Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Molecular plasticity of herpesvirus nuclear egress analysed in situ


The viral nuclear egress complex (NEC) allows herpesvirus capsids to escape from the nucleus without compromising the nuclear envelope integrity. The NEC lattice assembles on the inner nuclear membrane and mediates the budding of nascent nucleocapsids into the perinuclear space and their subsequent release into the cytosol. Its essential role makes it a potent antiviral target, necessitating structural information in the context of a cellular infection. Here we determined structures of NEC–capsid interfaces in situ using electron cryo-tomography, showing a substantial structural heterogeneity. In addition, while the capsid is associated with budding initiation, it is not required for curvature formation. By determining the NEC structure in several conformations, we show that curvature arises from an asymmetric assembly of disordered and hexagonally ordered lattice domains independent of pUL25 or other viral capsid vertex components. Our results advance our understanding of the mechanism of nuclear egress in the context of a living cell.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Visualizing stages of PrV nuclear egress.
Fig. 2: PrV NEC forms a locally ordered lattice.
Fig. 3: Initial stages of primary envelopment.
Fig. 4: Subvolume averaging of PrV capsids in different subcellular locations reveals the compartment-dependent assembly of penton vertex components.
Fig. 5: Subvolume averaging of the PrV NEC.
Fig. 6: A comparison of different PrV NEC lattice structures, created by fitting PDB 4Z3U chains C/D into various structures.

Similar content being viewed by others

Data availability

Maps for the following structures have been deposited in the Electron Microscopy Data Bank: PrV–ΔUS3 capsids under accession codes EMD-17974 (cytosolic), EMD-17975 (perinuclear) and EMD-17976 (nuclear); PrV WT capsids under EMD-18479 (cytosolic) and EMD-18480 (nuclear); HSV-1 WT capsids under EMD-18481 (cytosolic), EMD-18482 (perinuclear) and EMD-18483 (nuclear); PrV–ΔUS3 NEC under EMD-18474 (spherical form) and EMD-18473 (helical form); and HSV-1 NEC under EMD-18484 (spherical form). All other data supporting the conclusions of this study can be found within the Article, extended data and Supplementary Information. Source data are provided with this paper.

Code availability

Custom scripts used for this study are available via GitHub at


  1. Homa, F. L. et al. Structure of the pseudorabies virus capsid: comparison with herpes simplex virus type 1 and differential binding of essential minor proteins. J. Mol. Biol. 425, 3415–3428 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Panté, N. & Kann, M. Nuclear pore complex is able to transport macromolecules with diameters of about 39 nm. Mol. Biol. Cell 13, 425–434 (2002).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Mettenleiter, T. C., Müller, F., Granzow, H. & Klupp, B. G. The way out: what we know and do not know about herpesvirus nuclear egress. Cell. Microbiol. 15, 170–178 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Klupp, B. G. & Mettenleiter, T. C. The knowns and unknowns of herpesvirus nuclear egress. Annu. Rev. Virol. (2023).

    Article  PubMed  Google Scholar 

  5. Peng, L., Ryazantsev, S., Sun, R. & Zhou, Z. H. Three-dimensional visualization of gammaherpesvirus life cycle in host cells by electron tomography. Structure 18, 47–58 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Fuchs, W., Granzow, H. & Mettenleiter, T. C. A pseudorabies virus recombinant simultaneously lacking the major tegument proteins encoded by the UL46, UL47, UL48 and UL49 genes is viable in cultured cells. J. Virol. 77, 12891–12900 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zmasek, C. M., Knipe, D. M., Pellett, P. E. & Scheuermann, R. H. Classification of human Herpesviridae proteins using Domain-architecture Aware Inference of Orthologs (DAIO). Virology 529, 29–42 (2019).

    Article  CAS  PubMed  Google Scholar 

  8. Burke, B. & Stewart, C. L. The nuclear lamins: flexibility in function. Nat. Rev. Mol. Cell Biol. 14, 13–24 (2013).

    Article  CAS  PubMed  Google Scholar 

  9. Cibulka, J., Fraiberk, M. & Forstova, J. Nuclear actin and lamins in viral infections. Viruses 4, 325–347 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hagen, C. et al. Structural basis of vesicle formation at the inner nuclear membrane. Cell 163, 1692–1701 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hagen, C. et al. Correlative VIS-fluorescence and soft X-ray cryo-microscopy and tomography of adherent cells. J. Struct. Biol. 177, 193–201 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Klupp, B. G. et al. Vesicle formation from the nuclear membrane is induced by coexpression of two conserved herpesvirus proteins. Proc. Natl Acad. Sci. USA 104, 7241–7246 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Reynolds, A. E., Wills, E. G., Roller, R. J., Ryckman, B. J. & Baines, J. D. Ultrastructural localization of the herpes simplex virus type 1 UL31, UL34, and US3 proteins suggests specific roles in primary envelopment and egress of nucleocapsids. J. Virol. 76, 8939–8952 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Gao, J., Finnen, R. L., Sherry, M. R., Le Sage, V. & Banfield, B. W. Differentiating the roles of UL16, UL21, and Us3 in the nuclear egress of herpes simplex virus capsids. J. Virol. 94, e00738–20 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Yang, K., Wills, E., Lim, H. Y., Zhou, Z. H. & Baines, J. D. Association of herpes simplex virus pUL31 with capsid vertices and components of the capsid vertex-specific complex. J. Virol. 88, 3815–3825 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Leelawong, M., Guo, D. & Smith, G. A. A physical link between the pseudorabies virus capsid and the nuclear egress complex. J. Virol. 85, 11675–11684 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  18. Toropova, K., Huffman, J. B., Homa, F. L. & Conway, J. F. The herpes simplex virus 1 UL17 protein is the second constituent of the capsid vertex-specific component required for DNA packaging and retention. J. Virol. 85, 7513–7522 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Huet, A. et al. Extensive subunit contacts underpin herpesvirus capsid stability and interior-to-exterior allostery. Nat. Struct. Mol. Biol. 23, 531–539 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Fan, W. H. et al. The large tegument protein pUL36 is essential for formation of the capsid vertex-specific component at the capsid–tegument interface of herpes simplex virus 1. J. Virol. 89, 1502–1511 (2015).

    Article  PubMed  Google Scholar 

  21. Coller, K. E., Lee, J. I., Ueda, A. & Smith, G. A. The capsid and tegument of the alphaherpesviruses are linked by an interaction between the UL25 and VP1/2 proteins. J. Virol. 81, 11790–11797 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Newcomb, W. W. et al. The primary enveloped virion of herpes simplex virus 1: its role in nuclear egress. mBio 8, e00825–17 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Draganova, E. B., Zhang, J., Zhou, Z. H. & Heldwein, E. E. Structural basis for capsid recruitment and coat formation during HSV-1 nuclear egress. eLife 9, e56627 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Thorsen, M. K., Draganova, E. B. & Heldwein, E. E. The nuclear egress complex of Epstein–Barr virus buds membranes through an oligomerization-driven mechanism. PLoS Pathog. 18, e1010623 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Klupp, B. G., Granzow, H. & Mettenleiter, T. C. Nuclear envelope breakdown can substitute for primary envelopment-mediated nuclear egress of herpesviruses. J. Virol. 85, 8285–8292 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ryckman, B. J. & Roller, R. J. Herpes simplex virus type 1 primary envelopment: UL34 protein modification and the US3–UL34 catalytic relationship. J. Virol. 78, 399–412 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Benedyk, T. H. et al. pUL21 is a viral phosphatase adaptor that promotes herpes simplex virus replication and spread. PLoS Pathog. 17, e1009824 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Wild, P. et al. Herpes simplex virus 1 Us3 deletion mutant is infective despite impaired capsid translocation to the cytoplasm. Viruses 7, 52–71 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Malhas, A., Goulbourne, C. & Vaux, D. J. The nucleoplasmic reticulum: form and function. Trends Cell Biol. 21, 362–373 (2011).

    Article  CAS  PubMed  Google Scholar 

  30. Hoyt, C. C., Bouchard, R. J. & Tyler, K. L. Novel nuclear herniations induced by nuclear localization of a viral protein. J. Virol. 78, 6360–6369 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Mou, F., Wills, E. & Baines, J. D. Phosphorylation of the U(L)31 protein of herpes simplex virus 1 by the U(S)3-encoded kinase regulates localization of the nuclear envelopment complex and egress of nucleocapsids. J. Virol. 83, 5181–5191 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ye, G. J., Vaughan, K. T., Vallee, R. B. & Roizman, B. The herpes simplex virus 1 U(L)34 protein interacts with a cytoplasmic dynein intermediate chain and targets nuclear membrane. J. Virol. 74, 1355–1363 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wang, G. et al. Structures of pseudorabies virus capsids. Nat. Commun. 13, 1533 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Granzow, H., Klupp, B. G. & Mettenleiter, T. C. The pseudorabies virus US3 protein is a component of primary and of mature virions. J. Virol. 78, 1314–1323 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Quemin, E. R. J. et al. Cellular electron cryo-tomography to study virus–host interactions. Annu. Rev. Virol. 7, 239–262 (2020).

    Article  CAS  PubMed  Google Scholar 

  36. Bigalke, J. M. & Heldwein, E. E. Structural basis of membrane budding by the nuclear egress complex of herpesviruses. EMBO J. 34, 2921–2936 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Michael, K., Klupp, B. G., Mettenleiter, T. C. & Karger, A. Composition of pseudorabies virus particles lacking tegument protein US3, UL47, or UL49 or envelope glycoprotein E. J. Virol. 80, 1332–1339 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Briggs, J. A. et al. Structure and assembly of immature HIV. Proc. Natl Acad. Sci. USA 106, 11090–11095 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Schur, F. K. et al. Structure of the immature HIV-1 capsid in intact virus particles at 8.8 Å resolution. Nature 517, 505–508 (2015).

    Article  CAS  PubMed  Google Scholar 

  40. Zeev-Ben-Mordehai, T. et al. Crystal structure of the herpesvirus nuclear egress complex provides insights into inner nuclear membrane remodeling. Cell Rep. 13, 2645–2652 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Speese, S. D. et al. Nuclear envelope budding enables large ribonucleoprotein particle export during synaptic Wnt signaling. Cell 149, 832–846 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Klupp, B. G., Granzow, H. & Mettenleiter, T. C. Effect of the pseudorabies virus US3 protein on nuclear membrane localization of the UL34 protein and virus egress from the nucleus. J. Gen. Virol. 82, 2363–2371 (2001).

    Article  CAS  PubMed  Google Scholar 

  43. Schaffer, M. et al. Cryo-focused ion beam sample preparation for imaging vitreous cells by cryo-electron tomography. Bio Protoc. 5, e1575 (2015).

    Article  PubMed  Google Scholar 

  44. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  45. Hagen, W. J. H., Wan, W. & Briggs, J. A. G. Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging. J. Struct. Biol. 197, 191–198 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Zheng, S. et al. AreTomo: an integrated software package for automated marker-free, motion-corrected cryo-electron tomographic alignment and reconstruction. J. Struct. Biol. X 6, 100068 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    Article  CAS  PubMed  Google Scholar 

  48. Heumann, J. M., Hoenger, A. & Mastronarde, D. N. Clustering and variance maps for cryo-electron tomography using wedge-masked differences. J. Struct. Biol. 175, 288–299 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Heymann, J. B. Bsoft: image and molecular processing in electron microscopy. J. Struct. Biol. 133, 156–169 (2001).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  51. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).

    Article  CAS  PubMed  Google Scholar 

  52. Hunter, J. D. Matplotlib: a 2D graphics environment. Comput. Sci. Eng. 9, 90–95 (2007).

    Article  Google Scholar 

  53. Zhou, Q.-Y., Park, J. & Koltun, V. Open3D: a modern library for 3D data processing. Preprint at (2018).

  54. Cragnolini, T. et al. TEMPy2: a Python library with improved 3D electron microscopy density-fitting and validation workflows. Acta Crystallogr. D 77, 41–47 (2021).

    Article  CAS  Google Scholar 

  55. Virtanen, P. et al. SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat. Methods 17, 261–272 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Harris, C. R. et al. Array programming with NumPy. Nature 585, 357–362 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Kaplan, A. S. & Vatter, A. E. A comparison of herpes simplex and pseudorabies viruses. Virology 7, 394–407 (1959).

    Article  CAS  PubMed  Google Scholar 

  58. Bindels, D. S. et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat. Methods 14, 53–56 (2017).

    Article  CAS  PubMed  Google Scholar 

  59. Bohannon, K. P., Jun, Y., Gross, S. P. & Smith, G. A. Differential protein partitioning within the herpesvirus tegument and envelope underlies a complex and variable virion architecture. Proc. Natl Acad. Sci. USA 110, E1613–E1620 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Tinevez, J. Y. et al. TrackMate: an open and extensible platform for single-particle tracking. Methods 115, 80–90 (2017).

    Article  CAS  PubMed  Google Scholar 

  61. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  62. Bowman, B. R. et al. Structural characterization of the UL25 DNA-packaging protein from herpes simplex virus type 1. J. Virol. 80, 2309–2317 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


We gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG) under Germany’s Excellence Strategy EXC 2155 project number 390874280 and by the Wellcome Trust through a Collaborative Award (209250/Z/17/Z) as well as Hamburg-X and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—GRK2771—project number 453548970 (J.B.B. and K.G.), Wellcome Trust grants 107806/Z/15/Z and 209250/Z/17/ Z (K.G.), Wellcome Trust grants 099683/Z/12/Z and 225902/Z/22/Z (M.G.) and BMBF grants 05K18BHA and 031L0287A (K.G.). This research was funded in part by DFG INST 152/ 772-1, 774-1, 775-1, 777-1 FUGG (CSSB cryoEM facility) and a Wellcome Trust core award to The Wellcome Centre for Human Genetics (090532/Z/09/Z). V.P. was supported by a Nuffield Dept. of Medicine Prize Studentship. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.

Author information

Authors and Affiliations



C.H. and K.G. designed the study. J.B.B., K.G., M.G., C.H., J.M.H., Y.J., S.S., V.P. and D.V. designed experiments (laboratory or computational). C.H., Y.J., U.L., B.G.K., Y.M. and S.S. generated specimens. M.G., C.H., Y.J., B.G.K., U.L., Y.M. and S.S. collected data. M.G., C.H., Y.J., U.L., Y.M., V.P., S.S. and D.V. processed data. J.B.B., M.G., C.H., J.M.H., Y.J., Y.M., V.P., S.S. and D.V. analysed data. J.B.B., K.G., M.G., C.H., Y.M., V.P. and D.V. performed critical analysis of the findings. J.B.B., M.G., Y.J., Y.M., V.P., S.S. and D.V. made figures. M.G., Y.M., V.P. and D.V. wrote the paper. J.B.B., M.G., J.M.H., B.G.K., V.P., D.V. and K.G. performed a critical paper review.

Corresponding authors

Correspondence to Michael Grange or Kay Grünewald.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Microbiology thanks Jeremy Kamil, Phil Pellett and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

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

Extended data

Extended Data Fig. 1 Workflow for CryoFIB/SEM and CryoET.

a, A low magnification SEM image of PrV-ΔUS3-infected porcine epithelial cells. b, A higher magnification image of the box shown in a. c, An oblique SEM view illuminated by the FIB with yellow boxes indicating the area above and below the cell for targeting with the focused ion beam. d, SEM image of thinned cellular section (lamella) from the top and side (inset). Transmission electron microscope (TEM) image is also shown (right), with target area shown in e highlighted by a white box. e, A TEM image shown at 9500x nominal magnification. Details of the cell are visible at this magnification, allowing targeting of regions of interest for higher magnification tomographic data collection (red box). Shown are representative samples of 113 lamella from 3 biological replicates. cyt = cytosol, nuc = nucleus. f, A tomographic slice of the region indicated in e) at 35000x magnification, nominal. ONM, outer nuclear membrane; INM, inner nuclear membrane.

Extended Data Fig. 2 Quantification of nuclear egress events.

a, Size and curvature distribution of perinuclear vesicles. Shown are inner luminal diameters of individual vesicles (see Methods) with mean values represented by red bars. *The mean value and standard deviation of perinuclear vesicles obtained by expression pUL31/pUL3410 are shown for comparison. Vesicles (and the cargo within) were counted as perinuclear where envelopment was judged to be more than 50% completed (for example Fig. 1c would be considered perinuclear). This is because a significant proportion of vesicles (~150 nm diameter) were only partially contained within the 150–250 nm thick tomograms (that is cut off during FIB milling). b, Quantification of capsid types observed during nuclear egress in porcine epithelial (PrV) and African Green monkey cells (HSV-1). c, Quantification of perinuclear vesicles. Classes were assigned to vesicles based on cargo type. In contrast to vesicle diameter measurements shown in a, vesicles containing procapsids were included in the capsid class, whereas scaffolds were counted as non-capsid cargo (note that there were 6 perinuclear procapsids). Procapsids contain capsid proteins some of which could be involved in the initiation of envelopment and therefore fit better in the ‘capsid’ class. At the same time, all perinuclear procapsids were only partially assembled and consequently the perinuclear vesicles were smaller. d, Diagrams indicating capsid classification. A-capsids lack both nucleic acid and scaffold, B-capsids contain a scaffold, and C-capsids are capsids after completion of DNA packaging. Procapsids (not shown) are C-capsid precursors.

Source data

Extended Data Fig. 3 Putative flat NEC double-layer lattice.

a, b, c Orthogonal section through average volume of lattice shown in Fig. 1f. There is no density for the membrane bilayers due to the majority of particles having their six-fold symmetry aligned with the tomogram Z axis (and the missing wedge). A total of 1944 C6 symmetrised particles were included in the average. d, Tangential sections through the volume at 1.4 nm intervals. e, A slice through the raw tomogram indicating the position of the inner nuclear membranes relative to the lattice. The membrane is not visible around the majority of the lattice layer due to its orientation to the tomogram missing wedge. The approximate position of the outer nuclear membrane was inferred from the exclusion zone of cytosolic components (ribosomes, intermediate filaments, microtubules). f, Two NEC hexamers segmented from the spherical lattice (Fig. 5, here shown in yellow) were fitted into one repeating unit of the lattice. In this orientation, pUL31 would form the interface between the two lattice layers. g, h, i, j, The hexamer centres in the double-layer lattice (yellow) are spaced 2 nm further apart and are rotated by approximately 20° to the 6-2-6 axis compared to spherical NEC (blue) and flat lattice derived from the HSV-1 crystal structure (purple). k, Slice through several adjacent type-1 NR, with the lumen of one of these zippered by putative head-head interacting NEC (yellow). Inset shows an orthogonal slice through the centre of the highlighted area. l, Plotback of individual symmetry units (dodecamers). There is a slight curvature to the lattice with a break in the middle, presumably to accommodate the tighter curvature of the underlying nucleoplasmic reticulum membrane.

Extended Data Fig. 4 Subvolume averaging generates a plausible model of NEC lattice order.

Shown is a top view of a single perinuclear vesicle (also in Figs. 2, 3 and Supplementary Fig. 8). The surface (generated and visualised using Open3D) was coloured with the intersecting voxel densities of either a, volume where an NEC average volume was backplotted using subvolume averaging particle positions or b, c, the original data. Orange lines highlight the same areas in a and b where particles were removed due to their relatively low cross correlation coefficient. A long-range hexagonal order is apparent outside these areas. Assessing the nature of disordered regions is more challenging. To guide the reader’s eye, some densities in putative disordered regions were highlighted with red lines. A single hexagonal region was highlighted in blue for comparison. Note: A direct interpretation of tomogram densities on this scale can be misleading and should be used with caution. This example is intended to highlight that there is likely NEC in the disordered regions. What the structure of this lattice may be is not clear.

Extended Data Fig. 5 Questionable connecting densities between nuclear capsids and budding NEC.

The NEC subvolumes were classified by the distance to the nearest penton vertex (top panel) and vice versa (bottom panel). Each column shows sections through the resulting class average volume, with the maximum separation distance and the number of particles included in the average indicated above. All volumes were filtered using the same bandpass filter. The NEC layer is smeared in the penton-aligned averages and accordingly the capsids are smeared in the NEC-aligned averages, indicating that the two lattices are not aligned. There is a hint of a density originating from pentons closer than ~7 nm from the nearest NEC surface (orange arrows), but any interpretation of this would be highly questionable due to the small number of particles.

Extended Data Fig. 6 Putative NEC coat on negatively curved surfaces.

a, b, c, Slices through the same nucleoplasmic reticulum at different depths showing the distribution of the putative NEC layer. d, e, An enlarged section of panel c, showing top views of ring-like structures (highlighted in blue in e). f Overlay of the surface representation of the average volume of 121 ring-like particles from two tomograms and the spherical NEC lattice. Each ring could plausibly accommodate two concentric layers of pUL31/34 dimers. Averaging a more exhaustive (but less stringently picked) set of negatively curved lattice particles did not converge (and is therefore not shown), suggesting a high degree of variability. Notably, the membrane was not included as an alignment feature. g, The thickness and distance to the membrane of this layer are consistent with the spherical NEC lattice. Sections through the ring average volume with different C symmetries applied. Visually, C7 is the best match to C1 but it is possible these structures have no strict symmetry, as suggested by d, e. Note that symmetrisation in this case means addition of subvolumes at defined rotations (for example 5 subvolumes with 60° degree increments for C6 symmetry). Alignment was performed after the addition of symmetry related particles. The bottom left-most panel is a section through the spherical NEC lattice.

Extended Data Fig. 7 Localization of individual nuclear and cytoplasmic mScarlet-UL25 labelled capsids.

a, PK15 cells were infected with PrV-mScarlet-UL25 or b, PrV-mScarlet-UL25-∆US3 fixed at 7 or 10 hpi, respectively, and imaged using spinning disc microscopy. UL25-mScarlet (red); DNA-Hoechst (blue). One plane of the acquired volume is shown. c, Viral particles were detected in the 3D volumes using Trackmate in FIJI with an expected blob diameter of 0.4 microns and the quality threshold set to 10.0. c, Fluorescent signal in a single plane of a PK15 cell infected with PrV-mScarlet-UL25-∆US3 and fixed 10 hpi (top) and projection of all detected single particles (purple) of the volume in a nuclear ROI (yellow) onto one plane (bottom). d, e, The FIJI plugin Trackmate was used to detect and measure individual virus particle fluorescent intensities. For each condition, the total intensity of more than 4,000 particles was quantified and detected in more than 30 different cells, all using a single biological replicate.

Source data

Extended Data Fig. 8 PrV nuclear egress complex forms tubes with helical symmetry.

a, b, Comparison of the NEC curvature in perinuclear vesicles and tubes. b, Shown are sections through the average volumes of four tubes, with the respective diameters indicated in cyan. The NEC tubes have substantially smaller luminal cavities compared to spherical NEC vesicles. Notably, the two separate tubes with matching diameters and helical parameters were located within the same nucleoplasmic reticulum and may have originated from the same assembly. c, Tubular NEC forms tubes with different helical parameters, indicating a flexibility in the direction of largest curvature. Highlighted is the angle of the 6-2-6 axis to the helical symmetry axis. Scale bars indicate 50 nm.

Extended Data Fig. 9 NEC in HSV-1.

a, Slice through a tomogram depicting primary enveloped nucleocapsid egressing to the cytosol. The NEC coat of WT HSV1 (red) follows the same pattern as that of ΔUS3 PrV and could be identified on the INM and in perinuclear vesicles. Likewise ring-like structures similar to those in PrV (yellow in i. and iii., Fig. 3c and Extended Data Fig. 6) was identified near the hexagonal NEC lattice (red in i. and iii.). Red area in the inset ii. shows budding NEC. b, HSV-1 NEC crystal structure (PDB ID 4ZXS) fitted into the subvolume average of HSV-1 NEC from perinuclear vesicles. ONM, outer nuclear membrane; INM, inner nuclear membrane. c, Raw tomographic slices through C-capsids of WT HSV-1 in indicated subcellular locations. d, Slices through the average volumes of C-capsids (middle) and their surface representations (bottom). Arrowheads indicate the position of an additional density present in the cytosolic capsids. 15 tomograms from 5 preparations were used.

Extended Data Table 1 Herpesvirus protein nomenclature

Supplementary information

Supplementary Information

Supplementary Figs. 1–8, Supplementary Table 1 and sequence notes.

Reporting Summary

Supplementary Video 1

Tomographic volume of capsids, perinuclear vesicles and other structures observable in the nucleus. The orange arrowheads indicate scaffolds found within chromatin-deficient areas. The magenta arrowhead shows a partial capsid that may be undergoing assembly. The yellow arrowhead points towards assembling B-capsids that are yet to spool DNA. The cyan arrowhead shows a capsid in the process of spooling DNA, which is assumed due to the more loosely packed density within the capsid—individual DNA fibres are visible. The blue arrowheads show C-capsids in the nucleus containing densely packed DNA.

Supplementary Video 2

Tomographic volume of perinuclear vesicles in the lumen between the INM and ONM. The black arrowheads point towards openings of perinuclear vesicles at the ONM towards the cytosol.

Supplementary Video 3

Tomographic volume of NEC-associated structures in type 1 nucleoplasmic reticulum herniations and invagination of the INM by the NEC. The orange arrowheads represent scaffolds found, once again, within chromatin-deficient areas. In one area of the tomogram, a scaffold is visible in an NEC tube. The yellow arrowhead points towards assembling B-capsids that are yet to spool DNA while the blue arrowheads point towards fully formed C-capsids. The red arrowhead points to invagination sites caused by the NEC. On one of these occasions, no structures are apparent to cause such an invagination to occur (bottom). The green asterisk shows a perinuclear vesicle containing a B-scaffold. The blue arrow shows an NEC tube-like structure.

Supplementary Video 4

Tomographic volume of perinuclear vesicles in type-1 nucleoplasmic reticula, capsids budding into perinuclear vesicles and tubular NEC-associated structure. Red arrows point toward assembling heterodimeric NEC arrays at the INM that are associated with capsids and a vesicle-like structure (pink arrowhead). A green asterisk denotes a perinuclear vesicle that has formed and enveloped 2 individual capsids (an A-capsid and a C-capsid). The blue asterisk denotes an NEC-associated tube, the sub-volume average for which can be seen in Fig. 6. A white arrowhead shows a small opening toward the nucleus in a perinuclear vesicle that is almost fully formed.

Source data

Source Data Fig. 1

Source data for Figs. 2 and 5, Extended Data Fig. 2, Extended Data Fig 7 and Supplementary Fig. 6b.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pražák, V., Mironova, Y., Vasishtan, D. et al. Molecular plasticity of herpesvirus nuclear egress analysed in situ. Nat Microbiol 9, 1842–1855 (2024).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing