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Capping pores of alphavirus nsP1 gate membranous viral replication factories

Nature volume 589, pages 615–619 (2021)Cite this article

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Abstract

Positive-sense single-stranded RNA viruses, such as coronaviruses, flaviviruses and alphaviruses, carry out transcription and replication inside virus-induced membranous organelles within host cells1,2,3,4,5,6,7. The remodelling of the host-cell membranes for the formation of these organelles is coupled to the membrane association of viral replication complexes and to RNA synthesis. These viral niches allow for the concentration of metabolites and proteins for the synthesis of viral RNA, and prevent the detection of this RNA by the cellular innate immune system8. Here we present the cryo-electron microscopy structure of non-structural protein 1 (nsP1) of the alphavirus chikungunya virus, which is responsible for RNA capping and membrane binding of the viral replication machinery. The structure shows the enzyme in its active form, assembled in a monotopic membrane-associated dodecameric ring. The structure reveals the structural basis of the coupling between membrane binding, oligomerization and allosteric activation of the capping enzyme. The stoichiometry—with 12 active sites in a single complex—redefines viral replication complexes as RNA synthesis reactors. The ring shape of the complex implies it has a role in controlling access to the viral organelle and ensuring the exit of properly capped viral RNA. Our results provide high-resolution information about the membrane association of the replication machinery of positive-sense single-stranded RNA viruses, and open up avenues for the further characterization of viral replication on cell membranes and the generation of antiviral agents.

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Fig. 1: Cryo-EM structure of the spherule–pore complex of CHIKV nsP1.
Fig. 2: nsP1 oligomerization and membrane binding are coupled processes.
Fig. 3: Structure of the capping active site of nsP1.

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Data availability

Structure coordinates are available from the PDB with accession codes 6Z0V and 6Z0U for single and double rings, respectively. The electron density maps are available from the Electron Microscopy Data Bank (EMDB) under accession codes EMD-11024 and EMD-11023 for single and double rings, respectively. All other data generated or analysed in this study are available from the corresponding author upon reasonable request.

Viral amino acid sequences used for gene synthesis and sequence alignments were retrieved from the UniProt database with the following accession numbers: CHIKV S27 African prototype (UniProt: Q8JUX6); CHIKV (UniProt: Q5XXP4), Semliki Forest virus (UniProt: P08411), Venezuelan equine encephalitis virus (UniProt: P27282), Sindbis virus (UniProt: P03317), aura virus (UniProt: Q86924) and salmonid sleeping disease virus (UniProt: Q8QL53). MTase structures for structural superpositions were retrieved from the PDB with accession codes 2RI1 and 1RI1 for E. cuniculi MTase, and 1L9K for dengue virus MTase.

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Acknowledgements

We thank the European Synchrotron Radiation Facility for provision of beam time on CM01, and E. Kandiah for assistance; J. Martin-Benito and the CNB-CIB (CSIC) cryo-EM facility for granting us access to cryo-EM equipment through a technical support contract, and J. Chichon for technical support; INSTRUCT-Eric and R. Melero for access to the computing facilities at the CNB-CSIC (PID 7046 VID 13154); A. Goulet, S. Spinelli and the electron microscopy platform at the AFMB; I. Berger and A. Aubert for supplying material and technical advice regarding eukaryotic expression; and T. Ahola, A. Merits and F. Rico for critical reading of the manuscript. The nsP1 antibody was provided by A. Merits, and the m7G cap antibody by B. Coutard. This work has been supported by the Bettencourt Shueller Fondation and an ATIP-Avenir grant (CNRS/INSERM).

Author information

Authors and Affiliations

  1. Aix-Marseille Université, CNRS, AFMB UMR 7257, Marseille, France

    Rhian Jones, Gabriel Bragagnolo & Juan Reguera

  2. National Center of Biotechnology, CSIC, Madrid, Spain

    Rocío Arranz

  3. INSERM, AFMB UMR7257, Marseille, France

    Juan Reguera

Authors
  1. Rhian Jones
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  2. Gabriel Bragagnolo
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Contributions

R.J. performed sample production and purification, biochemical characterization and structure determination together with J.R. G.B. contributed to protein production and purification. R.A. assisted with sample preparation for cryo-EM. J.R. conceived and obtained the funding for the project. R.J. and J.R. designed the experiments and J.R. wrote the Article with the input of R.J.

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Correspondence to Juan Reguera.

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Peer review information Nature thanks Kyung Choi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Isolation and biochemical characterization of nsP1 ring complexes.

a, Size-exclusion profile of monomeric CHIKV nsP1 expressed in E. coli cells after purification by affinity chromatography. The elution volume corresponds to a molecular weight of approximately 61 kDa, corresponding to a nsP1 monomer. Fractions corresponding to the peak of the monomeric nsP1are shown in the offset SDS–PAGE with the molecular weight markers on the left. b, Western-blot-based activity test with CHIKV nsP1 purified from insect cells (H5) and E. coli with an antibody specific for m3G and m7G. The capping intermediate of the methylated GMP covalently bound to nsP1 is detected at 61 kDa for samples purified from insect cells when SAM and GTP are added in the reaction, and substantially more when m7GTP is added as substrate instead of GTP. Conversely, the monomeric nsP1 produced in E. coli shows no activity despite a substantially higher amount of protein used in the experiments. This is shown in the SDS–PAGE gel on the right. c, SDS–PAGE gels of fractions derived from a sucrose gradient flotation assay (Methods), in which extracts from E. coli cells (top gel) and Hi5 insect cells (bottom gel) expressing CHIKV nsP1 were subjected to flotation analysis. Total samples (In) were loaded in the 60% sucrose layer of the gradient (fractions 8 and 9), and during centrifugation membrane-associated proteins float to the 10–50% sucrose interface (fractions 3 and 4) and soluble proteins remain in the 60% layer. nsP1 expressed in Hi5 cells floats and that expressed in E. coli is mainly soluble (arrows). The sucrose concentration along the gradient is indicated, and molecular weight markers are in the second lane from the left. The fluorescence emission of soluble YFP, used for following insect cell expression with the MultiBac system16, was monitored as an internal control (graph below). As expected, the YFP migrates to the soluble fraction. d, Screening to identify a detergent suitable for solubilization of CHIKV nsP1 from Hi5 membranes. Isolated membranes were incubated with a range of detergents at 1% concentration and subjected to ultracentrifugation. The resulting soluble fractions were analysed by SDS–PAGE (top-right panel) and for nsP1 guanyltransferase activity in the presence of SAH and m7GMP by western blotting with the m3G and m7G antibody (bottom-right panel). Lanes are as follows: 1, crude membrane fraction; 2–11, soluble fractions with no detergent (2), anzergent 3-10 (3), cymal 5 (orange) (4), DG (5), β-OG (purple) (6), fos-choline 12 (blue) (7), DM (green) (8), SDS (9), DMNG (yellow) (10), DDM (grey) (11). e, Size-exclusion chromatograms of a subset of promising detergents that were used in scaled-up purifications (same colour scheme used as in d). f, Size-exclusion chromatogram of CHIKV nsP1 purified from insect cells with SDS–PAGE of elution fractions with molecular weight markers shown on the left. Fractions corresponding to the oligomeric peak (1) and monomeric peak (2) are indicated, as well as the fractions used for subsequent biochemical experiments with the oligomeric (O) and the monomeric (M) nsP1. g, Acyl capture experiment to assess palmitoylation of oligomeric and monomeric nsP1. After blocking of free thiols and cleavage of acyl thioester bonds, palmitoylated samples are retained by a resin that binds free thiols (cBF, cleaved bound fraction) and non-palmitoylated samples are washed away (cUF, cleaved unbound fraction). Paired negative controls in which thiols were not cleaved are also shown (pBF, protected bound fraction; pUF, protected unbound fraction). The input for the resin is labelled IF (input fraction). Only a very small proportion of oligomeric nsP1 is palmitoylated. h, Guanyltransferase activity tests of the oligomeric (O) and monomeric (M) species that were pooled after size exclusion. The top panels show a western blot with a nsP1 antibody. The lower panel shows the protein activity as monitored in b. Only the oligomers produce the intermediate of the guanyltransferase reaction with both GTP and m7GTP as substrate. Molecular weight markers are shown on the left. i, Negative-stain electron micrograph of the central fraction from the oligomeric peak shown in f. The micrograph shows top views of the ring (1), side views of single rings (2) and side views of double rings (3), all indicated by arrows. The magnification is indicated by the 100-nm-long bar below. All original gels can be found in Supplementary Fig. 1. All results from activity assays (b, d, h), acyl-capture experiments (g) and negative-stain electron-microscopy experiments (i) were successfully reproduced at least three times. Gels for sucrose flotation assays (c) are representative of two repeats.

Extended Data Fig. 2 Cryo-EM reconstruction of nsP1 single and double dodecameric rings.

a, Cryo-EM micrograph of nsP1 oligomers acquired with a Titan Krios microscope (ESRF-CM01), representative of 4,948 micrographs collected. Top views and side views of the single and double rings are visible. The power spectrum of the micrograph is shown in the offset. b, Two-dimensional class averages of nsP1. Classes correspond to single or double rings in a range of orientations. c, The cryo-EM image processing workflow. The 180,981 particles selected following 2D classification and image pruning were subjected to 3D classification using an ab initio model low-pass-filtered to 60 Å. Classification with different numbers of classes consistently yielded two main classes, corresponding to single rings (pink) and double rings (gold). For the single rings, a second round of 3D classification improved homogeneity and resolution of the final reconstruction. Final 3D reconstructions were performed with masking and imposing C12 and D12 symmetry for single and double rings, respectively, following a single round of particle polishing and per-particle CTF refinement. d, Angular distribution plot of all particles included in the final C12 symmetry reconstruction of the single rings (pink) and D12 symmetry reconstruction of the double rings (gold). The length and colour of the cylinders correspond to the number of particles at a given Euler angle. The plots demonstrate a higher distribution of particles in side orientations in the case of the double rings, and top orientations for single rings, but sufficient coverage of the symmetry space to avoid this being detrimental to the reconstruction. e, Final 3D reconstructions coloured according to local resolution for single rings (left) and double (right) rings. The resolution range is relatively narrow for both reconstructions (scale indicated), where resolution is highest in the core of the complexes around the pore aperture. Resolution is lower in the top and bottom of the rings, corresponding to the region interacting with the FC12 micelles, and the C-terminal helix (αk) respectively. f, Fourier shell correlation curves for the single ring (left) and double ring (right) reconstructions. Curves for the unmasked (gold), masked (pink), phase-randomized (red) and cryo-EM density maps corrected for mask convolution effects (blue) are indicated, and the inset shows the final atomic model and mask used for the reconstructions. The resolutions estimated according to the 0.143 threshold are indicated, corresponding to 2.6 Å for single rings and 2.9 Å for double rings.

Extended Data Fig. 3 Details of the density maps of the C12 nsP1 volume reconstruction.

The panels were created in PyMOL with residues represented as sticks and density as blue mesh at the indicated σ. The carbon backbone is coloured according to domains, following Figs. 1, 2. a–h, Electron densities. a, The Zn-binding site. Dashed black lines indicate the tetragonal coordination of the Zn atom. The amino acid side chains coordinating the Zn atom are shown as sticks. b, The weak electron density for the C-terminal α-helix αk. c, The long helix αj. d, Helix αZ, e, The α-turn of the pore-wall helices αh–αi. f, The β-sheet β7. g,The GTP-binding site. h, The SAM-binding site. i–k, Three decreasing contour levels of the electron density observed around the cysteine triad at the edge of the membrane-binding spikes.

Extended Data Fig. 4 nsP1–detergent interactions.

Micelle arrangement and membrane-binding regions of the double-ring structure. a, Lateral section of the double-ring map represented by a green mesh. A section of the map including one detergent micelle is highlighted in blue (left). The central panel shows two symmetry-related nsP1 dimers (n and n + 1) interacting with the FC12 micelle, as a cartoon model coloured in pink and orange. Right, the same as in central panel without the blue mesh, showing the interaction between the two neighbouring nsP1s from each ring and the three cysteines at the tip of the spike. The closest residue between the two rings is C417 from each dimer, with an approximate distance of 4.4 Å between their sulfur atoms. b, Same representation as in a of the entire map, with the view rotated by 90° about the indicated two-fold axis.

Extended Data Fig. 5 Superposition of the nsP1 complex in the FHV tomographic reconstruction of spherule necks.

a, Cytoplasmic view of the nsP1 complex (coloured by protomer) superposed into the tomography map reconstructed from FHV growing spherules (light grey surface)4. The volume is shown at two contour levels, 1.2 (left) and 0.9 (right), using ChimeraX. The superposition shows the 12-fold symmetry of both structures and that they are of a similar shape and size. The FHV central crown appears to be slightly larger. The panel on the left at lower contour level allows for visualization of the membrane around the crown. b, Lateral view of the superposition. The nsP1 fits into the lower part of the FHV crown in contact with the membrane. In the right panel, it is possible to appreciate the membrane bending carried out by the complex together with 12 external detached volumes of unidentified origin. From this view, an extension of the crown is visible at the top of the ring. This region probably corresponds to the polymerase domain of FHV protein A, which is in the same polypeptide chain as the capping enzyme that is homologous to alphavirus nsP1. c, Bottom view of the superposition. In this orientation, the inner shape of the viral spherule can be seen. The FHV volume was provided by P. Ahlquist, who has since published the same structure at higher resolution39.

Extended Data Fig. 6 Structural comparison of nsP1 with E. cuniculi and dengue virus MTases.

a, Left, topology diagram of CHIKV nsP1. The MTase conserved motifs are coloured in yellow. The CHIKV-specific features of the capping domain are coloured in orange. The RAMBO domain is coloured in teal. The MBO regions are coloured in red and labelled. The Zn-binding site and the SAM-binding regions are also indicated. Middle, right, two views of nsP1 as a cartoon model rotated by 180°. The same colour code is used as in the diagram on the left, and secondary structural elements are labelled. The Zn atom is shown as a grey sphere. b, Same representation as in a, for the MTase of E. cuniculi. (PDB code 1RI1), in which the SAH and GTP ligands are labelled. c, Same representation as in b, for the MTase of dengue virus (PDB code 1L9K). In dengue virus, the MTase and RNA-dependent RNA polymerase domain are in the same protein (NS5). The connection with the polymerase domain is indicated at the N terminus. This MTase has only one central β-sheet instead of the two β-sheet folding of E. cuniculi and nsP1. d, ConSurf representation of nsP1 sequence conservation within the alphavirus family according to the alignment of Extended Data Fig. 7. Two views of nsP1 are represented as cartoons and coloured according to the conservation score calculated by ConSurf. The lower bar indicates the conservation degree of each residue from non-conserved (blue) to fully conserved residues (purple). The GTP and SAH putative ligands are shown as red and green transparent spheres. The side chains of residues mutated in previous studies are shown as green sticks (Supplementary Information). The positions of residues suspected to bind to nsP4, T351 and N375 (missing in the structure), are shown as yellow spheres. The αg and β11 secondary structures, previously43 thought to be an amphipathic membrane-associated helix, are labelled.

Extended Data Fig. 7 Sequence alignment of alphavirus nsP1.

The sequence alignment of nsP1 sequences of CHIKV (UniProt: Q5XXP4), Semliki Forest virus (SFV) (UniProt: P08411), Venezuelan equine encephalitis virus (VEEV) (UniProt: P27282), sindbis virus (SINV) (UniProt: P03317), aura virus (UniProt: Q86924) and salmonid sleeping disease virus (SDV) (UniProt: Q8QL53). The secondary structural elements revealed by the nsP1 structure are indicated at the top of the alignment. Below the sequences, the residues forming the GTP-binding site are highlighted by blue spots, those forming the SAM-binding site by red spots and those involved in Zn binding by red stars. Underneath, the interface line indicates the residues involved in interactions with the n + 1 nsP1 protomer in pink and with the n − 1 in brown. The blue lines indicate those residues making contacts to n − 1 and n + 1. At the bottom, rectangles indicate the domain boundaries of the nsP1 fold, coloured as described in Fig. 1e. Three motifs are conserved in the amino acid sequences of alphaviruses and similar viruses such hepatitis E virus, rubella virus and more26,27: (i) the catalytic histidine (H37) responsible for the cap transfer; (ii) the DXG motif (in which X stands for any residue) (63DIG65 in CHIKV); and (iii) and the DXXR motif (89DPER92 in CHIKV)—the last two define the SAM-binding site. This suggests that all alphavirus nsP1 have similar capping enzymes. Driven by the structural alignment of nsP1 and E. cuniculi MTase, we can more generally assign the DXG motif to the SAM-dependent MTases motif D/EX(G)XGXGXDL14,18,19, which—in alphavirus—corresponds to 63DIGSAPXRR71.

Extended Data Fig. 8 Oligomerization interface of the nsP1 complex.

Cartoon representation of one protomer (n, coloured by the domains as outlined in Fig. 1f, Extended Data Fig. 6a) flanked by two protomers (n + 1 and n − 1, in grey) from the exterior view of the complex. The interaction interfaces are depicted as transparent surfaces. Contacts with the n − 1 protomer are maintained mainly with the capping domain, whereas the contacts with n + 1 are made mainly with the RAMBO domain. The side chains of the n protomer residues involved in n/n − 1 and n/n + 1 hydrogen bonds are represented as sticks. The Zn atom is shown as a grey sphere and labelled. The secondary structural elements involved in interactions, the active site and the MBO loops of the n protomer are labelled and indicated by arrows. MBO loop 2 folds under the n + 1 protomer and MBO loop 1 over the n − 1 protomer. The interface with nsP1n − 1 involves the MTase main β-sheet (including β6′, β6 and β7), the helix αZ and the N-terminal extension (mainly the loop between αa and β2′). The RAMBO domain is also engaged in the interaction by the strands β12, β15, β16 and the beginning of β13, the C terminus of the long helix αj and one side of αh at the pore-forming α-bundle. The interface with nsP1n − 1 is built by the capping domain small β-sheet (β9 to β11), αB and the C-terminal αk in the top of the ring and notably by the MBO loop 1, which builds up most of the interface in the membrane-binding region. A small interface between nsP1n − 1 and nsP1n + 1, which is sealing the ring, is formed by residues represented in blue sticks and labelled.

Extended Data Fig. 9 Structure of the nsP1 capping active site and comparison with E. cuniculi MTase.

a, Structural alignment of CHIKV capping domain and E. cuniculi MTase (PDB code 1RI1) showing the GTP-binding site. The structures are represented in cartoon model and coloured in yellow for CHIKV (left) and green for E. cuniculi (right). The superposed GTP visible moieties corresponding to the E. cuniculi MTase structures in complex with SAH and m7GpppG (PDB code 1RI1) and only m7GpppG, (PDB code 1RI2) are shown in both models as thin sticks with carbon atoms coloured in blue and red, respectively, and are labelled. The residues involved in GTP binding in E. cuniculi, and the residues positioned to maintain contacts with the superposed GTP in CHIKV are shown as sticks and labelled. The secondary structures are also labelled. b, Same representation as in a, showing the SAM-binding site. The SAH molecule of the E. cuniculi structure is superposed on CHIKV. c, Same representation as in a, b, focusing on the catalytic histidine H37, showing in dashed lines the distances with the GTP superposed phosphate α oxygen where covalent binding would occur for the GTase reaction. d, Surface electrostatics of the GTP-binding site by APBS are shown from −5 kT/e in red to 5 kT/e in blue as indicated in the bar below. The superposed GTP from PDB codes 1RI1 and 1RI2 are shown as in a. The direction towards the pore of the ring is indicated by a black arrow.

Extended Data Fig. 10 Model of replication-complex assembly, replication and spherule formation.

a, Our results demonstrate that the nsP1 active complex can assemble in the absence of other replication complex subunits. The complex could be formed by uncleaved nsP precursors as nsP12340; in this case, nsP123 would stay non-cleaved until step d. b, The nsP2, 3 and 4 subunits of the replication complex would be recruited on the spherule pore complex together with other cellular factors for the formation of active replication complexes. The negatively charged cone walls and the exposed loop between helix αi and helix αh could serve as anchoring points. c, The deep insertion of the amphipathic spikes into the membrane would have a wedging effect on the membrane, and the membrane phospholipid heads would interact with the concave positively charged patches around the waist and below the complex. Altogether, these interactions are prone to induce the first negative curvature of the membrane. Coupled with the synthesis of the negative RNA intermediate by nsP123+4, the spherule could grow below the pore, while maintaining a tight attachment to the complex. The direction of RNA synthesis into the pore is indicated by arrows, as the genomic RNA transcripts released from virions are located in the cytoplasm. d, After complete synthesis of the negative strand and growth of the spherule, transcription of capped viral RNAs may occur. The double-stranded RNA templates for viral RNA transcription must be read by the polymerase inside the spherule. A conformational change of the replication complex upon cleavage of nsP1 from nsP2 (for example, the internalization of the nsP4 proteins into the spherule) should allow the change of RNA product direction (indicated by arrows). Indeed, thermal-sensitive mutants at both sides of the pore α-bundle suggests that nsP4 interacts with both sides of the pore (see ‘Atomic structure of CHIKV nsP1 protein’). The forced traffic of the newly synthetized viral RNAs through the pore and capping active sites would ensure their capping before reaching the cytoplasm. e, The stoichiometry of the complex and size of the pore suggests that up to 12 capped viral RNA molecules could be capped simultaneously. f, After the 5′ of the viral RNA is capped the rest of the molecule will be expelled close to the MTase-domain GTP-binding site. This could facilitate the incorporation of internal methylations, a phenomenon that has previously been described for alphavirus viral RNA41,42, that confers stability to the RNA and prevents its recognition by the innate immune system.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
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Supplementary information

Supplementary Information

This file contains Supplementary Text, Supplementary Methods, Data Availability Statement and Supplementary References.

Reporting Summary

Supplementary Table 1

List of atom-atom interactions across CHIKV nsP1complex protein-protein interface. List generated by the PDBsum server.

Supplementary Figure 1

Raw data gels corresponding to Extended Data Fig. 1.

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Jones, R., Bragagnolo, G., Arranz, R. et al. Capping pores of alphavirus nsP1 gate membranous viral replication factories. Nature 589, 615–619 (2021). https://doi.org/10.1038/s41586-020-3036-8

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