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Structural basis for membrane anchoring and fusion regulation of the herpes simplex virus fusogen gB

Nature Structural & Molecular Biology volume 25, pages 416–424 (2018)Cite this article

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Abstract

Viral fusogens merge viral and cell membranes during cell penetration. Their ectodomains drive fusion by undergoing large-scale refolding, but little is known about the functionally important regions located within or near the membrane. Here we report the crystal structure of full-length glycoprotein B (gB), the fusogen from herpes simplex virus, complemented by electron spin resonance measurements. The membrane-proximal (MPR), transmembrane (TMD), and cytoplasmic (CTD) domains form a uniquely folded trimeric pedestal beneath the ectodomain, which balances dynamic flexibility with extensive, stabilizing membrane interactions. The postfusion conformation of the ectodomain suggests that the CTD likewise adopted the postfusion form. However, hyperfusogenic mutations, which destabilize the prefusion state of gB, target key interfaces and structural motifs that reinforce the observed CTD structure. Thus, a similar CTD structure must stabilize gB in its prefusion state. Our data suggest a model for how this dynamic, membrane-dependent ‘clamp’ controls the fusogenic refolding of gB.

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Fig. 1: The structure of gBΔ71.
Fig. 2: Residues involved in formation of the trimeric MPR-TMD-CTD pedestal.
Fig. 3: Orientation of the MPR-TMD-CTD segment in the membrane.
Fig. 4: Membrane interactions of the CTD C terminus.
Fig. 5: Interprotomer distances in the isolated CTD.
Fig. 6: Locations of fusion-altering mutations within the MPR-TMD-CTD structure.
Fig. 7: The clamp-and-wedge model of HSV fusion.

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Acknowledgements

We thank NE-CAT staff for help with X-ray data collection and H. Rogalin for help with the cell–cell fusion assay. We also thank P. G. Spear (Northwestern University), R. J. Eisenberg (University of Pennsylvania), and G. H. Cohen (University of Pennsylvania) for the gift of plasmids and J. M. Coffin (Tufts University) for the gift of CHO cells. This work was funded by NIH grant 1R21AI107171 (E.E.H.), the Burroughs Wellcome Fund (E.E.H.), and the NIH Ruth L. Kirschstein NRSA postdoctoral fellowship 1F32GM115060 (R.S.C.). The research of E.E.H. was supported in part by a Faculty Scholar grant from the Howard Hughes Medical Institute. ESR experiments were funded by NIH grants P41GM103521 (J.H.F., ACERT) and R01GM123779 (J.H.F. and E.R.G.). This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the NIH (P41GM103403). The Pilatus 6 M detector on the 24-ID-C beamline is funded by an NIH-ORIP HEI grant (S10RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract DE-AC02-06CH11357. All software was installed and maintained by SBGrid59.

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Authors and Affiliations

  1. Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, USA

    Rebecca S. Cooper & Ekaterina E. Heldwein

  2. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA

    Elka R. Georgieva, Peter P. Borbat & Jack H. Freed

  3. National Biomedical Center for Advanced Electron Spin Resonance Technology (ACERT), Cornell University, Ithaca, NY, USA

    Elka R. Georgieva, Peter P. Borbat & Jack H. Freed

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Contributions

R.S.C. designed experiments, cloned the constructs, produced recombinant proteins, crystallized gBdelta71, collected diffraction data, phased the data and determined the structure, built and refined the models, carried out cell-cell fusion assays, analyzed the data, and wrote the manuscript. E.R.G. designed ESR studies, carried out spin labeling and lipid reconstitution, collected, analyzed, and interpreted ESR data, and wrote the manuscript. P.P.B. collected, analyzed, and interpreted ESR data, and wrote the manuscript. J.H.F. wrote the manuscript. E.E.H. designed experiments, phased the data and determined the structure, built and refined the models, analyzed the data, and wrote the manuscript.

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Correspondence to Ekaterina E. Heldwein.

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Supplementary Figure 1 Purification and crystallization of gBd71.

a, SEC of gBΔ71 in 0.05% n-dodecyl-β-d-maltopyranoside (DDM) reveals a trimer with an apparent molecular weight of 415 kDa. b, SDS–PAGE analysis of the peak SEC fractions, separated from aggregated protein. c, Representative H32 crystals of gBΔ71 in 0.075% n-undecyl-β-d-maltopyranoside and 0.0075% A8-35 amphipol. d, Representative P321 gBΔ71 crystals in 0.05% n-dodecyl-β-d-maltopyranoside and 0.01% A8-35 amphipol. e, gBΔ71 packing within P321 crystals. The folded CTD core (blue) packs against the crown of the trimer below (orange), an interaction that presumably stabilizes both the CTD and TMD.

Supplementary Figure 2 Residue conservation in human herpesviruses.

a, Sequence alignment of eight human herpesviruses, with identical residues boxed in magenta and similar residues boxed in gray. Residues that contribute to potentially important HSV-1 CTD features, including its acidic face and membrane-binding basic belt, are indicated with stars. Nearby basic residues that may perform a similar function in other herpesviruses are boxed. The regions resolved in the crystal structure and by ESR are underlined in orange and green, respectively. Secondary structure elements are shown above the alignment. b,c, Top and side views of the MPR–TMD–CTD pedestal, with protomers shown in different shades of blue. Identical residues in the alignment are shown as magenta spheres. d, A close-up view of the TMD–h1a–h1b zigzag.

Supplementary Figure 3 Residue conservation in α-herpesviruses.

a, Sequence alignment of 13 α-herpesviruses. Residues that contribute to important HSV-1 CTD features, including its membrane-binding basic belt and acidic face, are indicated with stars. Nearby basic residues that may perform a similar function in other herpesviruses are boxed. b, Residues identical in these viruses, as well as among eight human herpesviruses from different subfamilies, are mapped onto side and top views of the MPR–TMD–CTD.

Supplementary Figure 4 Effect of the ordered CTD on TMD and MPR.

a, The well-ordered CTD of FL-gB (P321 crystals) packs against the crown of the trimer below (Supplementary Fig. 1e), which appears to stabilize the position of the TMD helices. b, When the CTD of FL-gB is disordered (H32 crystals), the C terminus of the TMD is unresolved and both the MPR–TMD and TMD–TMD angles increase.

Supplementary Figure 5 Interprotomer interactions in the CTD.

Four classes of interactions were identified using CCP4 Contact. Only interactions ≤ 4 Å are depicted for simplicity. The “van der Waals + mixed” category indicates that a pair of residues may be linked by either van der Waals interactions or a combination of hydrophobic and van der Waals interactions. Positions where mutations alter the rate of fusion are shaded. Bolded residues are conserved in α-herpesviruses. Box colors indicate hyperfusogenic mutations identified in clinical isolates (light orange) or engineered (light green) as well as a single slow-entry mutation (light blue). Box outline colors indicate whether the collective interactions of a residue are mediated by main chain atoms (red), side chain atoms (blue), or both (purple).

Supplementary Figure 6 Fusion activity of single-cysteine FL-gB mutants.

Cell–cell fusion between effector cells expressing gD, gH–gL, and single-cysteine gB mutants and target cells expressing gD receptor HVEM was tested using a luciferase assay. The previously identified hyperfusogenic mutant R858H was included as a control for each assay. Every mutant was tested in at least two biological replicates (n), each consisting of three technical replicates. Dots show the average fusion activity of each biological replicate relative to wild-type gB (100% fusion). Depending on the background plasmid for mutant construction (indicated in the source data available online), wild-type gB denotes either pPEP98 or pRC30. Error bars show 1 s.d. from the biological replicate average, which is marked with a wider central bar. The activity of E816C was not tested. Most mutants had a wild-type or mildly hyperfusogenic phenotype, indicating that the global structure of the h2–h3 region was not significantly altered by these substitutions. For K862C, V880C, M881C, and R882C, which have hyperfusogenic phenotypes, interaction of the native side chains with the membrane may be stronger than that of the cysteine and important for CTD stability.

Source data

Supplementary Figure 7 Accessibility and mobility of the CTD C terminus in the presence of membrane.

a, Accessibility (Π) of CTD residues 861–885 to 4.5 mM NiEDDA and O2. b, Mobility (1/ΔH) of CTD residues 861–885. c, The mobility of residues 876–884 follows a periodic pattern (inset) that is consistent with an amphipathic helix in which the movement of membrane-facing residues is restricted. The D878C mutant precipitated and was excluded from measurements. Measurements were collected once on each mutant, with the exception of V876C, M879C, R882C, and R884C, which were tested twice. Initial validation of the data collection protocol on these isolated mutants produced similar results, but only the complete range was used for depth calculations and subsequent analysis.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1 and 2, and Supplementary Note

Reporting Summary

Supplementary Table 3

Single-cysteine mutant cloning

Source Data for Supplementary Figure 6

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Cooper, R.S., Georgieva, E.R., Borbat, P.P. et al. Structural basis for membrane anchoring and fusion regulation of the herpes simplex virus fusogen gB. Nat Struct Mol Biol 25, 416–424 (2018). https://doi.org/10.1038/s41594-018-0060-6

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