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Functional refolding of the penetration protein on a non-enveloped virus

Abstract

A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell1. For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP42,3. Here we describe the use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an ‘upright’ to a ‘reversed’ conformation. The reversed structure projects a previously buried ‘foot’ domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses.

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Fig. 1: Rotavirus entry and cryo-EM structures of the penetration protein in upright and reversed conformations.
Fig. 2: Interactions of the penetration protein domains VP5* and VP8* with VP7 capsid subunits.
Fig. 3: Infectivity and structure of TLPs recoated with mutant (S567C/A590C) penetration proteins.
Fig. 4: Molecular rearrangements of the membrane-penetration protein during rotavirus entry.

Data availability

The maps of the cryo-EM reconstructions have been deposited in the Electron Microscopy Data Bank (EMDB) (accession numbers: EMD-21955, upright conformation; EMD-21956, intermediate conformation; EMD-21957, reversed conformation), and the refined coordinates in the Protein Data Bank (PDB) (PDB IDs: 6WXE, upright conformation; 6WXF, intermediate conformation; 6WXG, reversed conformation). We obtained previously published rotavirus structures from the PDB (PDB IDs: 4V7Q and 1SLQ). The rotavirus protein sequences used to prepare the sequence alignments (shown in Supplementary Data 13) were retrieved from GenBank46. The accession numbers for VP4 are: RRV, P12473; SA11, P12976; WA, P11193; S2, AQT31697; DS1, P11196; ST3, P11200; AU-1, P39033; 116E, Q09113; 69M, P26451; NCDV, P17465; UK, P12474; 993/83, Q08010; Gottfried, P23045; OSU, P11114; CH3, Q86184; L338, Q98636; EW, Q83450. The accession numbers for VP7 are: RRV, P12473; SA11, P12976; WA, P11193; S2, AQT31697; DS1, P11196; ST3, P11200; AU-1, P39033; 116E, Q09113; 69M, P26451; NCDV, P17465; UK, P12474; 993/83, Q08010; Gottfried, P23045; OSU, P11114; CH3, Q86184; L338, Q98636; EW, Q83450. The accession numbers for VP6 are: RRV, P12473; SA11, P12976; WA, P11193; S2, AQT31697; DS1, P11196; ST3, P11200; AU-1, P39033; 116E, Q09113; 69M, P26451; NCDV, P17465; UK, P12474; 993/83, Q08010; Gottfried, P23045; OSU, P11114; CH3, Q86184; L338, Q98636; EW, Q83450. All data are available from the corresponding author upon reasonable request.

Code availability

The software programs used to generate and analyse the data of this study are publicly available. Custom-written C shell and Python scripts used to run the programs are available from the corresponding author on reasonable request.

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Acknowledgements

We thank Z. Li, S. Sterling, R. Walsh and S. Rawson for assistance and guidance at the Harvard Medical School Cryo-EM Center for Structural Biology and the Harvard Medical School Molecular Electron Microscopy Suite; C. Xu for assistance at the Brandeis University cryo-EM facility; and H. B. Greenberg for the gift of the HS1 and HS2 antibodies. The work was supported by National Institutes of Health grant CA-13202 (to S.C.H.). S.C.H. is an Investigator in the Howard Hughes Medical Institute.

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Authors and Affiliations

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Contributions

T.H., E.N.S., D.N., S.J. and S.C.H. designed the experiments; T.H., R.T., E.N.S. and C.B. conducted the experiments and recorded data; T.H., R.T., D.S. and S.J. analysed the data; T.H. and S.J. determined structures and built models; T.H., S.J. and S.C.H. wrote the paper; and T.H., C.B., D.S., D.N., S.J. and S.C.H. revised and edited the paper.

Corresponding authors

Correspondence to Simon Jenni or Stephen C. Harrison.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks David Bhella, John Patton and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Sample preparation and cryo-EM data collection.

a, Schematic protocol for recoating of double-layer particles (DLPs) with recombinant VP4 and VP7. b, Time course of the digestion of wild-type recoated triple-layer particles (wt rcTLPs) with 5 μg ml−1 trypsin at 37 °C. Samples were analysed after the shown incubation times by SDS–PAGE. The experiment was repeated independently twice with similar results. For gel source data, see Supplementary Fig. 1. c, Representative micrograph (aligned and summed movie frames) of wt rcTLPs recorded with a Polara F30 electron microscope equipped with a K2 summit detector (magnification, 40,650). Scale bar, 100 nm. d, Power spectrum of the micrograph shown in c.

Extended Data Fig. 2 Data processing for local reconstructions.

Full workflow for local reconstructions of rotavirus spike proteins.

Extended Data Fig. 3 Resolution analysis of the local cryo-EM reconstructions.

Left, Fourier shell correlation (FSC) curves for the reconstructions and refined models calculated with phenix.mtriage54. Correlations for the two half maps are shown as solid blue lines after applying a mask encompassing the models. Correlations between the refined model and final map are shown as red solid lines. Nominal resolution estimates at conventional FSC values are indicated by arrows. Right, local resolution of the reconstructions calculated with ResMap55. a, Upright conformation. Dashed lines (left) are the FSC analysis for the reconstruction of the distal VP5*/VP8* dimeric density that was obtained through alignment by classification (see Methods). The images on the right show the local resolution of this reconstruction. b, Intermediate conformation. c, Reversed conformation. For source data of the FSC plots, see Supplementary Data 5.

Extended Data Fig. 4 Cryo-EM density and structure comparison.

a, Magnified views of representative regions of the cryo-EM density maps obtained by local reconstruction. Density is shown as grey mesh; polypeptide-chain backbone as ribbon; side-chain atoms as sticks (carbon, main color; nitrogen, blue; oxygen, red; sulfur, orange). b, Per-residue Cα distances after subunit-wise superposition of VP4, VP7 and VP6 subunits from the upright and reversed conformation structures.

Extended Data Fig. 5 Comparison of penetration protein conformations on rcTLPs and native TLPs.

Relative subparticle amounts, and corresponding cryo-EM reconstructions obtained from rcTLPs (top row) and native TLPs (bottom row). Local spike reconstructions of rcTLPs were obtained from two cryo-EM samples prepared from two independent recoating reactions. Local spike reconstructions of native TLPs were obtained from one cryo-EM sample.

Extended Data Fig. 6 Rearrangements of the VP8* and VP5* penetration proteins during transition from upright to reversed conformation on the virion surface.

a, Distinct domains of the VP8* and VP5* spike proteins are coloured separately to illustrate their conformational change during transition from upright (top row) to reversed (bottom row) conformation. Domains that were not observed in our cryo-EM maps because of flexible attachment are drawn schematically. VP8*, magenta; VP5*, red, orange and salmon. b, Formation of the trimeric coiled coil and extrusion of the foot domains. Top row, magnified views of the VP5* foot domain exit sites as observed in the intermediate conformation structure. A partially cut surface representation is shown. VP5*, red, orange and salmon; VP7, yellow. The last modelled residues of the VP5* β-barrel domains—482 (chain 1), 480 (chain 2) and 481 (chain 3)—are located on the outside. The connections to the first modelled VP5* foot domain residue—494 (chain 1), 494 (chain 2) and 498 (chain 3)—are indicated by dashed lines (fuzzy density in the cryo-EM map). Arrows indicate a suggested mechanism for foot-domain reversal, involving zipping-up of the trimeric coiled coil and unfolding and extrusion of foot-domain residues. Bottom row, proposed transition from the intermediate structure (left) to the reversed structure by zipping up of the coiled coil and unfolding and extrusion of the foot domains.

Extended Data Fig. 7 Molecular details of the VP5*–VP7 interfaces for upright and reversed conformations.

a, Upright conformation; b, reversed conformation. In each part of the figure, the left-hand image is a view from outside the TLP, with circles in black and blue corresponding to coloured outlines of the detailed, side-view panels on the right. VP5*, salmon; VP7, yellow. The following VP5* interface residues are conserved (Supplementary Data 1): N268, N376, R467, S469. The VP5* β-barrel N terminus (residues 248–250) is not strictly conserved, but the interaction is based on main-chain hydrogen bonds and can probably be maintained for different side chains as well. The following VP7 interface residues are conserved (Supplementary Data 2): S201, T210, L172–Y175.

Extended Data Fig. 8 Inducing penetration protein reversal at alkaline pH and VP8* association with TLPs.

a, Analysis of rotavirus particles without (lanes 1 and 2) and with (lanes 3 and 4) high pH-induced conformational change of VP8* or VP5* and without (lanes 1 and 3) and with (lanes 2 and 4) EDTA-induced uncoating. Pelleted fractions were analysed by SDS–PAGE and silver staining (left) and by western blotting with the VP8*-specific antibody HS127 (right) (see Methods). The experiment was repeated independently three times with similar results. For gel and western blot source data, see Supplementary Fig. 1. b, Relative VP4 subparticle amounts, and corresponding cryo-EM reconstructions obtained from wild-type rcTLPs. c, Relative VP4 subparticle amounts, and corresponding cryo-EM reconstructions obtained from rcTLPs containing VP4(S567C/A590C). Recoating reactions for all samples were carried out at the same time and with the same VP7 and DLP stock solutions. All cryo-EM samples were prepared in the same blotting session.

Extended Data Fig. 9 Cryo-ET analysis of RRVs entering BSC-1 cells.

a, Sections of the tomogram from which the reconstructions (icosahedral average of single virion subtomograms) in Fig. 4a were obtained. Left, virus with loose membrane contact indicated by an arrow. Right, virus with tight (close) membrane contact indicated by an arrow. Images were low-pass filtered and contrast enhanced for display. Scale bar, 100 nm. b, Tomographic slices of manually selected viruses (not including particle in a) from several tomograms with tight membrane contacts (yellow arrowheads). Images were low-pass filtered and contrast-enhanced for display. Those particles were selected to detect VP4 reversed conformations in additional viruses (other than particle in Fig. 4a). c, Sub-subtomogram classification of VP4 positions extracted from the RRV particles shown in b yielded a class with the upright conformation (left) and a class with the reversed conformation (right). VP4 positions were extracted from 26 selected particles chosen to have an easily identified region of close membrane contact. The particle shown in Fig. 4a was excluded from this selection. Note that the reconstructions shown in Fig. 4a are from single particles with imposed icosahedral symmetry, whereas we show here classified averaged individual volumes extracted at VP4 positions.

Extended Data Table 1 Cryo-EM data collection and model statistics

Supplementary information

Supplementary Information

This file contains the Supplementary Discussion.

Reporting Summary

Peer Review File

Supplementary Figure

Supplementary Figure 1: This file shows the Western blot and gel source data for Fig. 3d, Extended Data Fig. 1b, and Extended Data Fig. 8a.

Supplementary Data

Supplementary Data 1: Rotavirus VP4 multiple sequence alignment from GenBank accession identifiers: RRV, P12473; SA11, P12976; WA, P11193; S2, AQT31697; DS1, P11196; ST3, P11200; AU-1, P39033; 116E, Q09113; 69M, P26451; NCDV, P17465; UK, P12474; 993/83, Q08010; Gottfried, P23045; OSU, P11114; CH3, Q86184; L338, Q98636; EW, Q83450. Modeled residues are shown as solid bars.

Supplementary Data

Supplementary Data 2: Rotavirus VP7 multiple sequence alignment from GenBank accession identifiers: RRV, P12473; SA11, P12976; WA, P11193; S2, AQT31697; DS1, P11196; ST3, P11200; AU-1, P39033; 116E, Q09113; 69M, P26451; NCDV, P17465; UK, P12474; 993/83, Q08010; Gottfried, P23045; OSU, P11114; CH3, Q86184; L338, Q98636; EW, Q83450. Modeled residues are shown as solid bars.

Supplementary Data

Supplementary Data 3: Rotavirus VP6 multiple sequence alignment from GenBank accession identifiers: RRV, P12473; SA11, P12976; WA, P11193; S2, AQT31697; DS1, P11196; ST3, P11200; AU-1, P39033; 116E, Q09113; 69M, P26451; NCDV, P17465; UK, P12474; 993/83, Q08010; Gottfried, P23045; OSU, P11114; CH3, Q86184; L338, Q98636; EW, Q83450. Modeled residues are shown as solid bars.

Supplementary Data

Supplementary Data 4: This file contains the source data for the bar plot shown in Fig. 3a.

Supplementary Data

Supplementary Data 5: This file contains the source data for the Fourier shell correlation (FSC) plots shown in Extended Data Fig. 3.

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Herrmann, T., Torres, R., Salgado, E.N. et al. Functional refolding of the penetration protein on a non-enveloped virus. Nature 590, 666–670 (2021). https://doi.org/10.1038/s41586-020-03124-4

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