Structural basis for the membrane fusion step in Hantavirus entry

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 the viral 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. We determined 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 G C structure together with our functional data provides new mechanistic insights into class II membrane fusion proteins and reveals new targets for membrane fusion inhibitors against these important pathogens. Both similarities and differences to other class II membrane fusion proteins implies a revise paradigm for the evolution of these unique proteins

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

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.Work with mutant Gc proteins was funded by FONDECYT 1140050 and Basal PFB-16 grants from CONICYT (to NDT), YM was supported by a Senior Research Fellowship from the Wellcome Trust, grant no. 101908/Z/13/Z,This is the final version of the article. It first appeared from PLOS via https://doi.org/10.1371/journal.ppat.100594

Cooperativity and flexibility in enzyme evolution

Enzymes are flexible catalysts, and there has been substantial discussion about the extent to which this flexibility contributes to their catalytic efficiency. What has been significantly less discussed is the extent to which this flexibility contributes to their evolvability. Despite this, recent years have seen an increasing number of both experimental and computational studies that demonstrate that cooperativity and flexibility play significant roles in enzyme innovation. This review covers key developments in the field that emphasize the importance of enzyme dynamics not just to the evolution of new enzyme function(s), but also as a property that can be harnessed in the design of new artificial enzymes.The European Research Council has provided financial support under the European Community’s Seventh Framework Program (FP7/2007-2013)/ERC Grant Agreement No. 306474. This work was also funded by the Feder Funds, Grants from the Spanish Ministry of Economy and Competitiveness (BIO2015-66426-R and CSD2009-00088) and the Human Frontier Science Program (RGP0041/2017). A.P. is a Wenner-Gren Foundations Postdoctoral Fellow and S. C. L. K. is a Wallenberg Academy Fellow

The stem region stabilizes the G_C trimer.

(A) On the left an orientation overview of PUUV GC 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]. 2FO-FC 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 GC from PUUV and ANDV GC. 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 GC was detected with anti- GC 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 GC 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 GC in each fraction by western blot using anti-GC MAb. The molecular mass of each fraction was determined experimentally by a molecular marker and plotted against the log of its theoretical molecular mass. GC trimers have a molecular mass of 165 KDa. (E) Trimer stability of wild type or mutant GC. VLPs including wild type GN and wild type or mutant GC were treated at different pHs and next incubated for 30 min with trypsin. The trypsine resistance of GC was assessed by western blot analysis with anti-GC 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 GC treated at pH 5.5. ***, P < 0.00025; **, P < 0.0025;*, P < 0.025; ns, not significant.</p

Overall fold of the post-fusion PUUV sG_C.

<p>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.</p

Inter-protomer interactions unique to PUUV G_C.

<p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005948#ppat.1005948.g001" target="_blank">Fig 1</a> 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.</p

Neutralizing epitope mapping on the surface of PUUV G_C.

<p>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.</p

Crystallographic data collection and refinement statistics.

<p>Crystallographic data collection and refinement statistics.</p

Hinge motions in PUUV sG_C protomers.

<p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005948#ppat.1005948.g001" target="_blank">Fig 1</a>. (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.</p