Corrections

NEUROBIOLOGY Correction for “High-resolution structure of hair-cell tip links,” The authors note that Figure 3 appeared incorrectly. The by Bechara Kachar, Marianne Parakkal, Mauricio Kurc, Yi-dong corrected figure and its legend appear below. This error does not Zhao, and Peter G. Gillespie, which appeared in issue 24, affect the conclusions of the article. November 21, 2000, of Proc Natl Acad Sci USA (97:13336– 13341; 10.1073/pnas.97.24.13336). CORRECTIONS

Fig. 3. Upper and lower attachments of the tip link. (A and B) Freeze-etch images of tip-link upper insertions in guinea pig cochlea (A) and (left to right) two from guinea pig cochlea, two from bullfrog sacculus, and two from guinea pig utriculus (B). Each example shows pronounced branching. (C and D) Freeze- etch images of the tip-link lower insertion in stereocilia from bullfrog sacculus (C) and guinea pig utriculus (D); multiple strands (arrows) arise from the stereociliary tip. (E) Freeze-fracture image of stereociliary tips from bullfrog sacculus; indentations at tips are indicated by arrows. (Scale bars: A = 100 nm, B = 25 nm; C–E = 100 nm.)

www.pnas.org/cgi/doi/10.1073/pnas.1311228110

www.pnas.org PNAS | July 16, 2013 | vol. 110 | no. 29 | 12155–12156 Downloaded by guest on September 28, 2021 BIOCHEMISTRY BIOPHYSICS AND COMPUTATIONAL BIOLOGY, STATISTICS Correction for “X-ray structure of the arenavirus glycoprotein Correction for “Differential principal component analysis of GP2 in its postfusion hairpin conformation,” by Sébastien Igo- ChIP-seq,” by Hongkai Ji, Xia Li, Qian-fei Wang, and Yang net, Marie-Christine Vaney, Clemens Vonhrein, Gérard Bri- Ning, which appeared in issue 17, April 23, 2013, of Proc Natl cogne, Enrico A. Stura, Hans Hengartner, Bruno Eschli, and Acad Sci USA (110:6789–6794; first published April 8, 2013; Félix A. Rey, which appeared in issue 50, December 13, 2011, 10.1073/pnas.1204398110). of Proc Natl Acad Sci USA (108:19967–19972; first published The authors note the following statement should be added to November 28, 2011; 10.1073/pnas.1108910108). the Acknowledgments: “Q.-f.W. is supported by the Strategic The authors note that, due to a printer’s error, the author Priority Research Program of the Chinese Academy of Sciences name Clemens Vonhrein should instead appear as Clemens (Grant No. XDA01010305).” Vonrhein. The corrected author line appears below. The online version has been corrected. www.pnas.org/cgi/doi/10.1073/pnas.1311614110

Sébastien Igonet, Marie-Christine Vaney, Clemens Vonrhein, Gérard Bricogne, Enrico A. Stura, Hans Hengartner, Bruno Eschli, and Félix A. Rey

www.pnas.org/cgi/doi/10.1073/pnas.1311209110

12156 | www.pnas.org Downloaded by guest on September 28, 2021 X-ray structure of the arenavirus glycoprotein GP2 in its postfusion hairpin conformation

Sébastien Igoneta,b, Marie-Christine Vaneya,b, Clemens Vonrheinc, Gérard Bricognec, Enrico A. Sturad, Hans Hengartnere, Bruno Eschlie, and Félix A. Reya,b,1 aDépartement de Virologie, Unité de Virologie Structurale, Institut Pasteur, F-75724 Paris Cedex 15, France; bCentre National de la Recherche Scientifique, Unité de Recherche Associée 3015, F-75724 Paris Cedex 15, France; cGlobal Phasing Ltd., Cambridge CB3 0AX, United Kingdom; dCommissariat à l’Energie Atomique Saclay, Service d’Ingénierie et d’Etudes des Protéines, CE-Saclay F-91191 Gif-sur-Yvette Cedex, France; and eInstitute of Experimental Immunology, University Hospital, CH 8557 Zürich, Switzerland

Edited by Robert A. Lamb, Northwestern University, Evanston, IL, and approved October 10, 2011 (received for review June 19, 2011)

Arenaviruses are important agents of zoonotic disease worldwide. peptide (SSP, aa 1–58), the surface exposed GP1 (aa 59–265), The virions expose a tripartite envelope glycoprotein complex at and the transmembrane GP2 (266–498) to make a ðSSP∕GP1∕ their surface, formed by the glycoprotein subunits GP1, GP2 and GP2Þ3 trimeric complex. SSP is myristoylated (9) and rearranges the stable signal peptide. This complex is responsible for binding after signalase cleavage to translocate its C-terminal end back to to target cells and for the subsequent fusion of viral and host-cell the cytosolic side of the membrane, thus traversing the membrane membranes for entry. During this process, the acidic environment twice (10). Its interactions with the cytosolic domain of GP2 of the endosome triggers a fusogenic conformational change in are responsible for SSP retention as part of the mature GPC, the transmembrane GP2 subunit of the complex. We report here as shown for Junin virus (11). GP1 and GP2 are proteolytically the crystal structure of the recombinant GP2 ectodomain of the derived from the GPC ectodomain by subtilase SKI-1/S1P lymphocytic choriomeningitis virus, the arenavirus type species, (Fig. 1A) in the early Golgi compartments. This cleavage event at 1.8-Å resolution. The structure shows the characteristic trimeric is essential for productive infection and viral spread (12–15). GP1 coiled coil present in class I viral fusion proteins, with a central stut- is responsible for interactions with cellular receptors for entry. ter that allows a close structural alignment with most of the avail- Old World arenaviruses such as LCMV or Lassa fever virus use able structures of class I and III viral fusion proteins. The structure α-dystroglycan as entry receptor (16, 17), whereas the pathogenic further shows a number of intrachain salt bridges stabilizing the subgroup of New World arenaviruses uses the transferrin recep- postfusion hairpin conformation, one of which involves an aspartic tor-1 (TfR1) (18, 19). The interaction of GP1 with the receptor acid that appears released from a critical interaction with the stable results in virion uptake by the cell into an endosomal compart- signal peptide upon low pH activation. ment, the acidic environment of which triggers a fusogenic con- formational change in GP2 (20), the membrane fusion subunit. enveloped viruses ∣ membrane fusion ∣ RNA viruses ∣ emerging viruses GP2 contains a bipartite fusion peptide, with one of the segments located at the very N terminus of the protein, as observed in many BIOCHEMISTRY renaviruses are agents of emerging zoonotic diseases such as class I viral fusion proteins. GP2 also has a disulfide-bond- severe hemorrhagic fever. They cause chronic infections in A stabilized internal fusion loop located close to the N terminus rodents, the natural hosts, and transmission to humans often oc- (21), similar to the Ebola virus (22) and the avian sarcoma/ curs as a result of man-driven changes in the ecosystem that alter leucosis retrovirus fusion proteins (23). For Junin virus—and by the rodent population. Examples of recently emerged arenavirus extension, for all arenaviruses—the acid sensitivity was shown to infections in humans are those of Chapare and Lujo hemorrhagic fever viruses in Bolivia and South Africa, respectively (1, 2). be controlled, at least in part, by interactions between the GP2 The Arenaviridae family contains a single genus subdivided in ectodomain and the luminal loop connecting the two transmem- two separate serological groups, the Old World and the New brane (TM) segments of SSP (24). World arenaviruses. The lymphocytic choriomeningitis virus Amino acid sequence analyses and biochemical studies with (LCMV), Lujo virus, and the highly pathogenic Lassa fever virus, recombinant fragments have indicated that, despite the presence which is endemic of West Africa, are members of the first group. of a bipartite fusion loop, GP2 is a typical class I fusion protein, The second one includes several South American hemorrhagic forming an alpha helix-rich trimer in its postfusion state (25). α fever viruses—among them Machupo, Junin, Sabia, Guanarito, These studies identified two -helical regions (N- and C-term- and Chapare viruses. LCMV is the arenavirus type species and inal) with a heptad repeat pattern in the N-terminal helix (25, 26). is widely used as experimental model for the study of viral per- A loop containing a conserved disulfide bond connects the two sistence and pathogenesis (3). LCMV infections in humans occur helices. A model of the structure has been proposed by compar- worldwide, and, although often asymptomatic, they can cause a ison with the GP2 counterpart from Ebola virus (26), for which spectrum of illnesses ranging from isolated fever to meningitis the structure is known (27, 28). Ebola virus, however, belongs to a and encephalitis (4, 5). In particular, a number of fatal cases different viral family (the Filoviridae), and other than the pre- of transplant-associated LCMV infections have been recently sence of the characteristic heptad repeat pattern typical of coiled reported, illustrating the threat to patients undergoing immuno- suppressive treatment (6–8). Author contributions: B.E. and F.A.R. designed research; S.I. and M.-C.V. performed Arenavirus virions are pleomorphic enveloped particles con- research; H.H. and B.E. contributed new reagents/analytic tools; S.I., M.-C.V., C.V., G.B., taining a bisegmented, ambisense RNA genome with four open E.A.S., and F.A.R. analyzed data; and S.I. and F.A.R. wrote the paper. reading frames. The long (L) RNA segment encodes the viral The authors declare no conflict of interest. RNA-dependent RNA polymerase (protein L, 200 kDa) and a This article is a PNAS Direct Submission. small RING-finger protein (Z, 11 kDa) involved viral replication. Data deposition: Coordinates and structure factors have been deposited in the Protein The short (S) segment encodes the nucleoprotein (NP, 63 kDa) Data Bank, www.pdb.org (PDB ID code 3MKO). and the viral glycoprotein complex precursor (GPC, 75 kDa). 1To whom correspondence should be addressed. E-mail: [email protected]. Proteolytic maturation of the 498-amino acid GPC precursor This article contains supporting information online at www.pnas.org/lookup/suppl/ results in three noncovalently bound subunits: the stable signal doi:10.1073/pnas.1108910108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1108910108 PNAS ∣ December 13, 2011 ∣ vol. 108 ∣ no. 50 ∣ 19967–19972 coils—as well as a similar location of cysteine residues—shares which are in the fusion loop region. The experimental electron poor sequence identity (less than 20%) with the arenavirus GP2. density map was interpretable for residues 313–422 (red arrow- We report here the structure of the recombinant LCMV GP2 heads in Fig. 2A), with a break between amino acids 382–388 ectodomain at 1.8-Å resolution. As expected, it forms an alpha- (dots above the sequence in Fig. 2A), which are disordered in helical coiled coil similar to that of other class I viral fusion pro- the crystal, as are the C-terminal residues 423–438. teins in their postfusion conformations. The structure also shows a number of intrachain salt bridges stabilizing the postfusion Description of the Molecule. The structure shows that recombinant hairpin, one of them involving an aspartic acid that is believed LCMV GP2 is an elongated trimer about 75 Å long (Fig. 1C), in to interact with SSP in the prefusion conformation. Furthermore, which each protomer is folded in the typical “hairpin” postfusion comparison with the available structures of other viral fusion pro- conformation of viral fusion proteins. The protomer begins and teins reveals the striking conservation of a central stutter present ends as an alpha helix: a long amino-terminal helix spanning in the alpha-helical coiled coil of GP2. The stutter provides a amino acids 315–360 (N helix) and a carboxy-terminal helix com- register allowing for unambiguous structural alignment of these prising residues 408–420 (C helix), which interact with each other proteins along the coiled coil. We show that the shared central in the trimer. Amino acids 313 and 422, at the amino and carboxy location of the stutter in fusion proteins from many unrelated termini, respectively, are thus located at the same level, at one viruses—including those from class III, which also exhibit a cen- end of the trimeric rod (Fig. 1B). This conformation insures tral parallel 3-helix bundle—permits a direct comparison of the proximity of the two membrane-interacting regions (C-terminal relative location of their functional elements, revealing similari- TM segment and N-terminal fusion peptide/loop) in the full- ties and clear distinctions among them that have not been dis- length GP2 protein after the fusogenic conformational change, cussed previously. as shown for many other viral fusion proteins. Likewise, the N helix forms a central trimeric coiled coil, while the C helices pack Results and Discussion against the grooves formed in between N helices in the trimer. Structure Determination. The details of the construct used for The C helix makes four full turns and spans only about one third crystallization, the boundaries of which were delineated earlier of the length of the N helix, which makes 13 turns (Fig. 1C). An (25), are outlined in Fig. 1A. The details of the crystallization and extended polypeptide segment, spanning residues 391–407 and the structure determination procedures are described in SI Text. running “backwards” along the coiled coil grooves, connects In brief, we crystallized the fragment spanning amino acids 312– the C helix to the “turn” of the molecule—i.e., the region where 438 of the LCMV GPC precursor, lacking the bipartite fusion the chain reverts orientation at the C terminus of the N helix. peptide/loop region of GP2, which was omitted to avoid aggrega- Several highly conserved bulky aromatic or aliphatic side chains tion of the protein. In addition, to prevent artificial interchain (Trp392, Leu400, Phe405; see Fig. 2A) anchor this segment in an disulfide bond formation, we mutated Cys316 to serine because extended conformation into the interhelical grooves of the coiled this residue is believed to make a disulfide bond with one of the coil. At the end of the N helix, the chain reversal begins by a one- cysteines upstream (Cys285, Cys298, or Cys307; see Fig. 2A), turn 3/10 helix (η helix; Fig. 1B) running roughly at 90 degrees

Fig. 1. Overall structure of the LCMV GP2 trimer. (A) Diagram of the arenavirus GPC glycoprotein precursor (top) and the GP2 construct used in this study (bottom, full black line), numbered according to their position in the LCMV (WE-HPI strain) GPC sequence (Genbank accession no. CAC01231.1). An arrowhead marks the GP1/GP2 cleavage site. The boxes highlight the two fusion loops (N-FP and internal I-FP), the N and C helices, the T loop and the TM helix. IEGR indicates the Factor Xa cleavage site. (B) Ribbon diagram of the LCMV GP2 protomer. The conserved disulfide is displayed in yellow ball-and-stick. Residues involved in intrachain (B) and interchain (C) salt bridges are shown in orange. Full and empty arrowheads, respectively, mark conserved and nonconserved potential N-glycosylation sites in arenavirus GP2. (C) LCMV GP2 trimer. A vertical, central black line marks the coiled coil axis, with a magenta sphere indicating the location of a central Cl− ion. Green tubes mark the curved N-helix axes, with the stutter in red (highlighted with a full red arrow). (D) Blow up of the Cl− ion (magenta sphere) binding site, viewed down the coiled coil axis. The ion is chelated by Asn325 which also hydrogen bonds a structural water molecule (red spheres). (E) Interchain salt bridge stabilizing the base of the coiled coil mentioned in the text.

19968 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1108910108 Igonet et al. BIOCHEMISTRY

Fig. 2. Amino acid sequence alignment from representative arenaviruses. (A) Highly conserved residues are displayed in white font on red background, cysteines in yellow background, with two full orange triangles below the alignment marking the cysteines involved in the disulfide bond in the T loop. A black star above the alignment marks the cysteine mutated to serine in the construct. The N-terminal light-blue background highlights the consensus motif for the SKI-1/S1P cleavage site. A light-gray background marks segments of the GP2 sequence not included in the crystallized construct. The observed secondary structure is indicated above the sequence in black, while predicted secondary structures in the segments missing in the structure are in red. Red arrowheads on the sequence mark the limits of the atomic model built from this crystallographic study, with a dotted line indicating a disordered region in the T loop. Intra- and interchain salt bridges are numbered above the alignment in red and blue, respectively. A full red circle above the alignment marks Asn325, which chelates the central chloride ion. Conserved and nonconserved predicted N-glycosylation sites are indicated by full and open black circles, respectively, below the alignment. The “register” line provides the abcdefg heptad repeat assignment, with the x layer type stutter in red. Dashed lines under the sequences indicate the fusion peptide and loop (in blue at the N-terminal end) and the TM region (in red, at the C-terminal end). The sequences are truncated before the re- spective cytoplasmic tails. (B) Left: Central coiled coil region, showing residues with regular “knobs-into-holes” packing. Right: Region of the x layer type stutter (in red), with Val336 (position d, knob-into-knob packing) and Ala339 (position g) pointing toward the coiled coil axis. from the N helix. Then the polypeptide makes an excursion to tom half has a three-layered structure, with an inner core formed form a loop (the “T loop”), locked at its base by a highly con- by the C-terminal half of the central triple-stranded coiled coil, a served disulfide bond between Cys370 and Cys391. A third alpha middle layer formed by the extended segment between Cys391 helix (“T helix,” two turns) is present at the apex of the T loop and the C helix, and a third layer made by the T loop packing as (Fig. 1 B and C). There is a break in the electron density between a “flap” against the two inner layers. The fact that part of the flap the T helix and the disulfide at the base (dotted lines in the ribbon is disordered in the crystal indicates that it packs more loosely diagram, Fig. 1B). than the rest of the molecule and is held in place in part by As depicted in Fig. 1C, the GP2 trimer can be divided roughly the disulfide connecting it to the end of the extended segment in two halves, a membrane proximal “top” half and a “bottom” in the second layer (Fig. 1C). An interchain salt bridge, formed half (i.e., in the orientation of Fig. 1C, above and below the by residues Asp353 and Arg358 under the flap, stabilizes the end red arrow, respectively). The top half is a six-helix bundle with of the inner core (Fig. 1E). This salt bridge is strictly conserved in a central ion, presumably chloride as in other viral coiled coils, all arenavirus family members (Fig. 2A), suggesting a structural coordinated by the strictly conserved Asn325 (Fig. 1D). The bot- role. Although the recombinant protein used for crystallization

Igonet et al. PNAS ∣ December 13, 2011 ∣ vol. 108 ∣ no. 50 ∣ 19969 was produced in Escherichia coli and is therefore not glycosylated, hairpin conformation of GP2 during the membrane fusion re- the structure shows that the four predicted N-linked glycosylation action. sites in arenavirus GP2 (at positions 371, 379, 396, and 401, marked with circles under the sequence in Fig. 2A) cluster in A Stutter in the Coiled Coil Region. At the center of the GP2 trimer, the bottom half of the trimer (arrowheads in Fig. 1B). Removal the coiled coil is markedly open due to the presence of an x layer of glycosylation sites was shown to affect proper folding of the type stutter at the seventh turn of the N helix (red in Fig. 1C). The arenavirus envelope protein in the prefusion state, but the GP2 stutter is a perturbation of the heptad repeat pattern of the coiled ectodomain can obviously fold into its postfusion hairpin confor- coil in the form of a four-residue insertion, with a “defg” motif mation when produced in E. Coli, in the absence of glycans and/ intercalated in between two “abcdefg” repeats (32), as labeled in or ER chaperones, as was shown first for the influenza virus red under the corresponding sequence in Fig. 2A. Instead of the hemagglutinin HA2 protein (29). LCMV GP2 lacks the second regular “knob-into-hole” packing of side chains normally ob- arenavirus predicted glycosylation site (at position 379, empty served at a and d positions in the heptad, the side chain of the circle in Fig. 2A), which lies in the second turn of the T helix residue at the stutter (Val336) packs as “knob-into-knob” against (empty arrowhead in Fig. 1B). Of note, the first turn of this helix its counterparts from the other two helices, with the side chains contains the epitope (374-Lys-Phe-Trp-Tyr-Leu-378) of an arena- pointing toward the central threefold axis, as illustrated in Fig. 2B. virus broadly cross-reactive monoclonal antibody (mAb 83.6) The presence of the stutter induces a slight curvature of the N (30). This antibody was shown to be more reactive after the acid helices, creating an internal cavity within the coiled coil delimited pH-induced fusogenic conformational change of GP2 (20), in line above and below by two aromatic layers formed by the side chains with its location in the flap, at the membrane-distal end of the of Phe332 and Phe343, respectively. molecule. The presence of a glycan immediately downstream, at position 379, is likely to partially mask this epitope in many of the Stutter-Based Alignment of Viral Coiled Coils. We inspected the virulent arenaviruses. CC+ coiled coil database (33) and found the presence of an x The GP2 residues strictly conserved in the alignment of Fig. 2A layer type stutter in the coiled coil of the majority of class I and form two clusters in the GP2 structure, one at the top and the III viral fusion proteins, with the notable exception of those from other at the bottom of the molecule (Fig. S1). Inspection of lentiviruses (HIV-1 and SIV gp41). Further examination of the Fig. 2A indicates that this pattern is maintained in the region of 51 trimeric parallel coiled coils currently present in the database the top of the molecule that is missing from the structure (at the indicated that the stutter is present only in the structure of post- fusion viral fusogenic proteins, independent of the length of the N- and C-terminal ends of the ectodomain). The bottom cluster coiled coil helices. It is absent in the other parallel, trimeric coiled includes residues of all three layers of the molecule, including coils from either cellular or viral origin. the flap. The conservation of these residues highlights their im- We found that the location of the stutter allows for an unam- portance in maintaining a very stable postfusion conformation. biguous alignment of the central coiled coils of the corresponding fusion proteins (Fig. 3). The side chain at the stutter (Val336 in Intrachain Salt Bridges Stabilize the GP2 Postfusion Hairpin. In addi- LCMV) is branched in all the examples that we have analyzed tion to the hydrophobic interactions of the C helix and the (Fig. 3B). The corresponding superposition of the 3D structures extended segment packing against the central coiled coil, a num- revealed a common trimeric coiled coil core comprising 24 resi- ber of intraprotomer polar/electrostatic interactions are seen dues (i.e., GP2 residues 329–352 in the N helix, corresponding stabilizing the hairpin conformation, especially in the top half the to roughly seven turns and to 72 residues in the trimer). The trimer (Fig. 1B). There are two salt bridges between lysine side Cα atoms in this segment align with rmsd under 1 Å with all of chains in the N helix and aspartate and/or glutamate residues in the viral fusion proteins displayed in Fig. 3A. The matching seg- the C helix (Lys326 with Asp414, and Lys333 with Asp407 and ment is longer than this minimal core in the superposition with Glu410). The first salt bridge is highly conserved (Fig. 2A). In the GP2 counterparts from several viruses (green + yellow in particular, a mutation to alanine of the Asp414 counterpart Fig. 3A). For instance, influenza virus HA2 has 135 α-carbons in Junin virus (Asp400 in Junin GPC numbering) was shown to (i.e., 45 per protomer, corresponding to 13 turns or the entire restore the wild-type pH sensitivity of a Lys33Gln mutant in the LCMV GP2 coiled coil) matching within 1 Å rmsd. This very SSP subunit (24). This observation suggests that the polypeptide good match indicates that the packing of the alpha helices is region that becomes the C helix in the postfusion hairpin confor- almost identical in spite of the poor amino acid sequence identity mation makes electrostatic interactions with SSP in the prefusion in the same region. The SARS coronavirus and the Newcastle form, although we cannot rule out that a more global effect of disease virus (NDV, a paramyxovirus) also display matching these mutations on the protein affects the pH dependence of the segments longer than the minimal core (Fig. 3A). Superposition conformational change. on the stutter also brings into coincidence the Cl− ion observed in A third salt bridge, this time LCMV-specific, connects His341 LCMV GP2 with one of the two central Cl− ions identified in the in the N helix with Glu402 in the extended segment. Finally, a SARS coronavirus S protein (34) (see SI Text). The list of viral fourth salt bridge connects two residues close in the sequence, proteins with a structurally very similar coiled coil region includes Lys331 and Asp335, from two consecutive turns of the N helix the Epstein Barr virus (EBV) glycoprotein B (gB), which is a class (Fig. 1B). This interaction is part of a trimer contact, with the III fusion protein (Fig. 3A). Asp335 side chain making van der Waals interactions with Although the conserved coiled coil core of LCMV GP2 and Ser406 from the adjacent protomer. This salt bridge is conserved the viral proteins represented in Fig. 3 align very well, we found only in Old World arenaviruses. Junin and Machupo viruses lack that this is not the case with all members within the corresponding the equivalent to Lys331 (which becomes threonine), but at the viral families, in spite of the conservation of the stutter. This is same time the residue at position 406 is arginine, whereas aspar- illustrated in Fig. S2, which shows that the fusion (F) proteins tate or glutamate are maintained at position 335 (Fig. 2A). We from some of the other members of the Paramyxoviridae family therefore predict that in Machupo and Junin viruses there is an have a proline residue in the helical turn immediately down- interchain salt bridge connecting the residues at positions 335 and stream the stutter. This feature distorts the corresponding α-he- 406 (LCMV numbering). Importantly, it was shown that mutation lices such that the register of the heptad repeats is altered, of Arg406 to alanine abolished membrane fusion induced by resulting in a second stutter. This is reflected in a higher rmsd Junin virus (31). Taken together, these observations highlight the obtained after superposition to the core coiled coil (around 2 Å; functional implication of salt bridges stabilizing the postfusion Fig. S2A). Despite this distortion, the side-to-side comparisons of

19970 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1108910108 Igonet et al. BIOCHEMISTRY

Fig. 3. Stutter-based structural alignment of classes I and III viral fusion proteins. (A) Top: Side-to-side comparison of the postfusion structures of the indicated viral envelope proteins, after superposing the stutters (arrow). Horizontal dashed lines indicate the limits of the LCMV coiled coil, and horizontal dotted lines indicate the extent of the common core structure (in green). The remainder of the N helix is in blue and the C helices in red. Yellow indicates N-term and C-term extensions of the core, to highlight the region of each protein superposing within 1.0 Å rmsd onto LCMV GP2. Bottom: Cross-sections at the x layer type stutters (displaying “knob-into-knob” packing), indicated by black arrows in the alignment at the top. The table shows the corresponding rmsd values. Accession numbers for the structures used are provided in SI Text.(B) Stutter-based sequence alignment of viral fusion glycoproteins from classes I and III. The position of the x layer type stutter is indicated by the dashed red frame. Positions a and d of the central residues in the coiled coil are indicated in green and yellow, respectively. the molecules presented in Fig. S2A shows that the conserved HSV-1 gB, baculovirus gp64 and VSV G appear more straight stutter provides the correct structural alignment of these homo- and less coiled around each other when compared to EBV gB logous molecules, which in turn validates their comparison to or LCMV GP2 (Fig. S4). Yet it is clear from Fig. S4 that the LCMV GP2. stutter provides a useful means of aligning them, highlighting In the Retroviridae family, the stutter is absent altogether in the for instance the differences at the turn of the hairpin and showing lentiviruses, and in that case the best alignment obtained when that VSV G is clearly the most distant. superposing the core segment of LCMV GP2 onto the coiled coil In summary, although the class I fusion proteins have been of SIV gp41 yields 1.8 Å rmsd, again a clearly poorer match than compared side to side before, the stutter provides a useful refer- those reported in Fig. 3. Concerning other members of this viral ence for superposing the trimeric molecules, with a single register family, the amino acid sequence alignment of HTLV-1 and along the heptad repeat. This alignment allows a comparison of MoMLVallows the prediction of the stutter in the MoMLV coiled the relative locations of the functional elements of the molecule coil (Fig. S3) in spite of its absence in the available structure, (i.e., fusion loop and TM region) with respect to the conserved which appears truncated. Similarly, the class III fusion proteins, core. For instance, it is clear that there is an extension, in the form which are present in viruses belonging to unrelated families of a longer coiled coil, between the coronavirus core and the (gB from herpes viruses, gp64 from baculovirus, and G from the membrane-interacting elements. This feature is shared only with vesicular stomatitis virus) all display the stutter and can be the paramyxovirus F protein, in addition to the presence of a big- aligned accordingly. However, these molecules align to EBV gB ger “head” of the molecule (absent form the structure of the cor- with a higher rmsd (between 1.3 Å and 2 Å; Fig. S4) than does onaviruses S protein, which was truncated). These similarities EBV gB to LCMV GP2 (Fig. 3). This indicates that the confor- between the two proteins could be an indication of homology be- mation of the helices in the coiled coil can also be affected by tween them, although more structural studies of the coronavirus their particular amino acid sequence and/or the local environ- S protein are needed to confirm this hypothesis. The alignment ment within the protein. Indeed, the central alpha helices in of Fig. 3 thus also indicates that the common core of the fusion

Igonet et al. PNAS ∣ December 13, 2011 ∣ vol. 108 ∣ no. 50 ∣ 19971 protein is positioned at a different distance from the fused mem- GP2 is the influenza virus HA2 protein. Finally, the broad con- brane for different viruses. Finally, it is clear from Fig. 3 that servation of the stutter in viral fusion proteins—and its absence in there are extensive variations in the region of the turn of the other proteins with a parallel, trimeric coiled coil—suggests a molecule, as well as toward the membrane interaction region functional implication of this architectural feature, which may (top) region, of the respective hairpins. provide clear advantages to a protein that folds initially in one conformation and adopts later a different one in order to induce Concluding Remarks. The structure of the recombinant LCMV membrane fusion. GP2 ectodomain points to a number of salt-bridges stabilizing its postfusion hairpin conformation. The availability of mutagen- Materials and Methods esis data on some of the residues making these interactions, We used a synthetic DNA (Entelechon) corresponding to LCMV strain WE-HPI which display fusion deficient phenotypes, highlights their func- (35), codon optimized for E. coli production, inserted into the pET-19b vector tional importance. The link in Junin virus between the Lys33Gln (Novagen). The gene was expressed in the cytoplasm of E. coli Rosetta-gami and Asp414Ala (LCMV numbering) mutants with the pH sensi- (DE3) strain and cells were broken by two passages through an Emulsiflex-C5 tivity of the fusion reaction (24) also suggest that the polypeptide homogenizer (Avestin) to recover the protein (25). The soluble fraction was segment that forms the C helix in the postfusion form may be separated by centrifugation, purified by Ni-nitrilotriacetic acid affinity chro- involved in interactions with SSP in the prefusion conformation. matography (Amersham Biosciences) followed by gel filtration with a Super- The structure further reveals that the stutter provides a common dex 75 16/60 column (GE Healthcare). The structure was determined to 1.8-Å register to align the trimeric coiled coils of many viral fusion resolution (Tables S1 and S2), as explained in the SI Text. All illustrations were proteins, a striking observation that had not been previously prepared with the PyMOL Molecular Graphics System (36). reported. For some viruses, the conservation of the central core appears to be the result of convergent evolution (for instance, ACKNOWLEDGMENTS. We thank Ahmed Haouz and Patrick Weber from the when comparing GP2 with the class III proteins, and probably robotized crystallization facility (PF6) at Institut Pasteur for help in crystal also with the paramyxovirus and coronavirus fusion proteins). screening and the staff at the Swiss Light Source (SLS) for assistance during In contrast, comparison with the Ebola and HTLV fusion pro- data collection. Diffraction data were collected with the Pilatus detector at the PXI beamline, SLS synchrotron (Villigen, Switzerland). F.A.R. acknowl- teins suggests divergent evolution from an ancestral gene—as — edges support from the European Union (VirApt consortium 018753), Institut pointed out before which could have further diverged in the Pasteur, Centre National de la Recherche Scientifique, and Merck Serono. case of the lentiviruses to the point that the stutter is lost. It is H.H. and B.E. acknowledge support from the Swiss National Science striking, however, that the closest structural homolog of LCMV Foundation.

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19972 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1108910108 Igonet et al.