to plates for 1 h at room temperature and unbound antibody was removed by washing five times with water. Antibody binding was revealed by incubation with a peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare) and subsequent addition of OPD (Sigma) as substrate as per the manufacturer's instructions. Electron microscopy Purified proteins were applied to glow-discharged carboncoated grids and negatively stained with either 1% sodium silicotungstate (pH 7.0) or 1% aqueous uranyl formate. Micrographs were recorded with either a Jeol 1200 electron microscope operated at 100 kV or a Tecnai 12 FEI microscope operated at 120 kV at a nominal magnification of 67,000  .

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Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation.

Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRS...
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