Crystal Structure of Glycoprotein C from a Hantavirus in the Post-fusion Conformation

Abstract

Hantaviruses are important emerging human pathogens and are the causative agents of serious diseases in humans with high mortality rates. Like other members in the Bunyaviridae family their M segment encodes two glycoproteins, GN and GC, which are responsible for the early events of infection. Hantaviruses deliver their tripartite genome into the cytoplasm by fusion of the viral and endosomal membranes in response to the reduced pH of the endosome. Unlike phleboviruses (e.g. Rift valley fever virus), that have an icosahedral glycoprotein envelope, hantaviruses display a pleomorphic virion morphology as GN and GC assemble into spikes with apparent four-fold symmetry organized in a grid-like pattern on the viral membrane. Here we present the crystal structure of glycoprotein C (GC) from Puumala virus (PUUV), a representative member of the Hantavirus genus. The crystal structure shows GC as the membrane fusion effector of PUUV and it presents a class II membrane fusion protein fold. Furthermore, GC was crystallized in its post-fusion trimeric conformation that until now had been observed only in Flavi- and Togaviridae family members. The PUUV GC structure together with our functional data provides intriguing evolutionary and mechanistic insights into class II membrane fusion proteins and reveals new targets for membrane fusion inhibitors against these important pathogens.

Fig 1. Overall fold of the post-fusion PUUV sG_C.

PUUV sG_C has the same three-domain architecture as other class II proteins. Domain I is shown in red, domain II in yellow with the fusion loop in orange, domain III in blue and the stem region in light pink. Residue numbers follow GPC numbering. The membrane proximal part of the stem, the transmembrane anchor and the cytoplasmic tail (grey) are missing in the structure. Secondary structure elements are indicated. Glycans are linked to N937. Disulfide bonds are in green. Gray rectangle represents the outer leaflet of the membrane. On the right, a cholesterol and phosphatidylethanolamine molecules are shown for scale. On the top is linear domain organization of PUUV G_C. Color scheme is as described for the structure. Gray indicates regions that were not observed in the structure. Numbers correspond to GPC numbering and in parenthesis is G_C numbering.

Fig 2. The putative fusion loop of PUUV G_C compared to fusion loops of other viral class II membrane fusion proteins.

(A) Structure-based multiple sequence alignment of the fusion loop regions from different class II members. Shading is in the same color scheme as in Fig 1. The PXD insertion in PUUV G_C is highlighted with a black box. Residues of PUUV G_C and RVFV G_C correspond to polyprotein precursor numbering. (B) Clockwise from the top left: PUUV G_C, RVFV G_C, DENV2 E and Semliki forest virus (SFV) E1 fusion loops (PDB codes 5J81, 4HJ1, 1OK8 and 1RER, respectively). The hydrophobic residues that anchor the protein to the cellular membrane are shown in stick representation and the disulfide bonds are shown in ball-and-stick representation. (C) CONSURF analysis [74] of unique hantavirus G_C sequences projected on the surface of PUUV sG_C crystal structure. A top view on the fusion loop, down the crystallographic tree-fold axis. The following uniprot (http://www.uniprot.org/) entries were used for the analysis: M9QRJ8, Q9QIZ1, M9QSR6, W5RRK8, Q5MYC0, Q9WJ31, Q2V8Y2, A0A068EN08, A0A0A7EQ65, Q66753, M9QY05, A0S5D7, O12371, Q9WSK6, Q806Y7, A0A0D5W3U2, F1T2C3, A8RRS6, G0WJH7, P27315, C7AGW1, B1NSM7, Q83887, A0A075IFP0, A0A0K0K9P4, Q9WMK6, A0A0F6T9U0, H8XZQ0, Q9DXJ5, Q9E158, Q91BQ9, A0A068ETZ4, A6MD75, B6DDK4, V9MFN9, H6WCQ9, Q99BV0, A0A077D3A4, P08668, F6KBJ3, A0RZG8, K4MYY7, R4JAI4, U5L2G2, O55348, H8ZHK6, H8ZHL5 (D) Hydrogen bond between W773 and the carbonyl of P781. Yellow dash line represents the distance between P781 carbonyl and W773.

Fig 3. Inter-protomer interactions unique to PUUV G_C.

(A) Strand A₀ at the N-terminus of domain I extends the B₀-I₀-H₀-G₀ β-sheet of the adjacent protomer. The donor protomer (protomer 1) is indicated in the same color scheme as in Fig 1 while the neighboring protomer (protomer 2) is shown in faded colors. (B) Inter-trimer salt bridge at the membrane proximal part of domain II. Ionic pairs are in sticks representation. The boundaries of each protomer are highlighted. (C) The glycosylation on N937 mediates interactions between protomers. Right: view of the trimer from the membrane, down the crystallographic three-fold axis. Left: Close-up view on the glycosylation groove between the protomers. N937 and the glycans are in sticks representation. 2F_O-F_C electron density map at 1σ is shown in light blue mesh.

Fig 4. Neutralizing epitope mapping on the surface of PUUV G_C.

Solvent accessible surface representation of the PUUV G_C protomer with the linear epitopes of 1C9 (residues 822–834) and 4G2 (residues 903–920) MAb highlighted. Dark-surface protomers are the crystal structure of PUUV G_C and bright-surface protomers are pre-fusion model based on PUUV G_C domains superimposed onto Semliki forest virus E1 in the pre-fusion conformation (PDB code 2ALA). Gray dashed line represents the movement of domain III between the pre- and post-fusion conformations. On the right, a cartoon representation of the two epitope sites in the context of the trimer. Residues of the linear epitopes are highlighted with sphere representation. View angles are represented by eye symbols.

Fig 5. Hinge motions in PUUV sG_C protomers.

(A) Superposition of individual domains of sG_C ^XF1 (color) and sG_C ^XF2 (light grey). Root mean square deviation (RMSD, calculated in PyMol) for domain I, II and III are 0.339 Å, 0.483 Å, 0.227 Å respectively. (B) B-factor putty representation of the two crystal structures of PUUV sG_C. Cold colors (blue-green) represent lower B-factors whereas warm colors (yellow-red) represent high B-factors. In the inset is a ribbon representation of sG_C ^XF1 (color) and sG_C ^XF2 (light grey) in the same orientation as the putty representation. In red is the crystallographic 3-fold axis. (C) A quantifying B-factor analysis of the two PUUV sG_C crystal forms. Analysis was executed using bavarage module in CCP4 program suite (61). In black is sG_C ^XF1 and red is sG_C ^XF2. Linear domain organization is shown for orientation. Color scheme and domains borders are as in Fig 1. (D) A view on the fusion loop down the three-fold axis of sG_C ^XF1 (color) and sG_C ^XF2 (light grey) superposition. Triangles represent the distances between the C_α ^W773 of the protomers. The distance in sG_C ^XF1 (pH 6.0) is 11.0 Å whereas in sG_C ^XF2 (pH 8.0) it is 14.9 Å. Triangles area for pH 6.0 and pH 8.0 are 52.4 Ų and 114.1 Ų, respectively. (E) E770-R902 inter-protomer salt bridge at the two crystal forms. Color scheme is as in panel B.

Fig 6. The stem region stabilizes the G_C trimer.

(A) On the left an orientation overview of PUUV G_C trimer. In the middle, a close-up view of the stem region of one of the protomers. Side-chains of the stem residues are shown in sticks representation. The surface electrostatic potential (red, -5 kT/e; blue, 5 kT/e) of domain II was calculated by APBS [72]. 2F_O-F_C electron density map at 1σ is shown in green mesh. On the right is a detailed view of the interactions of the stem region in sticks/cartoon representation. Color scheme is as in Fig 1. (B) Cell-cell fusion activity of wild type and mutant G_C from PUUV and ANDV G_C. Representative fluorescence micrographs of Vero E6 cells expressing wild type or R1074A mutant GPC from PUUV or ANDV, and treated at different pHs. The cell cytoplasm was labelled with 5-chloromethylfluorescein diacetate (CMFDA; green fluorescence), nuclei with DAPI (blue fluorescence) and G_C was detected with anti- G_C MAb (Alexa555; red fluorescence). Cells from a partial microscopy field are shown from a representative experiment. Mock indicates cells transfected with an empty expression plasmid. Arrows indicate syncytia. (200 x magnification). (C) Quantification of the cell-cell fusion activity of cells expressing wild type and mutant GPC. The mean fusion index was calculated by counting cells and nuclei and represents n ≥ 2 independent experiments. Fusion activity of G_C mutant R902A was similar to the wild type and serves as a positive control. (D) Homotrimer formation of wild type and R1074A mutant Gc from ANDV after low pH treatment. Sucrose gradient sedimentation of glycoproteins extracted from ANDV-like particles after their treatment at the indicated pHs. Detection of G_C in each fraction by western blot using anti-G_C MAb. The molecular mass of each fraction was determined experimentally by a molecular marker and plotted against the log of its theoretical molecular mass. G_C trimers have a molecular mass of 165 KDa. (E) Trimer stability of wild type or mutant G_C. VLPs including wild type G_N and wild type or mutant G_C were treated at different pHs and next incubated for 30 min with trypsin. The trypsine resistance of G_C was assessed by western blot analysis with anti-G_C MAb. Results were quantified by densitometry from n ≥ 2 experiments. As a control, the fusion active mutant R902A serves as trypsin-resistant control. The statistical evaluation of each data point was performed in relation to the wild type G_C treated at pH 5.5. ***, P < 0.00025; **, P < 0.0025;*, P < 0.025; ns, not significant.

Fig 7. Structural and evolutionary relationships in class II fusion proteins.

Cladograms representing the structural relationships between different class II fusion proteins. The coordinates of PUUV G_C were submitted to the DALI server. Atomic coordinates were obtained from the Protein Data Bank (PDB). Structure based alignment of the collected coordinates was performed with MUSTANG. A dendrogram was estimated based on a neighbor-joining analysis of the aligned sequences and guided by the BLOSUM62 substitution matrix. Abbreviations and their respective PDBs are as follows: dengue virus 2 (DENV-2, 1OK8-A), tick-borne encephalitis virus (TBEV, 1SVB-A), dengue virus 3 (DENV-3, 1UZG-A), Semliki forest virus (SFV, 1RER-A), West Nile virus (WNV, 2I69-A), Dengue virus 1 (DENV-1, 3G7T-A), Sindbis virus (SINV, 3MUU-A), Chikugunya virus (CHV, 3N41-F), Japanese encephalitis virus (JEV, 3P54-A), rubella virus (RV, 4ADG-A), St. Louis encephalitis virus (SLEV, 4FG0-A), Rift valley fever virus (RVFV, 4HJ1). C.elegans EFF1 (4OJC-A) was added as an out-group. Color scheme is per legend. Icosahedron icon represents icosahedral envelope, P- pleomorphic envelope. Genome type and transmission vectors are indicated by representative symbols.