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Structure and mutagenesis reveal essential capsid protein interactions for KSHV replication

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) causes Kaposi’s sarcoma1,2, a cancer that commonly affects patients with AIDS3 and which is endemic in sub-Saharan Africa4. The KSHV capsid is highly pressurized by its double-stranded DNA genome, as are the capsids of the eight other human herpesviruses5. Capsid assembly and genome packaging of herpesviruses are prone to interruption6,7,8,9 and can therefore be targeted for the structure-guided development of antiviral agents. However, herpesvirus capsids—comprising nearly 3,000 proteins and over 1,300 Å in diameter—present a formidable challenge to atomic structure determination10 and functional mapping of molecular interactions. Here we report a 4.2 Å resolution structure of the KSHV capsid, determined by electron-counting cryo-electron microscopy, and its atomic model, which contains 46 unique conformers of the major capsid protein (MCP), the smallest capsid protein (SCP) and the triplex proteins Tri1 and Tri2. Our structure and mutagenesis results reveal a groove in the upper domain of the MCP that contains hydrophobic residues that interact with the SCP, which in turn crosslinks with neighbouring MCPs in the same hexon to stabilize the capsid. Multiple levels of MCP–MCP interaction—including six sets of stacked hairpins lining the hexon channel, disulfide bonds across channel and buttress domains in neighbouring MCPs, and an interaction network forged by the N-lasso domain and secured by the dimerization domain—define a robust capsid that is resistant to the pressure exerted by the enclosed genome. The triplexes, each composed of two Tri2 molecules and a Tri1 molecule, anchor to the capsid floor via a Tri1 N-anchor to plug holes in the MCP network and rivet the capsid floor. These essential roles of the MCP N-lasso and Tri1 N-anchor are verified by serial-truncation mutageneses. Our proof-of-concept demonstration of the use of polypeptides that mimic the smallest capsid protein to inhibit KSHV lytic replication highlights the potential for exploiting the interaction hotspots revealed in our atomic structure to develop antiviral agents.

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Figure 1: Cryo-EM reconstruction and atomic modelling of KSHV capsid.
Figure 2: Structures of MCP and SCP.
Figure 3: Network interactions in the MCP floor and function of MCP N-lasso.
Figure 4: Structure of triplex and function of Tri1 N-anchor.

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Acknowledgements

This project was supported in part by grants from the National Institutes of Health (NIH) (DE025567, GM071940, AI094386, CA091791 and CA177322) and indirectly through a Clinical and Translational Science Institute core voucher award (UL1TR000124) from UCLA’s National Center for Advancing Translational Science. We acknowledge the use of instruments at the Electron Imaging Center for Nanomachines supported by UCLA and by instrumentation grants from the NIH (1S10OD018111, 1U24GM116792) and NSF (DBI-1338135, DMR-1548924).

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Contributions

Z.H.Z., X.D., D.G. and R.S. designed the project; Z.H.Z., R.S. and T.-T.W. supervised research; D.G. and X.D. prepared the samples; X.D. acquired cryo-EM data and determined the structure; X.D., H.L. and J.J. built atomic models; D.G. performed functional studies; X.D., D.G., Z.H.Z., R.S. and T.-T.W. interpreted the results; Z.H.Z., X.D. and D.G. wrote the paper; R.S. revised the paper; and all authors reviewed the paper.

Corresponding authors

Correspondence to Ren Sun or Z. Hong Zhou.

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Extended data figures and tables

Extended Data Figure 1 Cryo-EM imaging of KSHV particles and data processing strategy to minimize interference of the tegument layer.

a, A cryo-EM micrograph. Images recorded with a Gatan K2 Summit direct electron detector show largely intact KSHV virions in our sample preparation. Naked capsids were only occasionally observed. The defocus value of this micrograph was −1.3 μm. b, Plot of phase-residue value distribution of particles after initial determination of orientation and centre parameters. Particles in the second peak with high phase-residue values are regarded as bad particles, for which the parameters were not correctly determined owing to the interference of the thick, pleomorphic tegument layer or the low quality of the particle. These particles were discarded and not included for following refinement to avoid their contaminating the reconstruction. c, Plot of phase-residue value distribution of our previous film dataset (X.D. et al., unpublished data) showing even more bad particles than those recorded in b, probably owing to decreased contrast of the film dataset compared to that of our current K2 dataset (b). d, Plot of phase-residue value distribution of particles after the final round of parameter refinement. There is only one peak representing the good particles, because the bad particles were discarded at the beginning of the refinement procedure. Moreover, only the top 85% of these good particles were selected for reconstruction. White lines denote the phase-residue value cutoff to select particles for refinement (b, c) or reconstruction (d).

Extended Data Figure 2 Resolution assessment of the cryo-EM reconstruction and model refinement statistics.

a, Gold-standard FSC curve of the cryo-EM reconstruction. The average resolution of the final density map is 4.2 Å as determined by the FSC = 0.143 criterion44. b, c, Local resolution assessment by ResMap52. A 6403-voxel sub-volume of the final density map was subjected to ResMap processing. Four slices of the input volume (b) and the local resolution heat map (c) are shown. Note that many regions of the density map have better resolution than the FSC-measured average resolution of 4.2 Å. The penton tower region has the lowest resolution because of its flexibility. d, Model refinement statistics reported by the Phenix real space refinement program47.

Extended Data Figure 3 Density maps and atomic models of MCP and SCP.

Insets correspond to zoomed-in views of boxed regions and illustrate residue features in the density map.

Extended Data Figure 4 Density maps and atomic models of Tri1, Tri2A and Tri2B.

Insets correspond to zoomed-in views of boxed regions and illustrate residue features in the density map.

Extended Data Figure 5 Structure-guided point mutations of MCPud to identify essential amino acid interactions in MCP–SCP binding.

a, Specific amino acid interactions between SCP and MCPud. The SCP model is coloured according to the hydrophobicity of its residues. The MCPud is coloured pink, except for one helix (amino acids 763–778, shown in cyan) that borders the groove for the binding of SCP stem helix. The corresponding region (amino acids 767–781) in the highly homologous HSV-1 MCPud is a loop structure, which thus forms a relatively flat surface for SCP binding. b, c, Demonstration of specific interaction between SCP and MCPud (b) or between the SCP-mimicking polypeptide 3SH–Flag and MCPud (c); 293T cells were co-transfected with expression plasmids of MCPud (amino acids 478–1033, wild type or mutants) and SCP–Flag (b) or of MCPud and 3SH–Flag (c). Two days later, cell lysates were subjected to a co-immunoprecipitation assay using mouse anti-Myc antibody, and further analysed by western blotting with rabbit anti-Flag and anti-Myc antibodies. As expected, expressed wild-type SCP (b) and 3SH–Flag (c) both bound to expressed wild-type MCPud. Conversely, four out of the seven MCPud point mutations that substitute a hydrophobic residue with a hydrophilic residue disrupted these interactions for both wild-type SCP (b) and 3SH–Flag (c). These results suggest that SCP-mimicking polypeptides interact with MCPud in a similar way to wild-type SCP. Experiments were repeated independently twice with similar results.

Extended Data Figure 6 Structural differences between hexon and penton.

a, Superimposed models of a hexon MCP and a penton MCP, which shows the hinged tilting of the floor region of a penton MCP towards the centre of the capsid. bd, Comparison of channel constrictions between a bacteriophage HK97 hexon (b), a KSHV hexon (c) and a KSHV penton (d). The hexon channel in HK97 is tightly constricted by a loop in the A-domain (Fig. 2k) of the Johnson fold (b, top). In the crystal structure of HK97, this channel is completely blocked by a sulfate ion53 (b, bottom). The hexon channel in KSHV is not constricted by the HK97-like Johnson fold. The diameter of the channel at this position is 25 Å (c, middle). Note the large gap between adjacent Johnson folds, and also the absence of a long loop corresponding to the channel-constricting loop in HK97. Instead, the KSHV hexon channel is most constricted at the channel domain (c, top). Side view of the hexon (c, bottom) shows that the helix-hairpin domain insertion (blue) seals a hole at the root of the capsomer tower. The penton channel in KSHV is constricted by the Johnson-fold domain (d, middle). The diameter of the penton channel at this position is 5 Å (d, bottom). Owing to the hinged tilting of the floor region of penton MCP towards the centre of the capsid (a), adjacent Johnson-fold domains move closer to one another and constrict the penton channel (d, top).

Extended Data Figure 7 The MCP network in KSHV capsid.

a, The network forged by MCP N-lassoes in the KSHV capsid floor, illustrated with atomic models. A short continuous segment of MCP N-terminal region (amino acids 1–186) including the N-lasso, N-arm, E-loop and spine helix is shown in each MCP model. b, A schematic representation of part of the network. An analogy can be drawn between the ‘dancer’ in the schematic representation and the MCP atomic model as shown in the inset.

Extended Data Figure 8 Truncation mutagenesis of the MCP N-lasso or Tri1 N-anchor does not notably affect KSHV DNA replication or gene expression.

a, b, Viral genome copy number in cells replicating the wild type (WT), MCP-truncated (a) or Tri1-truncated (b) KSHV. Design of MCP truncations or Tri1 truncations is shown in Figs 3i and 4j, respectively. KSHV lytic replication was induced in cells harbouring the wild-type or the mutated KSHV genome. Total DNA was extracted from cells, and viral genome copy number was determined by real-time PCR. Data are mean ± s.e.m. (n = 3 biologically independent samples). c, d, Viral RNA expression in cells replicating the wild-type, MCP-truncated (c) or Tri1-truncated (d) KSHV. Total RNA was extracted from cells induced for KSHV lytic replication. Viral RNA transcripts were quantified by real-time PCR with reverse transcription and presented as fold changes over RNA level of wild-type virus. Data are mean ± s.e.m. (n = 3 or 4 biologically independent samples). e, f, Expression of viral and cellular proteins in cells replicating the wild-type, MCP-truncated (e) or Tri1-truncated (f) KSHV. Correct sizes of truncated Tri1 were verified by western blotting with an anti-Tri1 antibody as shown in f. Verification of truncated MCP was not carried out owing to the lack of anti-MCP antibody. Experiments were repeated independently twice with similar results.

Extended Data Figure 9 Hydrophobic interactions in the formation of triplexes and in the anchoring of triplexes to the capsid floor.

ad, Hydrophobic interactions have a major role in the formation of triplex heterotrimers. Surface representations of Tri2A (a), Tri2B (b) and Tri1 (c) monomers or the Tri2 dimer (d) were calculated and coloured according to hydrophobicity. Red, hydrophobic; white, neutral; blue, hydrophilic. Large patches of hydrophobic residues at the interface of the Tri2A and Tri2B embracing arm domains hold the Tri2 dimer together, and contribute to interactions with the Tri1 third-wheel domain to form the heterotrimer. eg, Triplexes are anchored to the capsid floor by the tripod-shaped Tri1 N-anchor (e, f) via hydrophobic interactions (g).

Extended Data Figure 10 Structural polymorphism in the Tri1 N-anchor.

a, b, Distribution of triplexes in the MCP network viewed from outside (a) or inside (b) the capsid. c, d, Zoomed-in views of triplex Ta (c) or Tb (d) from inside the capsid. e, Superimposed models of triplexes Ta and Tb reveal structural differences in their Tri1 N-anchor domains. f, The refolded Tri1 N-anchor in triplex Ta contributes to penton stabilization. The refolded helix in Ta Tri1 forms a hydrophobic cleft with the spine helix of a penton MCP, in which the refolded dimerization domain of an adjacent penton MCP (magenta) binds with a series of hydrophobic residues.

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

Supplementary information

Life Sciences Reporting Summary (PDF 72 kb)

Overall structure and high resolution features of the KSHV capsid reconstruction

This video shows the overall structure and high resolution features of the KSHV capsid reconstruction. (MP4 29651 kb)

Structure of a hexon MCP

The density map of a hexon MCP was segmented out from the 3D reconstruction, displayed as semitransparent grey surface, and superimposed with its atomic model (ribbon). (MP4 10697 kb)

Structure difference between hexon MCP and penton MCP

This video shows the structural difference between hexon MCP and penton MCP. (MP4 1517 kb)

Densities around a hexon MCP N-lasso region

Densities around hexon MCP E1 N-lasso (cyan), which lashes the N-arm of MCP C4 (gold) and E-loop of MCP C5 (magenta), were segmented out and superimposed with the corresponding atomic models. (MP4 3284 kb)

MCP network interactions in the capsid floor

MCP network interactions in the capsid floor. (MP4 28201 kb)

Structure of a triplex

The density map of triplex Tc was segmented out from the 3D reconstruction, displayed as semitransparent grey surface, and superimposed with its atomic model (ribbon). (MP4 8365 kb)

Structure difference between Tri2A and Tri2B

This video shows the structural difference between Tri2A and Tri2B. (MP4 10280 kb)

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Dai, X., Gong, D., Lim, H. et al. Structure and mutagenesis reveal essential capsid protein interactions for KSHV replication. Nature 553, 521–525 (2018). https://doi.org/10.1038/nature25438

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