Cryo-EM structures of herpes simplex virus type 1 portal vertex and packaged genome

Abstract

Herpesviruses are enveloped viruses that are prevalent in the human population and are responsible for diverse pathologies, including cold sores, birth defects and cancers. They are characterized by a highly pressurized pseudo-icosahedral capsid—with triangulation number (T) equal to 16—encapsidating a tightly packed double-stranded DNA (dsDNA) genome1,2,3. A key process in the herpesvirus life cycle involves the recruitment of an ATP-driven terminase to a unique portal vertex to recognize, package and cleave concatemeric dsDNA, ultimately giving rise to a pressurized, genome-containing virion4,5. Although this process has been studied in dsDNA phages6,7,8,9—with which herpesviruses bear some similarities—a lack of high-resolution in situ structures of genome-packaging machinery has prevented the elucidation of how these multi-step reactions, which require close coordination among multiple actors, occur in an integrated environment. To better define the structural basis of genome packaging and organization in herpes simplex virus type 1 (HSV-1), we developed sequential localized classification and symmetry relaxation methods to process cryo-electron microscopy (cryo-EM) images of HSV-1 virions, which enabled us to decouple and reconstruct hetero-symmetric and asymmetric elements within the pseudo-icosahedral capsid. Here we present in situ structures of the unique portal vertex, genomic termini and ordered dsDNA coils in the capsid spooled around a disordered dsDNA core. We identify tentacle-like helices and a globular complex capping the portal vertex that is not observed in phages, indicative of herpesvirus-specific adaptations in the DNA-packaging process. Finally, our atomic models of portal vertex elements reveal how the fivefold-related capsid accommodates symmetry mismatch imparted by the dodecameric portal—a longstanding mystery in icosahedral viruses—and inform possible DNA-sequence recognition and headful-sensing pathways involved in genome packaging. This work showcases how to resolve symmetry-mismatched elements in a large eukaryotic virus and provides insights into the mechanisms of herpesvirus genome packaging.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Structures of the portal vertex and dsDNA genome.
Fig. 2: pUL6 portal structure and interactions with dsDNA.
Fig. 3: Tentacle helices and the portal cap.
Fig. 4: Capsid accommodations at the portal vertex.

Data availability

The five cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-9860 (C5 portal vertex reconstruction), EMD-9861 (C1 portal vertex reconstruction), EMD-9862 (C12 portal reconstruction), EMD-9863 (C1 terminal DNA and portal vertex reconstruction) and EMD-9864 (C1 virion reconstruction). The atomic models for pUL6 and periportal capsid or CATC proteins have been deposited in the Protein Data Bank under accession numbers 6OD7 and 6ODM, respectively.

References

  1. 1.

    Bauer, D. W., Huffman, J. B., Homa, F. L. & Evilevitch, A. Herpes virus genome, the pressure is on. J. Am. Chem. Soc. 135, 11216–11221 (2013).

  2. 2.

    Zhou, Z. H. et al. Seeing the herpesvirus capsid at 8.5 Å. Science 288, 877–880 (2000).

  3. 3.

    Yu, X. K., Jih, J., Jiang, J. S. & Zhou, Z. H. Atomic structure of the human cytomegalovirus capsid with its securing tegument layer of pp150. Science 356, eaam6892 (2017).

  4. 4.

    Heming, J. D., Huffman, J. B., Jones, L. M. & Homa, F. L. Isolation and characterization of the herpes simplex virus 1 terminase complex. J. Virol. 88, 225–236 (2014).

  5. 5.

    Neuber, S. et al. Mutual Interplay between the human cytomegalovirus terminase subunits pUL51, pUL56, and pUL89 promotes terminase complex formation. J. Virol. 91, e02384-16 (2017).

  6. 6.

    Rao, V. B. & Feiss, M. Mechanisms of DNA Packaging by large double-stranded DNA viruses. Annu. Rev. Virol. 2, 351–378 (2015).

  7. 7.

    Tang, J. et al. DNA poised for release in bacteriophage ϕ29. Structure 16, 935–943 (2008).

  8. 8.

    Mao, H. et al. Structural and molecular basis for coordination in a viral DNA packaging motor. Cell Reports 14, 2017–2029 (2016).

  9. 9.

    Harjes, E. et al. Structure of the RNA claw of the DNA packaging motor of bacteriophage Φ29. Nucleic Acids Res. 40, 9953–9963 (2012).

  10. 10.

    Jiang, W. et al. Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus. Nature 439, 612–616 (2006).

  11. 11.

    Booy, F. P. et al. Liquid-crystalline, phage-like packing of encapsidated DNA in herpes simplex virus. Cell 64, 1007–1015 (1991).

  12. 12.

    Newcomb, W. W., Cockrell, S. K., Homa, F. L. & Brown, J. C. Polarized DNA ejection from the herpesvirus capsid. J. Mol. Biol. 392, 885–894 (2009).

  13. 13.

    Ray, K., Ma, J., Oram, M., Lakowicz, J. R. & Black, L. W. Single-molecule and FRET fluorescence correlation spectroscopy analyses of phage DNA packaging: colocalization of packaged phage T4 DNA ends within the capsid. J. Mol. Biol. 395, 1102–1113 (2010).

  14. 14.

    Mocarski, E. S. & Roizman, B. Structure and role of the herpes simplex virus DNA termini in inversion, circularization and generation of virion DNA. Cell 31, 89–97 (1982).

  15. 15.

    Tong, L. & Stow, N. D. Analysis of herpes simplex virus type 1 DNA packaging signal mutations in the context of the viral genome. J. Virol. 84, 321–329 (2010).

  16. 16.

    Umene, K. Cleavage in and around the DR1 element of the A sequence of herpes simplex virus type 1 relevant to the excision of DNA fragments with length corresponding to one and two units of the A sequence. J. Virol. 75, 5870–5878 (2001).

  17. 17.

    McVoy, M. A., Nixon, D. E., Adler, S. P. & Mocarski, E. S. Sequences within the herpesvirus-conserved pac1 and pac2 motifs are required for cleavage and packaging of the murine cytomegalovirus genome. J. Virol. 72, 48–56 (1998).

  18. 18.

    Wang, J. B., Nixon, D. E. & McVoy, M. A. Definition of the minimal cis-acting sequences necessary for genome maturation of the herpesvirus murine cytomegalovirus. J. Virol. 82, 2394–2404 (2008).

  19. 19.

    Kumar, R. & Grubmüller, H. Elastic properties and heterogeneous stiffness of the phi29 motor connector channel. Biophys. J. 106, 1338–1348 (2014).

  20. 20.

    Ogasawara, M., Suzutani, T., Yoshida, I. & Azuma, M. Role of the UL25 gene product in packaging DNA into the herpes simplex virus capsid: location of UL25 product in the capsid and demonstration that it binds DNA. J. Virol. 75, 1427–1436 (2001).

  21. 21.

    Huffman, J. B. et al. The C terminus of the herpes simplex virus UL25 protein is required for release of viral genomes from capsids bound to nuclear pores. J. Virol. 91, e00641-17 (2017).

  22. 22.

    Pasdeloup, D., Blondel, D., Isidro, A. L. & Rixon, F. J. Herpesvirus capsid association with the nuclear pore complex and viral DNA release involve the nucleoporin CAN/Nup214 and the capsid protein pUL25. J. Virol. 83, 6610–6623 (2009).

  23. 23.

    Dai, X. & Zhou, Z. H. Structure of the herpes simplex virus 1 capsid with associated tegument protein complexes. Science 360, eaao7298 (2018).

  24. 24.

    Liu, Y. T. et al. A pUL25 dimer interfaces the pseudorabies virus capsid and tegument. J. Gen. Virol. 98, 2837–2849 (2017).

  25. 25.

    Wang, J. et al. Structure of the herpes simplex virus type 2 C-capsid with capsid-vertex-specific component. Nat. Commun. 9, 3668 (2018).

  26. 26.

    Lokareddy, R. K. et al. Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation. Nat. Commun. 8, 14310 (2017).

  27. 27.

    Yang, K., Wills, E. & Baines, J. D. The putative leucine zipper of the UL6-encoded portal protein of herpes simplex virus 1 is necessary for interaction with pUL15 and pUL28 and their association with capsids. J. Virol. 83, 4557–4564 (2009).

  28. 28.

    Truebestein, L. & Leonard, T. A. Coiled-coils: the long and short of it. BioEssays 38, 903–916 (2016).

  29. 29.

    Rao, V. B. & Black, L. W. DNA Packaging in Bacteriophage T4 in Viral Genome Packaging Machines (Plenum, 2005).

  30. 30.

    Berndsen, Z. T., Keller, N. & Smith, D. E. Continuous allosteric regulation of a viral packaging motor by a sensor that detects the density and conformation of packaged DNA. Biophys. J. 108, 315–324 (2015).

  31. 31.

    Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005).

  32. 32.

    Li, X. M. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).

  33. 33.

    Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).

  34. 34.

    Ludtke, S. J., Baldwin, P. R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999).

  35. 35.

    Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

  36. 36.

    Scheres, S. H. W. A Bayesian view on cryo-EM structure determination. J. Mol. Biol. 415, 406–418 (2012).

  37. 37.

    Ilca, S. L. et al. Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes. Nat. Commun. 6, 8843 (2015).

  38. 38.

    DeRosier, D. J. Correction of high-resolution data for curvature of the Ewald sphere. Ultramicroscopy 81, 83–98 (2000).

  39. 39.

    Zhang, X. & Zhou, Z. H. Limiting factors in atomic resolution cryo electron microscopy: no simple tricks. J. Struct. Biol. 175, 253–263 (2011).

  40. 40.

    Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

  41. 41.

    Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

  42. 42.

    Scheres, S. H. Processing of structurally heterogeneous cryo-EM data in RELION. Methods Enzymol. 579, 125–157 (2016).

  43. 43.

    Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

  44. 44.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

  45. 45.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

  46. 46.

    Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. E. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).

  47. 47.

    Trabuco, L. G., Villa, E., Mitra, K., Frank, J. & Schulten, K. Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. Structure 16, 673–683 (2008).

Download references

Acknowledgements

We thank W. Liu for assistance in molecular dynamic flexible fitting. This research has been supported in part by grants from the National Key R&D Program of China (2017YFA0505300 and 2016YFA0400900) and the US National Institutes of Health (GM071940/DE025567/DE028583/AI094386). We acknowledge the use of instruments at the Electron Imaging Center for Nanomachines supported by UCLA and by instrumentation grants from NIH (1S10RR23057 and 1U24GM116792) and NSF (DBI-1338135 and DMR-1548924). We thank the Bioinformatics Center of the University of Science and Technology of China, School of Life Science, for providing supercomputing resources for this project.

Reviewer information

Nature thanks Sarah Butcher, Venigalla Rao and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Z.H.Z. conceived the project; Z.H.Z. and G.-Q.B. supervised research; X.D. recorded the data; Y.-T.L. processed the data; J.J. built atomic models and made illustrations and videos; Z.H.Z., J.J. and Y.-T.L. analysed the results and wrote the paper. All authors edited and approved the paper.

Correspondence to Z. Hong Zhou.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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 Sequential localized classification and sub-particle reconstruction.

Flow chart illustrates the identification and resolution of symmetry-mismatched structures of the unique portal vertex.

Extended Data Fig. 2 Resolution verification.

a, b, Resolution of reconstructions determined by gold-standard FSC at the 0.143 criterion. cg, Density slices coloured by local resolution estimated from ResMap41.

Extended Data Fig. 3 pUL6 secondary structure and disorder prediction.

ac, pUL6 monomer coloured by domain for reference (a) and key (b) used to annotate a secondary structure and disorder prediction of pUL6 amino acid sequence (c) obtained from Phyre246.

Extended Data Fig. 4 Reconstruction of terminal DNA with surrounding portal.

a, b, C1 reconstruction of terminal DNA with surrounding portal colour-zoned by pUL6 domains and tentacle helices. c, Sequence of terminal DNA mapped onto our fitted terminal DNA model. d, Enlarged view of the trailing end of terminal DNA, where concatemeric cleavage occurs. e, Enlarged view of terminal the disordered leading end of DNA, which extends down through the portal aperture towards the interior of the capsid. fh, Slab views of C1 density showing interaction of terminal DNA with tentacle helices (f), portal clip (g) and the portal aperture (h).

Extended Data Fig. 5 pUL6 portal protein homologues.

ad, HSV-1 pUL6 and pUL6 homologues coloured analogously by pUL6 domain. eh, HSV-1 pUL6 portal complex and homologues coloured in rainbow (red (N terminus) to blue (C terminus)). Respective insets illustrate the conserved left-handed corkscrew of stem helices in the portal channel beneath the clip.

Extended Data Fig. 6 Comparison of periportal and peripenton capsid proteins.

ac, Comparison of periportal and peripenton P1 MCPs (a) reveal conformational differences in their dimerization domains (b, c). df, Comparison of periportal and peripenton P6 MCPs (d) reveal conformational differences in their N-lassos (e, f). gi, Comparison of periportal and peripenton Tri1s (g) reveal differences in a trunk loop where periportal Tri1 interfaces with tentacle helices (h) and a visible N-anchor helix in periportal Tri1 (i).

Extended Data Table 1 Cryo-EM parameters and statistics

Supplementary information

Reporting Summary

Video 1

Structures of the HSV-1 virion and portal vertex. Depicts the global reconstructed features of the HSV-1 virion and portal vertex. Related to Figure 1.

Video 2

pUL6 dodecameric portal. Depicts the atomic model of pUL6 protein and features of the dodecameric portal. Related to Figure 2.

Video 3

Terminal DNA in the portal vertex channel. Depicts fitted model of terminal DNA and its interactions with structures of the DNA translocation channel. Related to Figures 2 and 3.

Video 4

Capsid/CATC structures at the portal vertex. Depicts portal vertex-specific features of the capsid and CATC. Related to Figure 4.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liu, Y., Jih, J., Dai, X. et al. Cryo-EM structures of herpes simplex virus type 1 portal vertex and packaged genome. Nature 570, 257–261 (2019). https://doi.org/10.1038/s41586-019-1248-6

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.